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Dual Action of Eeyarestatin 24 on Sec-Dependent Protein Secretion and Bacterial DNA
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  • Ann-Britt Schäfer
    Ann-Britt Schäfer
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden
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  • Maurice Steenhuis
    Maurice Steenhuis
    Molecular Microbiology, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands
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  • Kin Ki Jim
    Kin Ki Jim
    Department of Medical Microbiology and Infection Prevention, Amsterdam University Medical Centers - Location Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands
    Amsterdam Institute for Infection and Immunity, Amsterdam University Medical Centers, 1081 HZ Amsterdam, The Netherlands
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  • Jolanda Neef
    Jolanda Neef
    Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, The Netherlands
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  • Sarah O’Keefe
    Sarah O’Keefe
    School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, United Kingdom
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  • Roger C. Whitehead
    Roger C. Whitehead
    School of Chemistry, Faculty of Science and Engineering, University of Manchester, Manchester M13 9PL, United Kingdom
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    Orcidhttps://orcid.org/0000-0003-2910-4033
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    Eileithyia Swanton
    School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, United Kingdom
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  • Biwen Wang
    Biwen Wang
    Bacterial Cell Biology and Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH Amsterdam, The Netherlands
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  • Sven Halbedel
    Sven Halbedel
    FG11 Division of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, 38855 Wernigerode, Germany
    Institute for Medical Microbiology and Hospital Hygiene, Otto von Guericke University Magdeburg, 39120 Magdeburg, Germany
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  • Stephen High
    Stephen High
    School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, United Kingdom
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  • Jan Maarten van Dijl
    Jan Maarten van Dijl
    Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, The Netherlands
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    Orcidhttps://orcid.org/0000-0002-5688-8438
  • Joen Luirink*
    Joen Luirink
    Molecular Microbiology, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands
    *Email: [email protected]
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    Michaela Wenzel
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden
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    Orcidhttps://orcid.org/0000-0001-9969-6113
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ACS Infectious Diseases

Cite this: ACS Infect. Dis. 2023, 9, 2, 253–269
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https://doi.org/10.1021/acsinfecdis.2c00404
Published January 13, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Eeyarestatin 24 (ES24) is a promising new antibiotic with broad-spectrum activity. It shares structural similarity with nitrofurantoin (NFT), yet appears to have a distinct and novel mechanism: ES24 was found to inhibit SecYEG-mediated protein transport and membrane insertion in Gram-negative bacteria. However, possible additional targets have not yet been explored. Moreover, its activity was notably better against Gram-positive bacteria, for which its mechanism of action had not yet been investigated. We have used transcriptomic stress response profiling, phenotypic assays, and protein secretion analyses to investigate the mode of action of ES24 in comparison with NFT using the Gram-positive model bacterium Bacillus subtilis and have compared our findings to Gram-negative Escherichia coli. Here, we show the inhibition of Sec-dependent protein secretion in B. subtilis and additionally provide evidence for DNA damage, probably caused by the generation of reactive derivatives of ES24. Interestingly, ES24 caused a gradual dissipation of the membrane potential, which led to delocalization of cytokinetic proteins and subsequent cell elongation in E. coli. However, none of those effects were observed in B. subtilis, thereby suggesting that ES24 displays distinct mechanistic differences with respect to Gram-positive and Gram-negative bacteria. Despite its structural similarity to NFT, ES24 profoundly differed in our phenotypic analysis, which implies that it does not share the NFT mechanism of generalized macromolecule and structural damage. Importantly, ES24 outperformed NFT in vivo in a zebrafish embryo pneumococcal infection model. Our results suggest that ES24 not only inhibits the Sec translocon, but also targets bacterial DNA and, in Gram-negative bacteria, the cell membrane.

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Antimicrobial resistance is a major threat to public health, with some countries reporting resistance rates as high as 92.9% against commonly used antibiotics. (1) Both Gram-positive and Gram-negative bacteria contribute to this problem, (2) yet the development of innovative drugs, especially with broad-spectrum activity, is still lacking. In particular, the number of antibiotics in clinical development that belong to truly novel structural or mechanistic classes is limited. (3) Considerable efforts have been undertaken to find such new classes, and the preclinical end of the pipeline has been filled with compounds with untapped mechanisms. (4) While it is promising that a number of these drug candidates target Gram-negative bacteria, which are difficult to treat because of their highly impermeable outer membrane, only a few have broad-spectrum activity. (4)
Eeyarestatin 24 (ES24) is a recently discovered small molecule with broad-spectrum antibacterial activity and a novel mechanism of action. It is derived from eeyarestatin 1 (ES1), a compound that was first discovered in a high-throughput screen for inhibitors of the mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for the disposal of misfolded proteins in a quest to find new therapeutics for diseases that are characterized by protein degradation defects. (5,6) ES1 inhibits two major ER-based cellular processes: the ER-associated degradation (ERAD) of misfolded proteins (7−10) and the Sec61-dependent translocation of nascent polypeptides into the ER. (11−13) Recently, a cryogenic electron microscopy (cryo-EM) structure of Sec61 in complex with ES1 has been solved. (14) ES24 is a smaller derivative of ES1, which retains its activity against Sec61 but not the ERAD system. (11) Since the eukaryotic Sec61 complex is homologous to the bacterial SecYEG translocon, (15) ES1 and ES24 have also sparked interest as potential inhibitors of bacterial protein secretion, a pathway that is essential in both Gram-positive and Gram-negative bacteria but has not yet been exploited as an antibiotic target. (16−19)
While ES1 was unable to cross the Gram-negative outer membrane and exhibited significant mammalian cell toxicity, ES24 showed broad-spectrum antibacterial activity and was significantly less toxic to HEK293 cells. (11,16) It was recently shown using the Gram-negative Escherichia coli as a model system that ES24 indeed inhibits SecYEG-dependent protein translocation, both in terms of inner membrane protein insertion and protein secretion into the periplasm. In line with this observation, stress response profiling revealed an activation of the σ32-dependent heat shock response. (16)
ES24 contains a nitrofuran group and, thus, shares structural similarity with the long-established antibiotic nitrofurantoin (NFT) (Figure 1A), which is commonly prescribed for the treatment of urinary tract infections. (20) Despite being widely used since 1953, resistance against this drug is relatively uncommon. (20) NFT is a prodrug. Upon activation by bacterial nitroreductases, reactive derivatives are formed that damage cellular components, in particular, the DNA and cell membrane, in a manner similar to oxidative stress. (21−24) Interestingly, ES24 also requires the presence of the nitroreductases NsfA and NsfB to inhibit the growth of E. coli, which suggests that it undergoes a similar activation process. (16) Notably, ES24 displayed higher activity than NFT against different Gram-positive and Gram-negative test strains. (16) Comparative stress response profiling revealed a significant overlap between the two compounds, in particular, activation of SOS and oxidative stress responses, yet nitrofurantoin had no effect on SecYEG-dependent protein translocation. (16) This raised the question of whether the activation of ES24 by cellular nitroreductases may also result in reactive compounds that cause cellular damage, in addition to its inhibitory effect on protein secretion. Moreover, both ES24 and NFT have led to cell elongation, thereby indicating potential additional or downstream effects on cell division. (16)

Figure 1

Figure 1. Summary of B. subtilis transcriptomic stress profiling results. (A) Chemical structures of ES24 and NFT. The nitrofuran ring is highlighted in red. (B) Number of regulated transcripts per regulon. Sec components and proteases potentially indicating secretion stress are highlighted. NfrAB is a nitro/flavin reductase under transcriptional control of the Spx regulator and among the most strongly activated transcripts for both ES24 and NFT treatment. (C) Number of induced transcripts per functional category of the respective gene products. Functional categories were assigned according to function, regulation, interaction, and expression data available on http://subtiwiki.uni-goettingen.de/. Transcripts that showed a log2-fold change of ≥3 (p < 0.05) were considered as differentially regulated.

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These observations left several open questions, most importantly whether ES24 is truly specific for SecYEG or if it displays any additional activities in bacteria, possibly related to the formation of reactive derivatives or the downstream effects of impaired protein translocation. Moreover, ES24 was notably more active against Gram-positive species [30-fold lower minimal inhibitory concentration (MIC) against Bacillus subtilis compared with E. coli), but its mechanism of action against this class of bacteria remains to be elucidated. Since Gram-positive bacteria lack a membrane-enclosed periplasm, the effects of secretion inhibition by ES24 may differ from observations made in E. coli. Finally, even though ES24 was more active against bacterial pathogens than NFT and less cytotoxic than its parent compound ES1, its in vivo efficacy has not yet been assessed.
Here, we characterized the mechanism of action of ES24 in the Gram-positive model organism B. subtilis and compared it with NFT. Stress response profiling revealed the activation of oxidative stress, DNA damage, and membrane stress responses. Following up on these different leads, we performed phenotypic analyses using different bacterial cytological profiling techniques. We found that ES24 indeed affects SecYEG-dependent protein translocation in B. subtilis but also induces DNA damage. ES24 was notably different from NFT in all assays, which suggests that it does not generate the same reactive derivatives, at least not at concentrations sufficient to inhibit bacterial growth. Strikingly, ES24 outperformed NFT in vivo in a zebrafish embryo pneumococcal infection model, thereby highlighting the clinical promise of this compound.

Results

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Transcriptomic Stress Response Profiling

We started out with transcriptomic stress response profiling as an unbiased global approach. To this end, we first determined suitable antibiotic concentrations for physiological stress experiments in our model strain B. subtilis 168CA. Minimal inhibitory concentrations (MICs) against this strain were 0.75 μM for ES24 and 15 μM for NFT. This is similar to previous results reporting ES24 to be 25 times more active against B. subtilis than NFT. (16)
Transcriptomics experiments were performed according to the conditions previously used for E. coli to achieve optimal comparability. (16) Thus, cultures were grown to mid-log phase, diluted to an OD600 of 0.05, and subsequently treated with 3 μM ES24 or 25 μM NFT for 15 min prior to RNA extraction and sample analysis. Figure 1B,C shows an overview of the transcriptomic data set. Tables S1–S3 summarize all upregulated transcripts. The full raw data sets can be found in the Supporting Information.
We first analyzed the regulons of differentially regulated transcripts, as previously done for E. coli (Figure 1B). (16) This analysis revealed that the major up- and downregulated regulons are rather similar for ES24 and NFT (Figure 1B), which follows the same trend previously observed in E. coli. (16) Affected regulons reflected general and large-scale stress adaptations, such as sporulation, stringent response, general stress response, chemotaxis, and motility. This is characteristic for compounds that affect multiple downstream processes.
Both compounds showed induction of transcripts regulated by LexA, thereby controlling the SOS response to DNA damage, which is consistent with the results obtained for E. coli. (16) While B. subtilis did not show a clear heat-shock response, as previously observed in E. coli, we did observe the upregulation of proteases, which is consistent with secretion inhibition, yet may also be caused by general stress. Notably, ES24, but not NFT, led to downregulation of Sec components (Figure 1B). Another marked difference was observed regarding the cell envelope stress-responsive σW regulon, which was induced by ES24 but repressed by NFT.
In B. subtilis, a significant number of genes is part of more than one regulatory mechanism. Therefore, we performed a deeper analysis of the functions of upregulated transcripts. To this end, we sorted all transcripts into functional categories by taking into account function, expression, regulation, and interaction data from the SubtiWiki database (www.subtiwiki.uni-goettingen.de) (25) (Figure 1C, Tables S1–S3). This analysis revealed largely overlapping stress responses to ES24 and NFT, with sporulation-related genes being the largest upregulated group. It also showed DNA repair and proteolysis as major responses to both compounds, while cell envelope stress was more prominent in ES24-treated samples. Additionally, we observed the upregulation of genes encoding membrane transport proteins, which could reflect an attempt to compensate for impaired protein secretion. Similar to other observations, this response was stronger in ES24-treated samples. Interestingly, we also observed that both ES24 and NFT induced a large number of transcripts that are typically found upregulated upon oxidative stress.
Taken together, the transcriptome analysis led us to the following hypotheses: (1) both ES24 and NFT cause the formation of reactive species that elicit an oxidative stress response; (2) both compounds induce DNA damage; (3) ES24 induces membrane stress, possibly because of disturbed protein secretion; and (4) ES24 impairs the Sec translocon. In the following sections, we tested each of these hypotheses with independent assays.

Antibiotic Concentrations for Phenotypic Analyses

To this end, we used a bacterial cytological profiling approach, making use of different fluorescent protein fusions and dyes that have been successfully employed in previous mode of action studies. (26−28) We first determined optimal stressor concentrations for these assays by examining the effects of acute antibiotic stress in log phase (Figure S1). We chose concentrations that led to a temporary halt of culture growth but did not result in cell death. This ensures that the stress applied is enough to elicit a phenotype while avoiding effects that are merely a consequence of cell death. These conditions were met at 4 μM for ES24 and 25 μM for NFT (Figure S1). Since the similarity in the transcriptome profiles led us to hypothesize that ES24 could possibly have a dual mechanism of inhibiting protein secretion and generating reactive species, we additionally included a lower concentration of 2 μM to assess any concentration-dependent effects. These concentrations were then used throughout all phenotypic experiments using B. subtilis. As additional controls, we used gramicidin (an antibiotic that forms a transmembrane ion channel and consequently perturbs a variety of cellular functions) and, where indicated, hydrogen peroxide (H2O2, producing hydroxyl radicals) and paraquat (producing superoxide) as controls for oxidative stress.

Oxidative Stress

Nitrofurantoin has been suggested to generate reactive oxygen species (ROS), (29) and both ES24 and NFT induced a high number of transcripts that indicate oxidative stress (Figure 1C) and are typically induced by hydrogen peroxide. (30) Therefore, we examined whether ROS are present in the antibiotic-treated samples using the fluorescent reporter Oxyburst Green (Figure S2). (31) However, we did not observe a marked increase in fluorescence signal for any of the samples compared with 0.3% hydrogen peroxide (Figure 2A). To confirm this result, we determined MICs against B. subtilis in the absence and presence of the superoxide scavenger tiron and the hydroxyl radical scavenger thiourea. (32,33) While tiron moderately, and thiourea strongly, increased the MIC of the positive control hydrogen peroxide, neither scavenger affected the MICs of ES24 or NFT (Figure 2B), thereby supporting the notion that no ROS are formed by these antibiotics.

Figure 2

Figure 2. Oxidative stress assays. (A) Oxyburst Green assay for detecting ROS in B. subtilis 168CA. The fluorescent probe shows a marked increase in fluorescence when ROS are present. It should be noted that Oxyburst Green has been reported to be specific for the detection of superoxide, yet we have found that it reacts to different sources of ROS (see Figure S2). (B) Influence of ROS scavengers on antibiotic activity against B. subtilis 168CA. MICs were determined in the absence or presence of the superoxide scavenger tiron or the hydroxyl radical scavenger thiourea. If one of these ROS species contributes to the activity of the compound, the MIC should increase in the presence of the respective scavenger.

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DNA Damage

While we did not find evidence for the presence of ROS, it is possible that nitroreductases metabolize NFT and/or ES24 into other reactive species, for example, nitric oxide or other reactive nitrogen species, that may cause similar cell damage and, hence, elicit a similar stress response without necessarily being detected by ROS-specific probes or scavengers. This hypothesis is supported by the upregulation of DNA damage repair mechanisms, such as the LexA-regulated SOS response, which are typically induced upon oxidative DNA damage (Figure 1B,C). Therefore, we examined the effects of the compounds on bacterial DNA in more detail. We first examined nucleoid morphology with a DAPI stain and used the membrane dye Nile red as a counter stain (Figure 3, Figure S3). It was previously observed that the treatment of B. subtilis with high NFT concentrations led to a temporary condensation of the nucleoid followed by an entirely dispersed DAPI signal after 30 min. (21) Here, despite using lower concentrations of NFT, the same effect was observed, thereby indicating strong effects on nucleoid integrity. ES24 caused similar, yet overall slower and milder, effects. Thus, we observed nucleoid condensation after 30 min followed by relaxation after 60 min, yet no complete dispersion of the DAPI signal. Notably, these phenotypes did not resemble the nucleoid morphology observed after treatment with hydrogen peroxide or paraquat (Figure S4), thereby supporting our notion that ROS do not account for these effects.

Figure 3

Figure 3. Bacterial cytological profiling of B. subtilis 168CA treated with ES24 or NFT. Red arrows indicate membrane aberrations, and blue arrows indicate nucleoid aberrations. See Figure S4 for corresponding images of cells treated with hydrogen peroxide and paraquat. Scale bar, 2 μM.

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We verified that the observed effects on nucleoid morphology are indeed indicative of DNA damage by employing a green fluorescent protein (GFP) fusion to the DNA damage reporter RecA (Table 1, Figure 4). This DNA repair protein is uniformly associated with the nucleoid and, upon DNA damage, forms foci over the damaged sites. (34) Indeed, RecA foci were observed for both ES24 and NFT-treated cells, thereby indicating that both compounds induce DNA damage. While we observed more cells with distinct RecA foci upon treatment with NFT, ES24-treated cells showed a clear, concentration-dependent increase of RecA foci. In both cases, the RecA response was strongest after 10 min of treatment and decreased over time (Figure S5).

Figure 4

Figure 4. Localization of RecA-GFP (B. subtilis UG10) after treatment with ES24 or NFT. Gramicidin (Gra) was used as additional control. Yellow arrows indicate clustered protein. Scale bar, 2 μM.

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Table 1. Overview of GFP Localization Results, With the Ion Pore-Forming Peptide Antibiotic Gramicidin (Gra), Which Affects Nearly All GFP Fusion Proteins, Used as a Positive Controla
reporter forproteinlocalization pattern
  untreated0.53 μM Grab15 μM NFTc2 μM ES244 μM ES24
DNA damageRecAcytosolic: dispersedcytosolic: dispersednucleoid-associated focibnucleoid-associated focibnucleoid-associated focib
replicationDnaNcytosolic: nucleoid-associatedcytosolic: dispersedbcytosolic: dispersedccytosolic: nucleoid-associatedcytosolic: nucleoid-associated
transcriptionRpoCcytosolic: nucleoid-associatedcytosolic: dispersedbcytosolic: dispersedbcytosolic: nucleoid-associatedcytosolic: nucleoid-associated
translationRpsBcytosolic: excluded from nucleoidcytosolic: dispersedbcytosolic: dispersedccytosolic: excluded from nucleoidcytosolic: excluded from nucleoid
membrane potential, cell divisionDivIVAmembrane-associated: septal/polarcytosolic: dispersedbcytosolic: dispersed, membrane: clustersbmembrane-associated: septal/polarmembrane-associated: septal/polar
membrane potential, cell divisionMinDmembrane-associated: septal/polarcytosolic: dispersedbcytosolic: dispersed, membrane: clusterscmembrane-associated: septal/polarmembrane-associated: septal/polar
ATP synthesisAtpAmembrane-associated: focicytosolic: dispersedbcytosolic: dispersed, membrane: clusterscmembrane-associated: focimembrane-associated: foci
protein secretionSecAmembrane-associated: focicytosolic: dispersedbcytosolic: dispersed, membrane clusterscmembrane: clusterscmembrane: clustersc
a

Fusions showing an effect are in bold.

b

Immediate effects.

c

Effects visible after 30–120 min.

We gained insight into whether this DNA damage and the concomitant changes to nucleoid morphology affect other nucleoid-associated proteins by examining the localization of the DNA polymerase DnaN and the RNA polymerase RpoC. Complementarily, we included the ribosomal protein RpsB, which is excluded from the nucleoid (Table 1, Supplementary Figures S6–S8). While NFT showed strong effects on the localization of all three proteins, ES24 did not show any pronounced phenotype in these assays, which is in line with its milder effects on nucleoid morphology and a less pronounced RecA response (Figure 3 and 4).

Membrane Stress

Our transcriptomic analysis revealed that ES24 induced more transcripts that are functionally connected to membrane stress than NFT. Yet, none of the common membrane stress markers that are typically induced by membrane-interacting compounds, like gramicidins, (35) were upregulated by ES24. If ES24 indeed inhibits the Sec translocon in B. subtilis, an accumulation of normally secreted proteins in the cell membrane may explain such an “atypical” membrane stress response. If that is, indeed, the case, we should not observe phenotypes typically associated with direct membrane damage. If, however, reactive species are formed that attack the cell membrane, at least some effects should be observed, as recently shown for NFT. (21) We investigated this by examining membrane parameters that are typically affected by membrane-interacting compounds: membrane morphology, membrane potential, membrane fluidity, and membrane protein localization.
We first examined the Nile red membrane stain for morphological changes of the cell membrane. While ES24 had no visible effect on this membrane stain, NFT caused aberrant membrane foci (Figure 3, Figure S3), which is in line with an earlier study. (21) Similar membrane defects were observed after treatment with hydrogen peroxide and paraquat (Figure S4), which suggests that they may be due to membrane damage caused by reactive species. Next, we tested whether the compounds affect the membrane potential using the voltage-sensitive dye DiSC(3)5. Neither ES24 nor NFT showed a notable effect in this assay (Figure 5A,B). We then examined the effects of the compounds on membrane fluidity, which has recently emerged as a key factor in the activities of membrane-active antibiotics. (26−28,36,37) We used the fluidity-sensitive fluorescence dye laurdan (38,39) and observed no change in membrane fluidity in ES24-treated cells. In contrast, NFT caused a clear membrane fluidization (Figure 5C). However, this effect disappeared during extended treatment, thereby suggesting that cells are able to re-establish their original membrane fluidity (Figure 5D). We hypothesized that this transient fluidization could be a consequence of lipid peroxidation due to reactive species. However, when we measured the membrane fluidity of cells treated with peroxide and paraquat, only very minor effects were observed, which suggests that lipid peroxidation does not lead to a similar fluidization effect (Figure S9). Thus, it is a more likely explanation that the transient fluidization observed with NFT is due to a temporary disturbance of membrane function when the compound crosses the bilayer.

Figure 5

Figure 5. Effects of ES24 and NFT on B. subtilis membrane potential and fluidity. (A) Kinetic DiSC(3)5 spectroscopy measurements of B. subtilis 168CA treated with ES24, NFT, or positive control gramicidin (Gra). An increase in fluorescence intensity indicates membrane depolarization. The arrow indicates the time point of antibiotic addition. (B) End point measurements under the same conditions as in panel A up to 90 min. (C) Kinetic laurdan generalized polarization (GP) spectroscopy measurements of B. subtilis 168CA treated with ES24, NFT, or positive control benzyl alcohol (BA). A decrease in GP indicates membrane fluidization. The arrow indicates the time point of antibiotic addition. (D) End point measurements under the same conditions as in panel C up to 120 min. The p-values were calculated using a paired two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001.

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To corroborate our findings, we examined the localization of the peripheral membrane proteins MinD and DivIVA, both of which are involved in cell division site regulation and depend on the membrane potential for correct localization, (28,40) and AtpA, a subunit of the transmembrane ATP synthase complex that is insensitive to fluctuations in both membrane potential and fluidity. (28) ES24 did not affect the localization of any of these proteins (Table 1, Figures S10–S12). In contrast, NFT affected all three proteins, with DivIVA delocalizing in a shorter time frame than MinD and AtpA.

Inhibition of Protein Secretion

To examine whether ES24 inhibits the Sec translocon in B. subtilis, we first examined the localization of a GFP fusion to the Sec component SecA. Prolonged treatment with ES24 caused a clear accumulation of SecA at specific sites in the cell membrane (Table 1, Figure 6, Figure S13). While NFT and gramicidin also affected SecA localization, they caused the protein to dissociate into the cytosol, an effect commonly observed for peripheral membrane proteins upon membrane damage. (26−28,41) The accumulation of SecA at specific sites, as observed with ES24, rather indicates a disturbance of the Sec translocon itself. We excluded that the observed accumulation of SecA may be related to delayed transition into stationary phase in treated cells (Figure S1) by sampling the localization of SecA at different time points over the growth curve of an untreated control culture (Figure S14). While SecA did lose its membrane binding after cells entered the late stationary “death phase,” we did not detect any accumulation into large foci, as observed for ES24-treated cells at any stage during the growth curve.

Figure 6

Figure 6. Localization of SecA-GFP after treatment with ES24 or NFT. Gramicidin (Gra) was used as additional control. Yellow arrows: clustered protein. Green arrows: dispersed protein. Scale bar, 2 μM.

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We then analyzed the capability of B. subtilis cultures to secrete the extracellular amylase AmyM using Western blotting. To this end, we used a B. subtilis strain expressing amyM from the constitutive PamyQ promoter. As shown in Figure 7, ES24 caused a clear, concentration-dependent inhibition of AmyM secretion, while no effect was observed with NFT (see Figures S15 and S16 for additional controls), thereby indicating that ES24 indeed inhibits SecYEG-dependent protein secretion in Gram-positive bacteria. As an additional control, we included the cytosolic protein PycA in the Western blot analysis. Neither ES24 nor NFT affected the levels of this control protein. We corroborated our results by additionally investigating the secretion of LipA, an extracellular lipase that is secreted by the Sec translocon. (42) Indeed, we observed that 2 μM ES24 caused a 20–30% reduction in LipA secretion compared with bacteria cultured in the absence of ES24 or bacteria treated with only 1 μM ES24. In contrast, NFT at concentrations of up to 12 μM, had no detectable effect on LipA secretion (Figure S17).

Figure 7

Figure 7. Effect on AmyM secretion. B. subtilis BWB09 (ΔxynA ΔamyE) carrying pCS73 (PamyQ–amyM) to express amyM from the constitutive PamyQ promoter was grown until mid-log phase and treated with different concentrations of ES24 or NFT for 3 h prior to SDS-PAGE and Western blotting. (A) Western blot analysis of culture supernatant using an α-AmyM antibody (top) and of cell lysate using an α-streptavidin antibody detecting the cytosolic protein PycA (bottom). (B) Quantification of pixel intensity in AmyM bands. **p < 0.01.

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Mechanistic Differences in E. coli

Taken together, our data on B. subtilis suggest that NFT causes general cell damage through reactive species while ES24 also acts through secretion inhibition. However, the effects of secretion inhibition may have different consequences in Gram-negative bacteria because of the presence of a periplasm. Thus, it is known that secretion stress activates different membrane stress responses in E. coli. (43) Therefore, we used DiSC(3)5 microscopy (44) to perform membrane potential measurements in E. coli. Indeed, we could observe a significant reduction in membrane potential after prolonged treatment with both ES24 and NFT (Figure 8A). A similar, yet less pronounced, trend was observed with paraquat but not hydrogen peroxide, which suggests that the formation of superoxide may also lead to a partial loss of membrane potential (Figure S18).

Figure 8

Figure 8. Effects of ES24 on cell membrane function in E. coli. (A) DiSC(3)5 measurements of E. coli MC4100 carrying pABCON2-fhuA ΔC/Δ4L treated with ES24 or NFT. Polymyxin B (PolB) was used as positive control. p-Values were calculated using a heteroscedastic, two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001. (B) MinD oscillation in E. coli RC1 carrying pFX9 treated with ES24 or NFT. The proton ionophore CCCP, which is known to abolish MinD oscillation due to membrane depolarization, (40) was used as positive control. (C) Quantification of FtsZ delocalization. Cells displaying an entirely cytosolic GFP signal, GFP clusters in the cell membrane, double bands, or spirals of FtsZ were counted as delocalized. Cells displaying single FtsZ bands, two opposing foci, or one focus at midcell (indicative of a closing septum) were counted as normal.

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We next examined whether the observed membrane depolarization has an effect on membrane proteins in E. coli by performing timelapse fluorescence microscopy with a strain expressing GFP-labeled MinD. (45) In E. coli, MinD oscillates from pole to pole, and this oscillation is aborted when the membrane potential is dissipated by ionophores, such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP). (40) This property has made fluorescent fusions to MinD a popular reporter for membrane depolarization. (35,44,46) However, no significant effects on the number of oscillating cells were observed with ES24, thereby suggesting that the partial membrane depolarization observed in the DiSC(3)5 assay is not sufficient to abort MinD oscillation. In contrast, the number of oscillating cells was significantly reduced in NFT-treated cultures and further decreased during prolonged incubation (Figure 8B, Supplementary Figure S19). When examining our time lapse data in more detail, we observed that while MinD still oscillated in ES24-treated samples, this oscillation appeared to slow down in many ES24-treated cells compared with the untreated control. However, the high cell-to-cell variability in oscillation speed did not allow for meaningful analysis and quantification of this observation.
Therefore, we examined the localization of another cell division protein, FtsZ. This tubulin homologue is the major bacterial cell division protein and is anchored to the cell membrane through FtsA. (47) Like MinD, FtsA is sensitive to dissipation of the membrane potential, yet appears to be more sensitive than MinD. (40,48) Since FtsA is the only membrane anchor of FtsZ in E. coli, FtsA delocalization inevitably causes FtsZ delocalization. In fact, we observed a clear and time-dependent delocalization of FtsZ in both ES24 and NFT-treated E. coli. In line with the DiSC(3)5 data, this effect was both time-dependent and stronger for NFT (Figure 8C, Figure S20). We also observed cell elongation, which was previously observed for ES24-treated E. coli (16) and can now be explained by FtsZ delocalization.

Efficacy in a Zebrafish Embryo Infection Model

ES24 shows promising potential as an antibiotic, as demonstrated by its novel mechanism of action, low cytotoxicity, and broad-spectrum antibacterial activity, which is equal to or better than that of NFT against common uropathogens. (11,16) We further examined its suitability as a future antibiotic drug by assessing its in vivo efficacy against Streptococcus pneumoniae, a common Gram-positive pathogen, in a zebrafish embryo infection model. (49) To this end, we first confirmed its bactericidal activity against a S. pneumoniae D39 serotype 2 strain in vitro. Complete killing was achieved at 6.25 μM, similar to NFT (Figure 9A). After confirming its bactericidal activity, we proceeded with S. pneumoniae infection experiments in two-day-old zebrafish embryos by injecting 150 colony-forming units (CFU) of the bacteria into the bloodstream. One hour post infection, the embryos were treated with either ES24 or NFT by adding 5 μM of the respective compound to the water. While ES24 did not eradicate the infection, it significantly increased the survival time and clearly outperformed NFT in these experiments (Figure 9B), thereby underlining its clinical promise.

Figure 9

Figure 9. In vivo efficacy of ES24 in an S. pneumoniae zebrafish embryo infection model. (A) Viability of S. pneumoniae D39 after overnight incubation with a concentrations series of ES24 or NFT measured with resazurin. (B) Survival of 2 days postfertilization zebrafish embryos infected with S. pneumoniae D39. Embryos were injected with 150 CFU of bacteria in the caudal vein and subsequently treated with 5 μM ES24 or 5 μM NFT by adding the compounds directly to the water 1 h post infection (hpi). ES24 treatment improved the survival rate significantly in zebrafish embryos infected with S. pneumoniae D39 compared with NFT-treated embryos (p < 0.0001) or nontreated infected embryos (p < 0.0001). NFT also improved the survival rate compared with nontreated infected embryos, albeit not significantly (p = 0.4139). Data represents the mean ± SEM. Experiments were performed in biological triplicates with 10 fish per condition in each replicate. Survival rates were compared using log-rank statistics.

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Discussion

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ES24 is a promising antibiotic candidate with broad-spectrum activity and a novel mechanism of action. In the present study, we have further evaluated its mechanism of action and compared its effects on Gram-negative and Gram-positive bacteria. Figure 10 summarizes our major findings and takes into account previous results on E. coli. (16) We could show that ES24 indeed inhibits Sec-dependent protein translocation in both Gram-negative and Gram-positive bacteria. Given recent structural evidence that ES1 intercalates in the lateral gate/plug region of the Sec61 secretion system, thus blocking the translocation channel, (14) it is reasonable to assume that ES24 may bind similarly to the homologous bacterial SecYEG channel. Additionally, ES24 induces DNA damage. Interestingly, in Gram-negative bacteria, ES24 also partially dissipates the membrane potential and leads to cell elongation. We could further show that, despite its structural similarity to NFT and a similar stress response profile, ES24 has a profoundly different mechanism of action, which does not involve generalized macromolecule damage, as observed with NFT. Importantly, ES24 outperformed NFT in a zebrafish embryo pneumococcal infection model.

Figure 10

Figure 10. Current model of ES24 mechanism of action in Gram-positive and Gram-negative bacteria in comparison with NFT. NFT affects different cellular processes, including DNA packing; localization of DNA-binding proteins; and membrane integrity, membrane fluidity, and membrane protein binding. No clear differences were observed in its mechanism against E. coli and B. subtilis. ES24 affects Sec-mediated protein secretion and causes DNA damage in both organisms, but only affects cell division in E. coli. Reporter protein functions: MinD and DivIVA, cell division site regulation; FtsZ, cell division; SecA, Sec-dependent protein secretion; AtpA, ATP synthesis; RecA, DNA damage repair; DnaN, replication; RpoC, transcription; RpsB: translation.

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Differences in Effects on Gram-Negative and Gram-Positive bacteria

When compared with Gram-negative bacteria, ES24 has stronger antibacterial activity against Gram-positive bacteria, (16) which may indicate a higher affinity for the Sec-translocon of the latter group of bacteria. Alternatively, the absence of an outer membrane may enhance the ability of ES24 to access its target. Consistent with the second explanation, the MIC value for E. coli is reduced when outer membrane permeability is increased by the expression of a mutant β-barrel protein. (16) Since eukaryotic cells also possess a Sec homologue, cytotoxicity is a potential concern. While ES24 displays a significantly lower cytotoxicity than its parent compound ES1 (11) and did not display acute toxicity in our zebrafish embryo model, a larger therapeutic window may still increase its safety. Thus, while displaying broad-spectrum activity, ES24 may be of higher relevance as future treatment for Gram-positive, versus Gram-negative, infections.
While we could confirm that ES24 does impair Sec-dependent protein translocation in both E. coli and B. subtilis, we also found marked mechanistic differences between both organisms in our phenotypic analysis. ES24 did not affect the cytoplasmic membrane of B. subtilis in terms of membrane potential, fluidity, or morphology, and did not affect the localization of any tested membrane-bound protein other than SecA. In contrast, we did observe a gradual dissipation of the membrane potential and concomitant delocalization of FtsZ in E. coli. FtsZ localization depends on the membrane potential since its membrane anchor FtsA is sensitive to depolarization and both proteins lose their membrane binding upon treatment with ionophores. (40) It was previously observed that E. coli cells elongate after prolonged treatment with ES24, (16) which can now be explained by delocalization of the key cell division protein FtsZ. In B. subtilis, both MinD and FtsA are sensitive to dissipation of the membrane potential and so are FtsZ, its secondary membrane anchor SepF, and the cell division site regulation protein DivIVA. (28,40,50) Thus, membrane depolarization can likewise lead to cell elongation in this organism. (40) However, since ES24 did not affect the membrane potential in B. subtilis, it makes sense that we did not observe any effects on the localization of MinD or DivIVA. In line, we did not observe any change in the cell length of ES24-treated B. subtilis cells (Figures 3, 4, 6, and Figures S21 and S22).
The reason why ES24 dissipates the membrane potential in E. coli but not in B. subtilis is not yet clear. It is possible that the presence of a periplasm makes Gram-negative bacteria more sensitive to secretion defects. Indeed, defects in the Sec translocon induce expression of membrane stress response systems in E. coli. (43) However, this would not explain why the same observation was made for NFT, which did not inhibit protein secretion in either organism. It could be speculated that both compounds, being structurally similar, have a higher affinity for Gram-negative than Gram-positive cell membranes, which could, for example, lead to stronger membrane disturbances upon passing through the lipid bilayer, possibly caused by an extended translocation time, as previously suspected for membrane-active atypical tetracyclines. (21,51)
In contrast to our phenotypic assays, no apparent membrane stress response was found in the E. coli transcriptome analysis, (16) while in B. subtilis at least some membrane stress responses were activated (Figure 1). However, the most typical markers for membrane stress in B. subtilis, which are induced upon membrane depolarization, changes in membrane fluidity, or membrane protein delocalization, were not found to be upregulated in the transcriptome. (35,36,52) The absence of these markers is consistent with the absence of any of these effects in ES24-treated cells. While we cannot fully explain the absence of membrane stress markers in E. coli, despite a clear effect on both membrane potential and membrane protein localization, the transcriptome analysis was performed after 15 min of treatment while first phenotypic effects set in after 30 min. Thus, it is possible that transcriptome profiling would show a corresponding membrane stress response when analyzed after longer antibiotic exposure.

Differences between ES24 and NFT

Both ES24 and NFT carry a nitrofuran ring (Figure 1A), and both compounds require activation by bacterial nitroreductases. (16,23,24) Therefore, a first notion was that they may have a similar mechanism of action. Transcriptomic profiling of both E. coli and B. subtilis did show a significant overlap between the stress responses to ES24 and NFT (Figure 1B,C). (16) However, it was previously shown that only ES24, but not NFT, inhibited protein secretion in E. coli, (16) and we have now shown the same for B. subtilis (Figure 7). Moreover, our phenotypic analysis consistently showed major differences between ES24 and NFT in almost every assay performed, which suggests a rather specific mechanism of ES24 on the Sec translocon and more general macromolecule and structural damage by NFT.
Both ES24 and NFT elicited a DNA damage response in both E. coli (16) and B. subtilis (Figure 1B,C). Using a B. subtilis RecA-GFP strain, we could now show that, indeed, both antibiotics induce DNA damage (Figure 4). This was further corroborated by DAPI staining, which revealed morphological changes to the nucleoid after prolonged treatment with 4 μM ES24 (Figure 3). In contrast, NFT led to full dispersion of the DAPI signal, most likely because of complete structural disintegration of the nucleoid, as previously suggested by both fluorescence and electron microscopy imaging. (21) Moreover, NFT caused large-scale effects on the localization of the DNA-associated proteins DnaN and RpoC, as well as RpsB, which is excluded from the nucleoid, while ES24 did not affect any of these reporters (Figures S6–S8). Taken together, both compounds induce DNA damage, yet NFT appears to be much more aggressive and structurally disintegrates the nucleoid.
A similar pattern was observed for the cell membrane in B. subtilis. While NFT caused membrane damage, which was apparent in the Nile red stain and laurdan-based fluidity measurements, and delocalized all tested membrane proteins, ES24 had no effect in any membrane assay with the exception of SecA localization. While this was clearly different in E. coli, where ES24 did affect the cell membrane, NFT displayed stronger effects on the membrane potential, MinD oscillation, and FtsZ localization than ES24 (Figure 8, Figures S18–S20), thereby confirming the notion that the mechanism of NFT is broader and more aggressive than that of ES24.
NFT has previously been proposed to cause oxidative damage to cellular macromolecules, in particular DNA and cell membrane lipids. (21−24) However, it has not yet been clear whether it leads to the formation of ROS, such as superoxide and hydroxyl radicals, or whether activation by cellular nitroreductases generates reactive products that then oxidize cellular components. Here, we found no evidence for ROS playing a role for NFT activity, thereby supporting the latter explanation (Figure 2).
This raises the question as to whether the same, or similar, derivative species are formed upon nitroreductase-dependent activation of ES24 and NFT. For instance, it is conceivable that these compounds both lead to the formation of nitric oxide or other reactive nitrogen species, but to different extents. (53) This could explain the observed SOS response and RecA activation, as well as the overall similar, general stress response to ES24 and NFT. It would also fit with our observation that the DNA damage and nucleoid morphology changes induced by ES24 appear to be concentration-dependent. Since such a mechanism would depend on the number of reactive species released, and the activity of ES24 and NFT differs by a factor of 10, this explanation would also fit well with the generally more pronounced effects observed with NFT. It is conceivable that equimolar concentrations of ES24 would cause similar effects as NFT; however, this hypothesis could not be tested since 8 μM of ES24 already completely killed B. subtilis cultures (Figure S1).

Conclusion

Taken together, we propose that ES24 primarily acts by inhibiting the Sec translocation machinery together with damaging DNA through the generation of reactive species. We propose that in Gram-negative bacteria, membrane damage caused by aberrant protein insertion additionally contributes to its activity. Strikingly, ES24 outcompeted NFT in our zebrafish embryo infection model using the human pathogen S. pneumoniae, which suggests that its higher in vitro activity corresponds to better in vivo efficacy. These results highlight ES24 as a promising new lead structure for further drug development.

Methods

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Antimicrobial Compounds

ES24 was synthesized according to Gamayun et al. (11) Nitrofurantoin and benzyl alcohol were purchased from Acros Organics; gramicidin and CCCP were purchased from Alfa Aesar; chloramphenicol and hydrogen peroxide were purchased from Fisher Bioreagents; polymyxin B was purchased from Duchefa; and paraquat was purchased from Sigma-Aldrich. Stock solutions were prepared in sterile water (chloramphenicol, hydrogen peroxide) or sterile DMSO (all other compounds). Aliquots were kept at 4 °C (hydrogen peroxide) or −20 °C (all other compounds) until further use. Benzyl alcohol dilutions were freshly prepared for each day of experiments. Unless stated otherwise, the following antibiotic concentrations were used throughout the study: 2 μM and 4 μM ES24, 25 μM nitrofurantoin, 0.53 μM gramicidin, 100 mM benzyl alcohol, 0.3% hydrogen peroxide, and 500 μM paraquat for B. subtilis and 6.25 μM ES24, 0.72 μM polymyxin B, and 100 μM CCCP for E. coli.

Strains and Growth Conditions

Strains and plasmids used in this study are listed in Table S4. All B. subtilis and E. coli strains were aerobically grown in lysogeny broth (LB) supplemented with appropriate inducer concentrations where indicated (see Table S4). Unless stated otherwise, E. coli and B. subtilis cultures were grown at 30 °C. S. pneumoniae was grown in competence medium plus yeast (C+Y medium) at 37 °C. (54) Unless stated otherwise, experiments were performed in biological triplicates, and error bars represent the standard deviations of independent replicate experiments.

Strain Construction

Primers used for strain construction are listed in Table S5. Strain B. subtilis BSN101 (secA::Pxyl-secA-gfp cat) was constructed by restriction cloning using the pSG1164 plasmid backbone. (55) The secA gene was amplified from B. subtilis 168CA chromosomal DNA using the primer pair SV77/SV78 and cloned into pSG1164 using Asp718 and SalI restriction sites. The resulting plasmid pSH54 was transformed into B. subtilis 168CA using a standard starvation protocol. (56) Campbell integration into the native locus secA was confirmed by PCR, resistance pattern, and fluorescence microscopy.
Strain BWB09 was constructed by marker-free deletion of the xynA and amyE genes in the tryptophan–prototrophic background strain BSB1 (57) following the method by Morimoto et al. (58) Purified amplicons of the xynA upstream (primer pair BW45/BW46) and downstream sequences (BW41/42), the SpR-mazF cassette (BW05/BW06), and the xynA gene (BW47/BW48) were fused by overlap extension PCR (BW45/BW48) to create the xynA deletion cassette. The resulting recombinant DNA fragment was used to transform competent B. subtilis BSB1, followed by selection for spectinomycin resistance (150 μg/mL). Subsequently, expression of the mazF toxin was induced by the addition of 1 mM IPTG, which resulted in excision of the deletion cassette and, thus, marker-free deletion of xynA (strain BWB06). In a second analogous step, the amyE gene was deleted. Purified amplicons of the amyE upstream (BW49/BW51) and downstream sequences (BW50/BW52), the SpR-mazF cassette (BW05/BW06), and the amyE gene (BW53/BW54) were fused by overlap extension PCR (BW49/BW54), and the resulting deletion cassette was used to transform competent BWB06. Selection for spectinomycin resistance followed by IPTG-forced excision of the deletion cassette resulted in a marker-free double deletion of xynA and amyE (strain BWB09). The strains were confirmed by sequencing.

Minimal Inhibitory Concentration and Growth Experiments

Minimal inhibitory concentrations (MICs) were determined in a serial microdilution assay according to guidelines issued by the Clinical Laboratory Standardization Institute (CLSI), as described previously. (27) In short, serial 2-fold dilutions of the compound of interest were prepared in sterile 96-well plates and inoculated with 5 × 105 CFU of B. subtilis 168CA per mL. MIC plates were incubated at 37 °C under steady agitation for 16 h. Optical density readings were taken at 600 nm. Growth experiments aimed at finding an appropriate stress level for physiological experiments were performed with B. subtilis 168CA, as described previously. (26) MICs and growth experiments were performed in biological duplicates.

Transcriptomics

B. subtilis 168CA cells were grown at 30 °C to mid-log phase, diluted to an OD600 of 0.05, and subsequently treated with 0.5% DMSO (untreated control), 3 μM ES24, or 25 μM nitrofurantoin for 15 min. Cultures were then pelleted by centrifugation (5000 × g, 5 min), and total RNA was extracted using the Qiagen RNA purification kit according to the manufacturer’s instructions. Transcriptomics and differential expression analysis were performed by Macrogen (Seoul, Korea). The experiments were performed in duplicates. Transcripts that showed a log2-fold change of ≥3 (p < 0.05) were considered differentially regulated.

Fluorescence Light Microscopy

Unless stated otherwise, all microscopy experiments were performed on a Nikon Eclipse Ti2 equipped with a CFI Plan Apochromat DM Lambda 100× Oil objective (N.A. 1.45, W.D. 0.13 mm), a Photometrics PRIME BSI camera, a Lumencor Sola SE II FISH 365 light source, and an Okolab temperature incubation chamber. Images were obtained using the NIS-Elements AR software version 5.21.03 and analyzed with ImageJ. (59) Quantification of the microscopy images was performed using the ImageJ plugins ObjectJ (60) and MicrobeJ. (61)

Oxyburst Green Assay

Oxyburst Green H2DCFDA succinimidyl ester was purchased from Thermo Fisher and dissolved in sterile DMSO. Aliquots were covered with nitrogen gas and stored at −20 °C until further use. B. subtilis 168CA was inoculated from overnight cultures in fresh LB medium to an OD600 of 0.05 and allowed to grow until early log phase (OD600 = 0.4) prior to the addition of antibiotics. Samples were taken after 10, 30, 60, and 120 min of antibiotic incubation, respectively. Staining with 10 μM Oxyburst Green was performed for 160 min, with the respective antibiotic incubation times included. After staining and antibiotic treatment, cells were washed once with phosphate-buffered saline (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH 7.4), immobilized on glass slides covered with a thin film of 1.2% agarose, (44) and imaged immediately. Images were analyzed with MicrobeJ. (61) The parameters for bacterial recognition were set to an area of 1.5 max, length of 1 max, width of 0.5–2.5, curvature of 0–1.5, and an angularity of 0–0.5 for detection in phase contrast. All other parameters remained at default settings. For detecting the fluorescence intensity, parameters were set to an area of 0.7–4.5, length of 1.2–4, and width of 2.5.

ROS Scavenger Assay

Stock solutions of tiron (Acros Organics) and thiourea (Alfa Aesar) were prepared in sterile water at 1 and 2.5 M, respectively. Serial 2-fold dilutions of antimicrobial compounds were prepared in LB containing either 10 mM tiron, 150 mM thiourea, both 10 mM tiron and 150 mM thiourea, or no scavenger. B. subtilis 168CA was added to a final CFU count of 5 × 105 CFU/mL. Cells were incubated for 16 h at 30 °C. Optical density was measured by absorbance readings at 600 nm using a BMG Clariostar Plus plate reader.

Bacterial Cytological Profiling

Bacterial cytological profiling was performed according to Wenzel et al. (26) In short, B. subtilis 168CA was grown until an OD600 of 0.3 prior to antibiotic addition, and samples were taken after 10, 30, 60, and 120 min of antibiotic treatment. Samples were then stained with 0.5 μM Nile red or MitoTracker Green (membrane stains, Invitrogen) and 1 μg/mL of DAPI (DNA stain, Invitrogen) for 1 min, spotted on 1.2% agarose films, and imaged immediately. Images were processed with ImageJ and analyzed with ObjectJ. (60)

DiSC(3)5 Spectroscopy

DiSC(3)5 is a dye that accumulates in polarized cell membranes. Because of its self-quenching properties, a release of the dye from the cell membrane following depolarization results in an increased fluorescence in the cell suspension, which can be observed spectroscopically. (44) Stock solutions of DiSC(3)5 (Anaspec) were prepared at 100 μM in sterile DMSO and stored at −20 °C until further use. The membrane potential in B. subtilis 168CA was determined by DiSC(3)5 spectroscopy. To this end, cultures were grown to an OD600 of 0.3 in the presence of 50 μg/mL of BSA. Kinetic measurements were performed on a Biotek Synergy MX plate reader, as described previously. (26) Kinetic measurements longer than 30 min become imprecise because of photobleaching. Therefore, end point measurements were performed to assess membrane potential after prolonged treatment times. To this end, samples were taken after 10, 30, 60, and 90 min of antibiotic treatment and subsequently stained with DiSC(3)5. Fluorescence was measured at an excitation wavelength of 610–30 nm and an emission wavelength of 675–50 nm in a BMG Clariostar Plus plate reader. Three technical replicates were performed for each biological replicate.

DiSC(3)5 Microscopy

Membrane potential measurements of E. coli MC4100 carrying plasmid pABCON2-fhuA ΔC/Δ4L (62) were performed by DiSC(3)5 microscopy according to te Winkel et al. (44) In contrast with spectroscopic measurements, where an increase in the total signal of the sample is observed upon depolarization because of dequenching of the dye, microscopic measurements show a decrease in single-cell fluorescence upon release of the dye. This is due to the remaining signal of the quenched fluorescence still being strong enough to be detected microscopically with a good signal to background ratio. Detachment of the dye from the cell membrane results in a clearly reduced fluorescence signal in single cells. (44) The expression of FhuA increases outer membrane permeability, (62) results in better uptake of DiSC(3)5, and subsequently, better sensitivity and signal stability in this assay. Cells were grown to an OD600 of 0.3 prior to antibiotic treatment. DiSC(3)5 staining was carried out for 10 min immediately prior to microscopy. Samples were taken after 10, 30, 60, and 90 min of antibiotic stress; spotted on glass slides covered with 1.2% agarose; and imaged immediately. Images were analyzed with MicrobeJ. (49) The parameters for bacterial recognition were set to an area of 0.7–4.65, length of 1.7–4.9, width of 0.6–2.5, circularity of 0.45–1, curvature of 0–1.5, and an angularity of 0–1 for detection in phase contrast. All other parameters remained at default settings. For detecting the fluorescence intensity, parameters were set to an area of 0.7–4.5, length of 1.2–4, and width of 2.5.

MinD Oscillation Microscopy

MinD oscillation in E. coli PFX9 expressing GFP-MinD (63) was observed by timelapse microscopy. Cells were induced at an OD600 of 0.1 by the addition of 10 μM IPTG and allowed to grow until an OD600 of 0.2 prior to the addition of antibiotics. Samples were taken after 10, 30, and 60 min of antibiotic treatment; immobilized on glass slides covered with a 1.2% agarose film; and immediately imaged. Images were taken for 4 min at 15 s intervals. The counting of oscillating and nonoscillating cells was done manually.

Laurdan Spectroscopy

Stock solutions of laurdan (Anaspec) were prepared at 10 mM in sterile dimethylformamide and stored at −20 °C until further use. Kinetic membrane fluidity measurements were performed as described previously (39) with minor modifications. In short, cells were grown in the presence of 0.2% glucose until an OD600 of 0.5 and subsequently stained with 10 μM laurdan for 5 min. The cells were washed 5 times with laurdan buffer (PBS, 0.2% glucose, 1% DMF) and resuspended in the same buffer to a final OD600 of 0.8. Samples of 100 μL were withdrawn and added to a prewarmed black 96-well polystyrene microplate (Corning). Laurdan fluorescence was measured at an excitation of 350–15 nm and emission of 460–15 nm and 500–15 nm in a BMG Clariostar Plus plate reader. After recording the untreated baseline for 10 min, 100 μL of prewarmed laurdan buffer containing the respective antibiotics was added, and measurements were continued for 30 min. Longer kinetic measurements become imprecise because of photobleaching. Therefore, additional end point measurements were performed to assess membrane fluidity after prolonged treatment times. End point measurements were obtained by following the same workflow until the resuspension step. Cell suspensions were adjusted to an OD600 of 0.4 prior to antibiotic addition and further incubated at constant temperature and agitation. Samples of 200 μL were taken after 10, 30, 60, and 120 min; transferred to microplates; and measured as described above. Laurdan general polarization values were calculated according to ref (39).

Protein Localization

Protein localization studies were performed as previously described. (26) In short, B. subtilis and E. coli strains expressing the different GFP fusion proteins were grown in the presence of appropriate inducer concentrations (Table S4) until an OD600 of 0.3 and subsequently stressed with antibiotics. Samples were taken after 10, 30, and 120 min of treatment; spotted on glass slides coated with a thin film of 1.2% agarose; and immediately imaged using a Nikon Eclipse Ti microscope equipped with a CFI Plan Apochromat DM 100× oil objective, an Intensilight HG 130 W lamp, a C11440-22CU Hamamatsu ORCA camera, and an Okolab temperature incubation chamber. Images were recorded with NIS elements software, version 4.20.01, and processed with ImageJ.

AmyM Secretion

B. subtilis BWB9 carrying pCS73 (PamyQ-amyM) was grown in LB medium at 37 °C until mid-log phase, followed by washing twice in fresh LB medium and resuspension to a final OD600 of 0.05. Subsequently, the cells were treated with ES24 (2 μM, 1 μM, 0.5 μM), NFT (12 μM, 6 μM, 3 μM), or DMSO (0.5%) as a negative control, and growth was continued for 3 h. The cells were collected by centrifugation at 5000 × g for 5 min, and the culture supernatant was subjected to trichloroacetic acid precipitation at 4 °C overnight. Both the cell lysate and supernatant were analyzed by 9% SDS-PAGE, followed by Coomassie staining and Western blotting. AmyM was detected using a rabbit α-AmyM primary antibody (64) in 1:20 000 dilution and a horseradish peroxidase-conjugated goat α-rabbit secondary antibody (Rockland) in 1:10 000 dilution. PycA was detected with a streptavidin horseradish peroxidase conjugate (Sigma-Aldrich) in 1:2000 dilution. Sample loading on the protein gel was adjusted on the basis of the OD600 of the culture prior to collection for analysis.

LipA Secretion

B. subtilis TEB1030 (ΔlipA) carrying pBSlipA (42) to express the lipA gene from the constitutive PhpaII promoter was grown in LB broth. After reaching midexponential growth phase, the cells were collected and washed twice with LB growth medium and resuspended in LB to a final OD600 of 0.05. Subsequently, different concentrations of ES24 or NFT were added, and cultures were incubated for 3 h under vigorous shaking (in the dark) until midexponential growth phase. Samples were collected, and the cells and growth medium fractions were separated by centrifugation. Bacterial cells were disrupted by bead-beating and, subsequently, proteins in the cell and growth medium fraction were separated by LDS-PAGE using 10% NuPage gels (Life Technologies). Gel loading of the samples was corrected on the basis of cellular protein content as determined with the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermofisher Scientific, USA). Gels were stained with InstantBlue Coomassie Protein Stain (AbCam). The experiment was performed in duplicate.

Resazurin Assay

The cell viability of S. pneumoniae D39 was measured in a standard resazurin bacterial viability assay in 96-well plates, as previously reported. (65) In brief, 20 μL of resazurin solution [0.025% (w/v) resazurin sodium] were added to each well containing bacterial cultures. After color conversion of the dye, bacterial viability was measured on the basis of fluorescence intensity using a BioTek plate reader (Synergy H1) with bottom reading mode (excitation/emission, 560 nm/590 nm).

Zebrafish Embryo Experiments

All methods were carried out in accordance with relevant guidelines and regulations. Danio rerio (zebrafish) were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (zfin.org). The breeding of zebrafish in authorized institutions such as the Amsterdam Animal Research Center of the Vrije Universiteit (VU) Amsterdam is in full compliance with the Dutch law on animal research. All animal experiments are supervised by the local Animal Welfare Body (Instantie voor Dierenwelzijn, IvD) of the VU and the VU Medical Center (IvD VU/VUmc). All used research protocols adhere to the international guidelines on the protection of animals used for scientific purposes, that is, the EU Animal Protection Directive 2010/63/EU, which allows zebrafish embryos to be used up to the moment that they are able to independently take up external food (5 days after fertilization) without additional approval by the Central Committee for Animal Experiments in The Netherlands (Centrale Commissie Dierproeven, CCD). Because the zebrafish embryos used in this study meet these criteria, this specific study was therefore approved by the IvD VU/VUmc. Casper zebrafish embryos were infected at 2 days postfertilization in the caudal vein by microinjection with 150 CFU of S. pneumoniae D39, as previously described. (49) The embryos were treated 1 h postinjection (hpi) by addition of 5 μM of either ES24 or NFT to the water. Infected embryos were monitored for survival at 24, 30, and 48 hpi. All experiments were performed in triplicate.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.2c00404.

  • Growth experiments, additional ROS assay controls, quantification and overview images of microscopy, peroxide and paraquat controls, protein localization assays, additional protein secretion experiments and controls, microscopy images of membrane potential and cell division assays in E. coli, cell length measurements, transcriptomics data tables, list of strains, plasmids, and primers, supplementary references (PDF)

  • Transcriptomics raw data (XLSX)

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Author Information

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  • Corresponding Authors
    • Joen Luirink - Molecular Microbiology, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands;  Email: [email protected]
    • Michaela Wenzel - Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden;  Orcidhttps://orcid.org/0000-0001-9969-6113;  Email: [email protected]
  • Authors
    • Ann-Britt Schäfer - Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden
    • Maurice Steenhuis - Molecular Microbiology, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands;  Present Address: Sanquin Diagnostic Services, 1066 CX Amsterdam, The Netherlands
    • Kin Ki Jim - Department of Medical Microbiology and Infection Prevention, Amsterdam University Medical Centers - Location Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands;  Amsterdam Institute for Infection and Immunity, Amsterdam University Medical Centers, 1081 HZ Amsterdam, The Netherlands
    • Jolanda Neef - Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, The Netherlands
    • Sarah O’Keefe - School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, United Kingdom;  Present Address: Institute of Biotechnology, HiLIFE, University of Helsinki, 00014 Helsinki, Finland
    • Roger C. Whitehead - School of Chemistry, Faculty of Science and Engineering, University of Manchester, Manchester M13 9PL, United Kingdom;  Orcidhttps://orcid.org/0000-0003-2910-4033
    • Eileithyia Swanton - School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, United Kingdom
    • Biwen Wang - Bacterial Cell Biology and Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH Amsterdam, The Netherlands
    • Sven Halbedel - FG11 Division of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, 38855 Wernigerode, Germany;  Institute for Medical Microbiology and Hospital Hygiene, Otto von Guericke University Magdeburg, 39120 Magdeburg, Germany;  Orcidhttps://orcid.org/0000-0002-5575-8973
    • Stephen High - School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, United Kingdom
    • Jan Maarten van Dijl - Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, The Netherlands;  Orcidhttps://orcid.org/0000-0002-5688-8438
  • Author Contributions

    A.-B.S. and M.S. contributed equally to this work. Conceptualization was performed by A.-B.S., M.S., K.K.J., J.N., R.C.W., E.S., S.High, J.M.v.D., J.L., and M.W. Data curation was performed by A.-B.S., M.S., K.K.J., J.N., J.M.v.D., J.L., and M.W. Formal analysis was performed by A.-B.S., M.S., K.K.J., J.N., S.O., R.C.W., E.S., J.M.v.D., J.L., and M.W. Funding acquisition was secured by M.S., S.High, and M.W. Investigation was completed by A.-B.S., M.S., K.K.J., J.N., S.O., R.C.W., E.S., B.W., and S.Halbedel. Project administration was conducted by J.M.v.D., J.L., and M.W. Methodology was determined by B.W. and S.Halbedel. Resources were procured by S.O., R.C.W., E.S., S.High, B.W., and S.Halbedel. Supervision was conducted by J.M.v.D., J.L., M.W. Validation was performed by A.-B.S., M.S., K.K.J., J.N., B.W., S.Halbedel, J.M.v.D., J.L., and M.W. Visualization was performed by A.-B.S., M.S., K.K.J., and M.W. Writing of the original draft was completed by A.-B.S., M.S., K.K.J., and M.W. The writing review and editing was performed by A.-B.S., M.S., K.K.J., J.N., S.O., R.C.W., E.S., B.W., S.Halbedel, S.High, J.M.v.D., J.L., and M.W.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We would like to thank Gregory Koningstein for technical assistance. This work was financially supported by the Chalmers foundation (MW), the NWO graduate program (022.005031, awarded to M.S.), and a Wellcome Trust Investigator Award in Science (204957/Z/16/Z, awarded to S.High). Funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

References

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This article references 65 other publications.

  1. 1
    Agnew, E.; Dolecek, C.; Hasan, R.; Lahra, M.; Merk, H.; Perovic, O.; Sievert, D.; Smith, R.; Taylor, A.; Turner, P. Global antimicrobial resistance and use surveillance system (GLASS) report; World Health Organization, 2021.
    Google Scholar
    There is no corresponding record for this reference.
  2. 2
    Tacconelli, E.; Carrara, E.; Savoldi, A.; Harbarth, S. Discovery, Research, and Development of New Antibiotics: The WHO Priority List of Antibiotic-Resistant Bacteria and Tuberculosis. Lancet Infect. Dis. 2018, 18 (3), 318– 327,  DOI: 10.1016/S1473-3099(17)30753-3
    Google Scholar
    2
    Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis
    Tacconelli Evelina; Carrara Elena; Savoldi Alessia; Harbarth Stephan; Mendelson Marc; Monnet Dominique L; Pulcini Celine; Kahlmeter Gunnar; Kluytmans Jan; Carmeli Yehuda; Ouellette Marc; Outterson Kevin; Patel Jean; Cavaleri Marco; Cox Edward M; Houchens Chris R; Grayson M Lindsay; Hansen Paul; Singh Nalini; Theuretzbacher Ursula; Magrini Nicola
    The Lancet. Infectious diseases (2018), 18 (3), 318-327 ISSN:.
    BACKGROUND: The spread of antibiotic-resistant bacteria poses a substantial threat to morbidity and mortality worldwide. Due to its large public health and societal implications, multidrug-resistant tuberculosis has been long regarded by WHO as a global priority for investment in new drugs. In 2016, WHO was requested by member states to create a priority list of other antibiotic-resistant bacteria to support research and development of effective drugs. METHODS: We used a multicriteria decision analysis method to prioritise antibiotic-resistant bacteria; this method involved the identification of relevant criteria to assess priority against which each antibiotic-resistant bacterium was rated. The final priority ranking of the antibiotic-resistant bacteria was established after a preference-based survey was used to obtain expert weighting of criteria. FINDINGS: We selected 20 bacterial species with 25 patterns of acquired resistance and ten criteria to assess priority: mortality, health-care burden, community burden, prevalence of resistance, 10-year trend of resistance, transmissibility, preventability in the community setting, preventability in the health-care setting, treatability, and pipeline. We stratified the priority list into three tiers (critical, high, and medium priority), using the 33rd percentile of the bacterium's total scores as the cutoff. Critical-priority bacteria included carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, and carbapenem-resistant and third-generation cephalosporin-resistant Enterobacteriaceae. The highest ranked Gram-positive bacteria (high priority) were vancomycin-resistant Enterococcus faecium and meticillin-resistant Staphylococcus aureus. Of the bacteria typically responsible for community-acquired infections, clarithromycin-resistant Helicobacter pylori, and fluoroquinolone-resistant Campylobacter spp, Neisseria gonorrhoeae, and Salmonella typhi were included in the high-priority tier. INTERPRETATION: Future development strategies should focus on antibiotics that are active against multidrug-resistant tuberculosis and Gram-negative bacteria. The global strategy should include antibiotic-resistant bacteria responsible for community-acquired infections such as Salmonella spp, Campylobacter spp, N gonorrhoeae, and H pylori. FUNDING: World Health Organization.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MzkvFOgsA%253D%253D&md5=14021ec57670b29fec6a88226a6a1fdd
  3. 3
    Butler, M. S.; Blaskovich, M. A.; Cooper, M. A. Antibiotics in the clinical pipeline at the end of 2015. J. Antibiot (Tokyo) 2017, 70, 3– 24,  DOI: 10.1038/ja.2016.72
    Google Scholar
    3
    Antibiotics in the clinical pipeline at the end of 2015
    Butler Mark S; Blaskovich Mark At; Cooper Matthew A
    The Journal of antibiotics (2017), 70 (1), 3-24 ISSN:.
    There is growing global recognition that the continued emergence of multidrug-resistant bacteria poses a serious threat to human health. Action plans released by the World Health Organization and governments of the UK and USA in particular recognize that discovering new antibiotics, particularly those with new modes of action, is one essential element required to avert future catastrophic pandemics. This review lists the 30 antibiotics and two β-lactamase/β-lactam combinations first launched since 2000, and analyzes in depth seven new antibiotics and two new β-lactam/β-lactamase inhibitor combinations launched since 2013. The development status, mode of action, spectra of activity and genesis (natural product, natural product-derived, synthetic or protein/mammalian peptide) of the 37 compounds and six β-lactamase/β-lactam combinations being evaluated in clinical trials between 2013 and 2015 are discussed. Compounds discontinued from clinical development since 2013 and new antibacterial pharmacophores are also reviewed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s7ks1amtA%253D%253D&md5=4baaeaed43677d32f5c68170c732a72b
  4. 4
    Theuretzbacher, U.; Outterson, K.; Engel, A.; Karlén, A. The global preclinical antibacterial pipeline. Nat. Rev. Microbiol 2020, 18, 275– 285,  DOI: 10.1038/s41579-019-0288-0
    Google Scholar
    4
    The global preclinical antibacterial pipeline
    Theuretzbacher Ursula; Outterson Kevin; Outterson Kevin; Engel Aleks; Karlen Anders
    Nature reviews. Microbiology (2020), 18 (5), 275-285 ISSN:.
    Antibacterial resistance is a great concern and requires global action. A critical question is whether enough new antibacterial drugs are being discovered and developed. A review of the clinical antibacterial drug pipeline was recently published, but comprehensive information about the global preclinical pipeline is unavailable. This Review focuses on discovery and preclinical development projects and has found, as of 1 May 2019, 407 antibacterial projects from 314 institutions. The focus is on Gram-negative pathogens, particularly bacteria on the WHO priority bacteria list. The preclinical pipeline is characterized by high levels of diversity and interesting scientific concepts, with 135 projects on direct-acting small molecules that represent new classes, new targets or new mechanisms of action. There is also a strong trend towards non-traditional approaches, including diverse antivirulence approaches, microbiome-modifying strategies, and engineered phages and probiotics. The high number of pathogen-specific and adjunctive approaches is unprecedented in antibiotic history. Translational hurdles are not adequately addressed yet, especially development pathways to show clinical impact of non-traditional approaches. The innovative potential of the preclinical pipeline compared with the clinical pipeline is encouraging but fragile. Much more work, focus and funding are needed for the novel approaches to result in effective antibacterial therapies to sustainably combat antibacterial resistance.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MfgvFyksA%253D%253D&md5=23ae61239f8792a0fa6ed5b48ae11dcf
  5. 5
    Fiebiger, E.; Hirsch, C.; Vyas, J. M.; Gordon, E.; Ploegh, H. L.; Tortorella, D. Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin. Mol. Biol. Cell 2004, 15, 1635– 1646,  DOI: 10.1091/mbc.e03-07-0506
    Google Scholar
    5
    Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin
    Fiebiger, Edda; Hirsch, Christian; Vyas, Jatin M.; Gordon, Eva; Ploegh, Hidde L.; Tortorella, Domenico
    Molecular Biology of the Cell (2004), 15 (4), 1635-1646CODEN: MBCEEV; ISSN:1059-1524. (American Society for Cell Biology)
    The mammalian endoplasmic reticulum (ER)-to-cytosol degrdn. pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degrdn. The ability to do so is hampered by the small no. of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I mols. We used this change in signal intensity as a readout to screen a chem. library of 16,320 compds. and identified two structurally related compds. (eeyarestatin I and II) that interfered with the degrdn. of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degrdn. of a second misfolded type I membrane protein, T-cell receptor α. Both compds. stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degrdn. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXivFalsrs%253D&md5=c6952152891fc81f5b18cd98030c32e6
  6. 6
    Sannino, S.; Brodsky, J. L. Targeting protein quality control pathways in breast cancer. BMC Biol. 2017, 15, 109,  DOI: 10.1186/s12915-017-0449-4
    Google Scholar
    6
    Targeting protein quality control pathways in breast cancer
    Sannino, Sara; Brodsky, Jeffrey L.
    BMC Biology (2017), 15 (), 109/1-109/20CODEN: BBMIF7; ISSN:1741-7007. (BioMed Central Ltd.)
    The efficient prodn., folding, and secretion of proteins is crit. for cancer cell survival. However, cancer cells thrive under stress conditions that damage proteins, so many cancer cells overexpress mol. chaperones that facilitate protein folding and target misfolded proteins for degrdn. via the ubiquitin-proteasome or autophagy pathway. Stress response pathway induction is also important for cancer cell survival. Indeed, validated targets for anti-cancer treatments include mol. chaperones, components of the unfolded protein response, the ubiquitin-proteasome system, and autophagy. We will focus on links between breast cancer and these processes, as well as the development of drug resistance, relapse, and treatment.
    >> More from SciFinder ®
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  7. 7
    Aletrari, M.-O.; McKibbin, C.; Williams, H.; Pawar, V.; Pietroni, P.; Lord, J. M.; Flitsch, S. L.; Whitehead, R.; Swanton, E.; High, S.; Spooner, R. A. Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways. PLoS One 2011, 6, e22713  DOI: 10.1371/journal.pone.0022713
    Google Scholar
    7
    Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways
    Aletrari, Mina-Olga; McKibbin, Craig; Williams, Helen; Pawar, Vidya; Pietroni, Paola; Michael Lord, J.; Flitsch, Sabine L.; Whitehead, Roger; Swanton, Eileithyia; High, Stephen; Spooner, Robert A.
    PLoS One (2011), 6 (7), e22713CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Background: The small mol. Eeyarestatin I (ESI) inhibits the endoplasmic reticulum (ER)-cytosol dislocation and subsequent degrdn. of ERAD (ER assocd. protein degrdn.) substrates. Toxins such as ricin and Shiga/Shiga-like toxins (SLTx) are endocytosed and trafficked to the ER. Their catalytic subunits are thought to utilize ERAD-like mechanisms to dislocate from the ER into the cytosol, where a proportion uncouples from the ERAD process, recovers a catalytic conformation and destroys their cellular targets. We therefore investigated ESI as a potential inhibitor of toxin dislocation. Methodol. and Principal Findings: Using cytotoxicity measurements, we found no role for ESI as an inhibitor of toxin dislocation from the ER, but instead found that for SLTx, ESI treatment of cells was protective by reducing the rate of toxin delivery to the ER. Microscopy of the trafficking of labeled SLTx and its B chain (lacking the toxic A chain) showed a delay in its accumulation at a peri-nuclear location, confirmed to be the Golgi by examn. of SLTx B chain metabolically labeled in the trans-Golgi cisternae. The drug also reduced the rate of endosomal trafficking of diphtheria toxin, which enters the cytosol from acidified endosomes, and delayed the Golgi-specific glycan modifications and eventual plasma membrane appearance of tsO45 VSV-G protein, a classical marker for anterograde trafficking. Conclusions and Significance: ESI acts on one or more components that function during vesicular transport, while at least one retrograde trafficking pathway, that of ricin, remains unperturbed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVKgtL7E&md5=4c81e54bee25c38f435995b57890967f
  8. 8
    McKibbin, C.; Mares, A.; Piacenti, M.; Williams, H.; Roboti, P.; Puumalainen, M.; Callan, A. C.; Lesiak-Mieczkowska, K.; Linder, S.; Harant, H.; High, S.; Flitsch, S. L.; Whitehead, R. C.; Swanton, E. Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response. Biochem. J. 2012, 442, 639– 648,  DOI: 10.1042/BJ20111220
    Google Scholar
    8
    Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response
    McKibbin, Craig; Mares, Alina; Piacenti, Michela; Williams, Helen; Roboti, Peristera; Puumalainen, Marjo; Callan, Anna C.; Lesiak-Mieczkowska, Karolina; Linder, Stig; Harant, Hanna; High, Stephen; Flitsch, Sabine L.; Whitehead, Roger C.; Swanton, Eileithyia
    Biochemical Journal (2012), 442 (3), 639-648CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    Selective small-mol. inhibitors represent powerful tools for the dissection of complex biol. processes. ESI (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addn. to acutely inhibiting ERAD (ER-assocd. degrdn.), ESI causes prodn. of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivV2iu74%253D&md5=ddc55c09736e307c81963bbc027a91f6
  9. 9
    Wang, Q.; Shinkre, B. A.; Lee, J.; Weniger, M. A.; Liu, Y.; Chen, W.; Wiestner, A.; Trenkle, W. C.; Ye, Y. The ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group. PLoS One 2010, 5, e15479  DOI: 10.1371/journal.pone.0015479
    Google Scholar
    9
    The ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group
    Wang Qiuyan; Shinkre Bidhan A; Lee Jin-gu; Weniger Marc A; Liu Yanfen; Chen Weiping; Wiestner Adrian; Trenkle William C; Ye Yihong
    PloS one (2010), 5 (11), e15479 ISSN:.
    BACKGROUND: Protein homeostasis in the endoplasmic reticulum (ER) has recently emerged as a therapeutic target for cancer treatment. Disruption of ER homeostasis results in ER stress, which is a major cause of cell death in cells exposed to the proteasome inhibitor Bortezomib, an anti-cancer drug approved for treatment of multiple myeloma and Mantle cell lymphoma. We recently reported that the ERAD inhibitor Eeyarestatin I (EerI) also disturbs ER homeostasis and has anti-cancer activities resembling that of Bortezomib. METHODOLOGY AND PRINCIPAL FINDINGS: Here we developed in vitro binding and cell-based functional assays to demonstrate that a nitrofuran-containing (NFC) group in EerI is the functional domain responsible for the cytotoxicity. Using both SPR and pull down assays, we show that EerI directly binds the p97 ATPase, an essential component of the ERAD machinery, via the NFC domain. An aromatic domain in EerI, although not required for p97 interaction, can localize EerI to the ER membrane, which improves its target specificity. Substitution of the aromatic module with another benzene-containing domain that maintains membrane localization generates a structurally distinct compound that nonetheless has similar biologic activities as EerI. CONCLUSIONS AND SIGNIFICANCE: Our findings reveal a class of bifunctional chemical agents that can preferentially inhibit membrane-bound p97 to disrupt ER homeostasis and to induce tumor cell death. These results also suggest that the AAA ATPase p97 may be a potential drug target for cancer therapeutics.
    >> More from SciFinder ®
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  10. 10
    Wang, Q.; Li, L.; Ye, Y. Inhibition of p97-dependent protein degradation by Eeyarestatin I. J. Biol. Chem. 2008, 283, 7445– 7454,  DOI: 10.1074/jbc.M708347200
    Google Scholar
    10
    Inhibition of p97-dependent Protein Degradation by Eeyarestatin I
    Wang, Qiuyan; Li, Lianyun; Ye, Yihong
    Journal of Biological Chemistry (2008), 283 (12), 7445-7454CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-assocd. degrdn. involves substrate retrotranslocation from the ER lumen into the cytosol for degrdn. by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which hydrolyzes ATP to dislocate polyubiquitinated substrates into the cytosol. Polypeptides extd. by p97 are eventually transferred to the proteasome for destruction. In mammalian cells, ERAD can be blocked by a chem. inhibitor termed Eeyarestatin I, but the mechanism of EerI action is unclear. Here we report that EerI can assoc. with a p97 complex to inhibit ERAD. The interaction of EerI with the p97 complex appears to neg. influence a deubiquitinating process that is mediated by p97-assocd. deubiquitinating enzymes. We further show that ataxin-3, a p97-assocd. deubiquitinating enzyme previously implicated in ER-assocd. degrdn., is among those affected. Interestingly, p97-assocd. deubiquitination is also involved in degrdn. of a sol. substrate. Our analyses establish a role for a novel deubiquitinating process in proteasome-dependent protein turnover.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjt1emurs%253D&md5=07da868005f68246bc48501c2aae0c6d
  11. 11
    Gamayun, I.; O’Keefe, S.; Pick, T.; Klein, M. C.; Nguyen, D.; McKibbin, C.; Piacenti, M.; Williams, H. M.; Flitsch, S. L.; Whitehead, R. C.; Swanton, E.; Helms, V.; High, S.; Zimmermann, R.; Cavalié, A. Eeyarestatin compounds selectively enhance Sec61-mediated Ca 2+ leakage from the endoplasmic reticulum. Cell Chem. Biol. 2019, 26, 571– 583, e6  DOI: 10.1016/j.chembiol.2019.01.010
    Google Scholar
    11
    Eeyarestatin Compounds Selectively Enhance Sec61-Mediated Ca2+ Leakage from the Endoplasmic Reticulum
    Gamayun, Igor; O'Keefe, Sarah; Pick, Tillman; Klein, Marie-Christine; Nguyen, Duy; McKibbin, Craig; Piacenti, Michela; Williams, Helen M.; Flitsch, Sabine L.; Whitehead, Roger C.; Swanton, Eileithyia; Helms, Volkhard; High, Stephen; Zimmermann, Richard; Cavalie, Adolfo
    Cell Chemical Biology (2019), 26 (4), 571-583.e6CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
    Eeyarestatin 1 (ES1) inhibits p97-dependent protein degrdn., Sec61-dependent protein translocation into the endoplasmic reticulum (ER), and vesicular transport within the endomembrane system. Here, we show that ES1 impairs Ca2+ homeostasis by enhancing the Ca2+ leakage from mammalian ER. A comparison of various ES1 analogs suggested that the 5-nitrofuran (5-NF) ring of ES1 is crucial for this effect. Accordingly, the analog ES24, which conserves the 5-NF domain of ES1, selectively inhibited protein translocation into the ER, displayed the highest potency on ER Ca2+ leakage of ES1 analogs studied and induced Ca2+-dependent cell death. Using small interfering RNA-mediated knockdown of Sec61a, we identified Sec61 complexes as the targets that mediate the gain of Ca2+ leakage induced by ES1 and ES24. By interacting with the lateral gate of Sec61a, ES1 and ES24 likely capture Sec61 complexes in a Ca2+-permeable, open state, in which Sec61 complexes allow Ca2+ leakage but are translocation incompetent.
    >> More from SciFinder ®
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  12. 12
    McKenna, M.; Simmonds, R. E.; High, S. Mechanistic insights into the inhibition of Sec61-dependent co- and post-translational translocation by mycolactone. J. Cell Sci. 2016, 129, 1404– 1415,  DOI: 10.1242/jcs.182352
    Google Scholar
    12
    Mechanistic insights into the inhibition of Sec61-dependent co- and post-translational translocation by mycolactone
    McKenna, Michael; Simmonds, Rachel E.; High, Stephen
    Journal of Cell Science (2016), 129 (7), 1404-1415CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
    The virulence factor mycolactone is responsible for the immunosuppression and tissue necrosis that characterize Buruli ulcer, a disease caused by infection with Mycobacterium ulcerans. In this study, we confirm that Sec61, the protein-conducting channel that coordinates entry of secretory proteins into the endoplasmic reticulum, is a primary target of mycolactone, and characterize the nature of its inhibitory effect. We conclude that mycolactone constrains the ribosome-nascent-chain-Sec61 complex, consistent with its broad-ranging perturbation of the co-translational translocation of classical secretory proteins. In contrast, the effect of mycolactone on the post-translational ribosome-independent translocation of short secretory proteins through the Sec61 complex is dependent on both signal sequence hydrophobicity and the translocation competence of the mature domain. Changes to protease sensitivity strongly suggest that mycolactone acts by inducing a conformational change in the pore-forming Sec61α subunit. These findings establish that mycolactone inhibits Sec61-mediated protein translocation and highlight differences between the co- and post-translational routes that the Sec61 complex mediates. We propose that mycolactone also provides a useful tool for further delineating the mol. mechanisms of Sec61-dependent protein translocation.
    >> More from SciFinder ®
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  13. 13
    Cross, B. C. S.; McKibbin, C.; Callan, A. C.; Roboti, P.; Piacenti, M.; Rabu, C.; Wilson, C. M.; Whitehead, R.; Flitsch, S. L.; Pool, M. R.; High, S.; Swanton, E. Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum. J. Cell Sci. 2009, 122, 4393– 4400,  DOI: 10.1242/jcs.054494
    Google Scholar
    13
    Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum
    Cross, Benedict C. S.; McKibbin, Craig; Callan, Anna C.; Roboti, Peristera; Piacenti, Michela; Rabu, Catherine; Wilson, Cornelia M.; Whitehead, Roger; Flitsch, Sabine L.; Pool, Martin R.; High, Stephen; Swanton, Eileithyia
    Journal of Cell Science (2009), 122 (23), 4393-4400CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
    Prodn. and trafficking of proteins entering the secretory pathway of eukaryotic cells is coordinated at the endoplasmic reticulum (ER) in a process that begins with protein translocation via the membrane-embedded ER translocon. The same complex is also responsible for the co-translational integration of membrane proteins and orchestrates polypeptide modifications that are often essential for protein function. We now show that the previously identified inhibitor of ER-assocd. degrdn. (ERAD) eeyarestatin 1 (ESI) is a potent inhibitor of protein translocation. We have characterized this inhibition of ER translocation both in vivo and in vitro, and provide evidence that ESI targets a component of the Sec61 complex that forms the membrane pore of the ER translocon. Further analyses show that ESI acts by preventing the transfer of the nascent polypeptide from the co-translational targeting machinery to the Sec61 complex. These results identify a novel effect of ESI, and suggest that the drug can modulate canonical protein transport from the cytosol into the mammalian ER both in vitro and in vivo.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmtVKrsg%253D%253D&md5=9c681f79f5af809d7c48a1ce0c749e22
  14. 14
    Itskanov, S.; Wang, L.; Junne, T.; Sherriff, R.; Xiao, L.; Blanchard, N.; Shi, W. Q.; Forsyth, C.; Hoepfner, D.; Spiess, M.; Park, E. A common mechanism of Sec61 translocon inhibition by small molecules. bioRxiv (Biochemistry) , August 11, 2022, 503542. DOI: 10.1101/2022.08.11.503542 .
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Denks, K.; Vogt, A.; Sachelaru, I.; Petriman, N.-A.; Kudva, R.; Koch, H.-G. The Sec translocon mediated protein transport in prokaryotes and eukaryotes. Mol. Membr. Biol. 2014, 31, 58– 84,  DOI: 10.3109/09687688.2014.907455
    Google Scholar
    15
    The Sec translocon mediated protein transport in prokaryotes and eukaryotes
    Denks, Kaert; Vogt, Andreas; Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-Georg
    Molecular Membrane Biology (2014), 31 (2-3), 58-84CODEN: MMEBE7; ISSN:0968-7688. (Informa Healthcare)
    A review. Protein transport via the Sec translocon represents an evolutionary conserved mechanism for delivering cytosolically synthesized proteins to extracytosolic compartments. The Sec translocon has a 3-subunit core, termed Sec61 in eukaryotes and SecYEG in bacteria. It is located in the endoplasmic reticulum (ER) of eukaryotes and in the cytoplasmic membrane of bacteria where it constitutes a channel that can be activated by multiple partner proteins. These partner proteins det. the mechanism of polypeptide movement across the channel. During signal recognition particle (SRP)-dependent co-translational targeting, the ribosome threads the nascent protein directly into the Sec channel. This pathway in bacteria is mainly dedicated for membrane proteins, but in eukaryotes it is also employed by secretory proteins. The alternative pathway, leading to post-translational translocation across the Sec translocon engages an ATP-dependent pushing mechanism by motor protein SecA in bacteria and a ratcheting mechanism by lumenal chaperone BiP in eukaryotes. Protein transport and biogenesis is also assisted by addnl. proteins at the lateral gate of SecY/Sec61α and in the lumen of the ER or in the periplasm of bacterial cells. The modular assembly enables the Sec complex to transport a vast array of substrates. Here, the authors summarize recent biochem. and structural information on the prokaryotic and eukaryotic Sec translocons and describe the remarkably complex interaction network of the Sec complexes.
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  16. 16
    Steenhuis, M.; Koningstein, G. M.; Oswald, J.; Pick, T.; O’Keefe, S.; Koch, H.-G.; Cavalié, A.; Whitehead, R. C.; Swanton, E.; High, S.; Luirink, J. Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogens. Mol. Microbiol. 2021, 115, 28– 40,  DOI: 10.1111/mmi.14589
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    16
    Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogens
    Steenhuis, Maurice; Koningstein, Gregory M.; Oswald, Julia; Pick, Tillman; O'Keefe, Sarah; Koch, Hans-Georg; Cavalie, Adolfo; Whitehead, Roger C.; Swanton, Eileithyia; High, Stephen; Luirink, Joen
    Molecular Microbiology (2021), 115 (1), 28-40CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
    Eeyarestatin 1 (ES1) is an inhibitor of endoplasmic reticulum (ER) assocd. protein degrdn., Sec61-dependent Ca2+ homeostasis and protein translocation into the ER. Recently, evidence was presented showing that a smaller analog of ES1, ES24, targets the Sec61-translocon, and captures it in an open conformation that is translocation-incompetent. We now show that ES24 impairs protein secretion and membrane protein insertion in Escherichia coli via the homologous SecYEG-translocon. Transcriptomic anal. suggested that ES24 has a complex mode of action, probably involving multiple targets. Interestingly, ES24 shows antibacterial activity toward clin. relevant strains. Furthermore, the antibacterial activity of ES24 is equiv. to or better than that of nitrofurantoin, a known antibiotic that, although structurally similar to ES24, does not interfere with SecYEG-dependent protein trafficking. Like nitrofurantoin, we find that ES24 requires activation by the NfsA and NfsB nitroreductases, suggesting that the formation of highly reactive nitroso intermediates is essential for target inactivation in vivo.
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  17. 17
    du Plessis, D. J. F.; Nouwen, N.; Driessen, A. J. M. The Sec translocase. Biochim. Biophys. Acta 2011, 1808, 851– 865,  DOI: 10.1016/j.bbamem.2010.08.016
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    17
    The Sec translocase
    du Plessis, David J. F.; Nouwen, Nico; Driessen, Arnold J. M.
    Biochimica et Biophysica Acta, Biomembranes (2011), 1808 (3), 851-865CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    A review. The vast majority of proteins trafficking across or into the bacterial cytoplasmic membrane occur via the translocon. The translocon consists of the SecYEG complex that forms an evolutionarily conserved heterotrimeric protein-conducting membrane channel that functions in conjunction with a variety of ancillary proteins. For post-translational protein translocation, the translocon interacts with cytosolic motor protein SecA that drives the ATP-dependent stepwise translocation of unfolded polypeptides across the membrane. For the cotranslational integration of membrane proteins, the translocon interacts with ribosome-nascent chain complexes and membrane insertion is coupled to polypeptide chain elongation at the ribosome. These processes are assisted by the YidC and SecDF(yajC) complex that transiently interacts with the translocon. This review summarizes the authors' current understanding of the structure-function relation of the translocon and its interactions with ancillary components during protein translocation and membrane protein insertion.
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  18. 18
    Yuan, J.; Zweers, J. C.; van Dijl, J. M.; Dalbey, R. E. Protein transport across and into cell membranes in bacteria and archaea. Cell. Mol. Life Sci. 2010, 67, 179– 199,  DOI: 10.1007/s00018-009-0160-x
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    18
    Protein transport across and into cell membranes in bacteria and archaea
    Yuan, Jijun; Zweers, Jessica C.; van Dijl, Jan Maarten; Dalbey, Ross E.
    Cellular and Molecular Life Sciences (2010), 67 (2), 179-199CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
    A review. In the three domains of life, the Sec, YidC/Oxa1, and Tat translocases play important roles in protein translocation across membranes and membrane protein insertion. While extensive studies have been performed on the endoplasmic reticular and Escherichia coli systems, far fewer studies have been done on archaea, other Gram-neg. bacteria, and Gram-pos. bacteria. Interestingly, work carried out to date has shown that there are differences in the protein transport systems in terms of the no. of translocase components and, in some cases, the translocation mechanisms and energy sources that drive translocation. In this review, we will describe the different systems employed to translocate and insert proteins across or into the cytoplasmic membrane of archaea and bacteria.
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  19. 19
    Neef, J.; van Dijl, J. M.; Buist, G. Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential. Essays Biochem 2021, 65, 187– 195,  DOI: 10.1042/EBC20200171
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    19
    Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential
    Neef, Jolanda; van Dijl, Jan Maarten; Buist, Girbe
    Essays in Biochemistry (2021), 65 (2), 187-195CODEN: ESBIAV; ISSN:1744-1358. (Portland Press Ltd.)
    Secreted recombinant proteins are of great significance for industry, healthcare and a sustainable bio-based economy. Consequently, there is an ever-increasing need for efficient prodn. platforms to deliver such proteins in high amts. and high quality. Gram-pos. bacteria, particularly bacilli such as Bacillus subtilis, are favored for the prodn. of secreted industrial enzymes. Nevertheless, recombinant protein prodn. in the B. subtilis cell factory can be very challenging due to bottlenecks in the general (Sec) secretion pathway as well as this bacterium's intrinsic capability to secrete a cocktail of highly potent proteases. This has placed another Gram-pos. bacterium, Lactococcus lactis, in the focus of attention as an alternative, non-proteolytic, cell factory for secreted proteins. Here we review our current understanding of the secretion pathways exploited in B. subtilis and L. lactis to deliver proteins from their site of synthesis, the cytoplasm, into the fermn. broth. An advantage of this cell factory comparison is that it identifies opportunities for protein secretion pathway engineering to remove or bypass current prodn. bottlenecks. Noteworthy new developments in cell factory engineering are the mini-Bacillus concept, highlighting potential advantages of massive genome minimization, and the application of thus far untapped 'non-classical' protein secretion routes. Altogether, it is foreseen that engineered lactococci will find future applications in the prodn. of high-quality proteins at the relatively small pilot scale, while engineered bacilli will remain a favored choice for protein prodn. in bulk.
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  20. 20
    Gardiner, B. J.; Stewardson, A. J.; Abbott, I. J.; Peleg, A. Y. Nitrofurantoin and fosfomycin for resistant urinary tract infections: old drugs for emerging problems. Aust Prescr 2019, 42, 14– 19,  DOI: 10.18773/austprescr.2019.002
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    Nitrofurantoin and fosfomycin for resistant urinary tract infections: old drugs for emerging problems
    Gardiner Bradley J; Stewardson Andrew J; Abbott Iain J; Peleg Anton Y; Gardiner Bradley J; Stewardson Andrew J; Abbott Iain J; Peleg Anton Y; Gardiner Bradley J; Stewardson Andrew J; Abbott Iain J; Peleg Anton Y
    Australian prescriber (2019), 42 (1), 14-19 ISSN:0312-8008.
    UNCOMPLICATED URINARY TRACT INFECTION IS ONE OF THE MOST COMMON INDICATIONS FOR ANTIBIOTIC USE IN THE COMMUNITY HOWEVER THE GRAM-NEGATIVE ORGANISMS THAT CAN CAUSE THE INFECTION ARE BECOMING MORE RESISTANT TO ANTIBIOTICS: MANY MULTIDRUG RESISTANT ORGANISMS RETAIN SUSCEPTIBILITY TO TWO OLD ANTIBIOTICS NITROFURANTOIN AND FOSFOMYCIN ADVANTAGES OVER NEWER DRUGS INCLUDE THEIR HIGH URINARY CONCENTRATIONS AND MINIMAL TOXICITY: FOSFOMYCIN IS A POTENTIAL TREATMENT OPTION FOR PATIENTS WITH UNCOMPLICATED URINARY TRACT INFECTION DUE TO RESISTANT ORGANISMS NITROFURANTOIN MAY BE MORE EFFECTIVE AND CAN BE USED FOR URINARY INFECTIONS IN PREGNANT WOMEN:
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  21. 21
    Wenzel, M.; Dekker, M. P.; Wang, B.; Burggraaf, M. J.; Bitter, W.; van Weering, J. R. T.; Hamoen, L. W. A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline. Commun. Biol. 2021, 4, 306,  DOI: 10.1038/s42003-021-01809-8
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    21
    A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline
    Wenzel, Michaela; Dekker, Marien P.; Wang, Biwen; Burggraaf, Maroeska J.; Bitter, Wilbert; van Weering, Jan R. T.; Hamoen, Leendert W.
    Communications Biology (2021), 4 (1), 306CODEN: CBOIDQ; ISSN:2399-3642. (Nature Research)
    Transmission electron microscopy of cell sample sections is a popular technique in microbiol. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphol. phenotypes due to the lack of sufficient nos. of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture vols. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphol. phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.
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  22. 22
    Tu, Y.; McCalla, D. R. Effect of activated nitrofurans on DNA. Biochim. Biophys. Acta 1975, 402, 142– 149,  DOI: 10.1016/0005-2787(75)90032-5
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    22
    Effect of activated nitrofurans on DNA
    Tu, Yu; McCalla, D. R.
    Biochimica et Biophysica Acta, Nucleic Acids and Protein Synthesis (1975), 402 (2), 142-9CODEN: BBNPAS; ISSN:0005-2787.
    Enzymically activated nitrofurazone (I) [59-87-0] reacted with covalently closed circular DNA (derived from Escherichia coli minicells carrying λdv) to give ≥2 kinds of damage: breaks which were detected on neutral sucrose gradients and alkali-labile lesions in DNA which were converted to breaks when the DNA was subsequently treated with alkali. DNA, isolated from minicells exposed to the drug, also contained lesions which were converted to breaks upon treatment with endonuclease prepns. obtained from Micrococcus luteus. Minicells repaired both breaks and nuclease-susceptible lesions within 2 hr but did not repair alkali labile lesions within that time. Expts. with 3 other nitrofurans showed that there were considerable differences in the degree to which DNA was damaged by activated metabolites of various derivs. and that the potency of the compds. as mutagens and carcinogens was correlated with the amt. of damage caused to minicell DNA.
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  23. 23
    Whiteway, J.; Koziarz, P.; Veall, J.; Sandhu, N.; Kumar, P.; Hoecher, B.; Lambert, I. B. Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli. J. Bacteriol. 1998, 180, 5529– 5539,  DOI: 10.1128/JB.180.21.5529-5539.1998
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    23
    Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli
    Whiteway, J.; Koziarz, P.; Veall, J.; Sandhu, N.; Kumar, P.; Hoecher, B.; Lambert, I. B.
    Journal of Bacteriology (1998), 180 (21), 5529-5539CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    Nitroheterocyclic and nitroarom. compds. constitute an enormous range of chems. whose potent biol. activity has significant human health and environmental implications. The biol. activity of nitro-substituted compds. is derived from reductive metab. of the nitro moiety, a process catalyzed by a variety of nitroreductase activities. Resistance of bacteria to nitro-substituted compds. is believed to result primarily from mutations in genes encoding oxygen-insensitive nitroreductases. We have characterized the nfsA and nfsB genes of a large no. of nitrofuran-resistant mutants of Escherichia coli and have correlated mutation with cell ext. nitroreductase activity. Our studies demonstrate that first-step resistance to furazolidone or nitrofurazone results from an nfsA mutation, while the increased resistance assocd. with second-step mutants is a consequence of an nfsB mutation. Inferences made from mutation about the structure-function relationships of NfsA and NfsB are discussed, esp. with regard to the identification of FMN binding sites. We show that expression of plasmid-carried nfsA and nfsB genes in resistant mutants restores sensitivity to nitrofurans. Among the 20 first-step and 53 second-step mutants isolated in this study, 65% and 49%, resp., contained insertion sequence elements in nfsA and nfsB. IS1 integrated in both genes, while IS30 and IS186 were found only in nfsA and IS2 and IS5 were obsd. only in nfsB. Insertion hot spots for IS30 and IS186 are indicated in nfsA, and a hot spot for IS5 insertion is evident in nfsB. We discuss potential regional and sequence-specific determinants for insertion sequence element integration in nfsA and nfsB.
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  24. 24
    Valle, A.; Le Borgne, S.; Bolívar, J.; Cabrera, G.; Cantero, D. Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2’-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biol. Appl. Microbiol. Biotechnol. 2012, 94, 163– 171,  DOI: 10.1007/s00253-011-3787-0
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    24
    Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2'-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties
    Valle, Antonio; Borgne, Sylvie; Bolivar, Jorge; Cabrera, Gema; Cantero, Domingo
    Applied Microbiology and Biotechnology (2012), 94 (1), 163-171CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Benzohydroxamic acids, such as 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chem. synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, Et 2-(2'-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroarom. compds., which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analyzed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, resp. In both cases, the biotransformation capacity of the strains was recovered (6.09 ± 0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33% ± 9.42% and 59.68% ± 2.0% resp.).
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  25. 25
    Pedreira, T.; Elfmann, C.; Stülke, J. The current state of SubtiWiki, the database for the model organism Bacillus subtilis. Nucleic Acids Res. 2022, 50, D875– D882,  DOI: 10.1093/nar/gkab943
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    25
    The current state of SubtiWiki, the database for the model organism Bacillus subtilis
    Pedreira, Tiago; Elfmann, Christoph; Stuelke, Joerg
    Nucleic Acids Research (2022), 50 (D1), D875-D882CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    Bacillus subtilis is a Gram-pos. model bacterium with extensive documented annotation. However, with the rise of high-throughput techniques, the amt. of complex data being generated every year has been increasing at a fast pace. Thus, having platforms ready to integrate and give a representation to these data becomes a priority. To address it, SubtiWiki was created in 2008 and has been growing in data and viewership ever since. With millions of requests every year, it is the most visited B. subtilis database, providing scientists all over the world with curated information about its genes and proteins, as well as intricate protein-protein interactions, regulatory elements, expression data and metabolic pathways. However, there is still a large portion of annotation to be unveiled for some biol. elements. Thus, to facilitate the development of new hypotheses for research, we have added a Homol. section covering potential protein homologs in other organisms. Here, we present the recent developments of SubtiWiki and give a guided tour of our database and the current state of the data for this organism.
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  26. 26
    Wenzel, M.; Rautenbach, M.; Vosloo, J. A.; Siersma, T.; Aisenbrey, C. H. M.; Zaitseva, E.; Laubscher, W. E.; van Rensburg, W.; Behrends, J.; Bechinger, B.; Hamoen, L. W. The multifaceted antibacterial mechanisms of the pioneering peptide antibiotics tyrocidine and gramicidin S. mBio 2018, 9, e00802-18  DOI: 10.1128/mBio.00802-18
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    There is no corresponding record for this reference.
  27. 27
    Saeloh, D.; Tipmanee, V.; Jim, K. K.; Dekker, M. P.; Bitter, W.; Voravuthikunchai, S. P.; Wenzel, M.; Hamoen, L. W. The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluidity. PLoS Pathog 2018, 14, e1006876  DOI: 10.1371/journal.ppat.1006876
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    27
    The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluidity
    Saeloh, Dennapa; Tipmanee, Varomyalin; Jim, Kin Ki; Dekker, Marien P.; Bitter, Wilbert; Voravuthikunchai, Supayang P.; Wenzel, Michaela; Hamoen, Leendert W.
    PLoS Pathogens (2018), 14 (2), e1006876/1-e1006876/35CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
    The acylphloroglucinol rhodomyrtone is a promising new antibiotic isolated from the rose myrtle Rhodomyrtus tomentosa, a plant used in Asian traditional medicine. While many studies have demonstrated its antibacterial potential in a variety of clin. applications, very little is known about the mechanism of action of rhodomyrtone. Preceding studies have been focused on intracellular targets, but no specific intracellular protein could be confirmed as main target. Using live cell, high-resoln., and electron microscopy we demonstrate that rhodomyrtone causes large membrane invaginations with a dramatic increase in fluidity, which attract a broad range of membrane proteins. Invaginations then form intracellular vesicles, thereby trapping these proteins. Aberrant protein localization impairs several cellular functions, including the respiratory chain and the ATP synthase complex. Being uncharged and devoid of a particular amphipathic structure, rhodomyrtone did not seem to be a typical membrane-inserting mol. In fact, mol. dynamics simulations showed that instead of inserting into the bilayer, rhodomyrtone transiently binds to phospholipid head groups and causes distortion of lipid packing, providing explanations for membrane fluidization and induction of membrane curvature. Both its transient binding mode and its ability to form protein- trapping membrane vesicles are unique, making it an attractive new antibiotic candidate with a novel mechanism of action.
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  28. 28
    Müller, A.; Wenzel, M.; Strahl, H.; Grein, F.; Saaki, TN V; Kohl, B.; Siersma, T.; Bandow, J. E.; Sahl, H.-G.; Schneider, T.; Hamoen, L. W. Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E7077– E7086,  DOI: 10.1073/pnas.1611173113
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    28
    Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains
    Mueller, Anna; Wenzel, Michaela; Strahl, Henrik; Grein, Fabian; Saaki, Terrens N. V.; Kohl, Bastian; Siersma, Tjalling; Bandow, Julia E.; Sahl, Hans-Georg; Schneider, Tanja; Hamoen, Leendert W.
    Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (45), E7077-E7086CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clin. importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different expts. have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To det. which model is correct, the authors carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. The authors found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although the authors found no evidence for altered membrane curvature, the authors confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, the authors showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-assocd. lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization obsd. with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to the authors' understanding of membrane-active antibiotics.
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  29. 29
    Garcia Martinez, P.; Winston, G. W.; Metash-Dickey, C.; O’Hara, S. C.; Livingstone, D. R. Nitrofurantoin-stimulated reactive oxygen species production and genotoxicity in digestive gland microsomes and cytosol of the common mussel (Mytilus edulis L.). Toxicol. Appl. Pharmacol. 1995, 131, 332– 341,  DOI: 10.1006/taap.1995.1076
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    29
    Nitrofurantoin-stimulated reactive oxygen species production and genotoxicity in digestive gland microsomes and cytosol of the common mussel (Mytilus edulis L.)
    Garcia Martinez P; Winston G W; Metash-Dickey C; O'Hara S C; Livingstone D R
    Toxicology and applied pharmacology (1995), 131 (2), 332-41 ISSN:0041-008X.
    The ability of nitrofurantoin (NF) to produce reactive oxygen species (ROS) was investigated in subcellular fractions of digestive gland of the mussel Mytilus edulis in terms of oxygen consumption and the formation of superoxide anion radical (O2-) (measured as SOD-sensitive cytochrome c reduction or SOD-sensitive sensitive .OH production), H2O2 (effect of catalase), and hydroxyl radical (.OH) (iron/EDTA-mediated oxidation of KMBA to ethylene). Additionally, the genotoxic effects of NF were examined using the Salmonella typhimurium umu mutagenicity assay. Microsomal NAD(P)H-dependent oxygen consumption was stimulated by NF, leading to the formation of H2O2. Stimulation of microsomal O2- production by NF was evident for NADH but not NADPH, confirming redox cycling at least with the former coenzyme. No stimulation of O2- production was obvious for cytosolic fraction with either coenzyme. NF stimulated microsomal NAD(P)H-dependent .OH production; the rates of .OH production were greater for NADH than NADPH; and the .OH was indicated to be formed, at least in part, by an iron-catalyzed Haber-Weiss reaction. A role was indicated for a free radical driven Fenton reaction in the NF-stimulated microsomal production of .OH from NADPH. The production of mutagenic species from NF was observed for cytosol but not for microsomes, and the former effects were greater for NADH than NADPH. Overall, the NAD(P)H-dependent microsomal generation of ROS, and the lack of correlation of ROS production with mutagenicity, are considered indicative of the potential of digestive gland to metabolize NF by both one-electron and two-electron reductive pathways. From this and other studies, enhanced ROS production by NF and other redox cycling xenobiotics is indicated to be a widespread phenomenon in aquatic organisms and a potential mechanism of pollutant-mediated toxicity.
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  30. 30
    Mols, M.; Abee, T. Primary and secondary oxidative stress in Bacillus. Environ. Microbiol 2011, 13, 1387– 1394,  DOI: 10.1111/j.1462-2920.2011.02433.x
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    30
    Primary and secondary oxidative stress in Bacillus
    Mols, Maarten; Abee, Tjakko
    Environmental Microbiology (2011), 13 (6), 1387-1394CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)
    A review. Coping with oxidative stress originating from oxidizing compds. or reactive oxygen species (ROS), assocd. with the exposure to agents that cause environmental stresses, is one of the prerequisites for an aerobic lifestyle of Bacillus spp. such as B. subtilis, B. cereus and B. anthracis. This minireview highlights novel insights in the primary oxidative stress response caused by oxidizing compds. including hydrogen peroxide and the secondary oxidative stress responses apparent upon exposure to a range of agents and conditions leading to environmental stresses such as antibiotics, heat and acid. Insights in the pathways and damaging radicals involved have been compiled based among others on transcriptome studies, network analyses and fluorescence techniques for detection of ROS at single cell level. Exploitation of the current knowledge for the control of spoilage and pathogenic bacteria is discussed.
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  31. 31
    Ameziane, El.; Hassani, R.; Dupuy, C. Detection of intracellular reactive oxygen species (CM-H2DCFDA). Bio-protocol 2013, 3, e313  DOI: 10.21769/BioProtoc.313
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    There is no corresponding record for this reference.
  32. 32
    Taiwo, F. A. Mechanism of tiron as scavenger of superoxide ions and free electrons. Spectroscopy 2008, 22, 491– 498,  DOI: 10.1155/2008/953692
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    32
    Mechanism of tiron as scavenger of superoxide ions and free electrons
    Taiwo, Fatai A.
    Spectroscopy (Amsterdam, Netherlands) (2008), 22 (6), 491-498CODEN: SPIJDZ; ISSN:0712-4813. (IOS Press)
    Sodium 4,5-dihydroxybenzene-1,3-disulfonate (tiron) has been reported to be an efficient chelator of certain metal ions, and a substrate in several enzyme reactions. Its small size facilitates cell entry and therefore modulates intracellular electron transfer reactions as an antioxidant by scavenging free radicals. Its redn. by electrochem. and enzymic methods gives identical products; a semiquinone detectable by EPR spectroscopy. In a test of its use as a spin trap, in comparison with DMPO, tiron does not form a mol. spin-adduct but proves more functional as an electron trap. Electron addn. to tiron is more facile than redn. of dioxygen as obsd. by the non-formation of DMPO-OOH spin-adduct in the system XO/HPX/O2/DMPO/tiron. Rather, it is the tiron semiquinone radical which is formed quant. with increasing concn. of hypoxanthine independent of oxygen concn. These results offer explanation for the action of tiron and its suitability for measuring electron release in hypoxic conditions, and also for mitigating redox-induced toxicity in drug regimes by acting as an electron scavenger.
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  33. 33
    Chueca, B.; Pagán, R.; García-Gonzalo, D. Differential mechanism of Escherichia coli inactivation by (+)-limonene as a function of cell physiological state and drug’s concentration. PLoS One 2014, 9, e94072  DOI: 10.1371/journal.pone.0094072
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    33
    Differential mechanism of Escherichia coli inactivation by (+)-limonene as a function of cell physiological state and drug concentration
    Chueca, Beatriz; Pagan, Rafael; Garcia-Gonzalo, Diego
    PLoS One (2014), 9 (4), e94072/1-e94072/7, 7 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    (+)-Limonene is a lipophilic antimicrobial compd., extd. from citrus fruits' essential oils, that is used as a flavouring agent and org. solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. The objective here was to det. whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 h. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2'-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was obsd. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as obsd. for bactericidal drugs. However, several differences have been obsd. between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, the results demonstrated that E. coli inactivation was influenced by its physiol. state and the drug's concn. e. Expts. with stationary-phase cells or 4000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. This study has also shown that drug's concn. is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compd., and in clarifying the controversy about the mechanism of inactivation by bactericidal antibiotics.
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  34. 34
    Lenhart, J. S.; Brandes, E. R.; Schroeder, J. W.; Sorenson, R. J.; Showalter, H. D.; Simmons, L. A. RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stress. J. Bacteriol. 2014, 196, 2851– 2860,  DOI: 10.1128/JB.01494-14
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    34
    RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stress
    Lenhart, Justin S.; Brandes, Eileen R.; Schroeder, Jeremy W.; Sorenson, Roderick J.; Showalter, Hollis D.; Simmons, Lyle A.
    Journal of Bacteriology (2014), 196 (15), 2851-2860, 11 pp.CODEN: JOBAAY; ISSN:1098-5530. (American Society for Microbiology)
    RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these 2 pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examd., including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an addnl. component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.
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  35. 35
    Wenzel, M.; Kohl, B.; Münch, D.; Raatschen, N.; Albada, H. B.; Hamoen, L.; Metzler-Nolte, N.; Sahl, H. G.; Bandow, J. E. Proteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membrane. Antimicrob. Agents Chemother. 2012, 56, 5749– 5757,  DOI: 10.1128/AAC.01380-12
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    35
    Proteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membrane
    Wenzel, Michaela; Kohl, Bastian; Muench, Daniela; Raatschen, Nadja; Albada, H. Bauke; Hamoen, Leendert; Metzler-Nolte, Nils; Sahl, Hans-Georg; Bandow, Julia E.
    Antimicrobial Agents and Chemotherapy (2012), 56 (11), 5749-5757CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
    Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides contg. lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane compn. The authors studied the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane.
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  36. 36
    Scheinpflug, K.; Wenzel, M.; Krylova, O.; Bandow, E. J.; Dathe, M.; Strahl, H. Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis. Sci. Rep 2017, 7, 44332,  DOI: 10.1038/srep44332
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    36
    Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis
    Scheinpflug Kathi; Krylova Oxana; Dathe Margitta; Wenzel Michaela; Bandow Julia E; Strahl Henrik
    Scientific reports (2017), 7 (), 44332 ISSN:.
    The synthetic cyclic hexapeptide cWFW (cyclo(RRRWFW)) has a rapid bactericidal activity against both Gram-positive and Gram-negative bacteria. Its detailed mode of action has, however, remained elusive. In contrast to most antimicrobial peptides, cWFW neither permeabilizes the membrane nor translocates to the cytoplasm. Using a combination of proteome analysis, fluorescence microscopy, and membrane analysis we show that cWFW instead triggers a rapid reduction of membrane fluidity both in live Bacillus subtilis cells and in model membranes. This immediate activity is accompanied by formation of distinct membrane domains which differ in local membrane fluidity, and which severely disrupts membrane protein organisation by segregating peripheral and integral proteins into domains of different rigidity. These major membrane disturbances cause specific inhibition of cell wall synthesis, and trigger autolysis. This novel antibacterial mode of action holds a low risk to induce bacterial resistance, and provides valuable information for the design of new synthetic antimicrobial peptides.
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  37. 37
    Omardien, S.; Drijfhout, J. W.; Vaz, F. M.; Wenzel, M.; Hamoen, L. W.; Zaat, S. A. J.; Brul, S. Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer. Biochim. Biophys. Acta 2018, 1860, 2404– 2415,  DOI: 10.1016/j.bbamem.2018.06.004
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    37
    Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer
    Omardien, Soraya; Drijfhout, Jan W.; Vaz, Frederic M.; Wenzel, Michaela; Hamoen, Leendert W.; Zaat, Sebastian A. J.; Brul, Stanley
    Biochimica et Biophysica Acta, Biomembranes (2018), 1860 (11), 2404-2415CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    Amphipathic cationic antimicrobial peptides (AMPs) TC19 and TC84, derived from the major AMPs of human blood platelets, thrombocidins, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide showed to perturb the membrane of Bacillus subtilis. We aimed to det. the means by which the three AMPs cause membrane perturbation in vivo using B. subtilis and to evaluate whether the membrane alterations are dependent on the phospholipid compn. of the membrane. Physiol. anal. was employed using Alexa Fluor 488 labeled TC84, various fluorescence dyes, fluorescent microscopy techniques and structured illumination microscopy. TC19, TC84 and BP2 created extensive fluidity domains in the membrane that are permeable, thus facilitating the entering of the peptides and the leakage of the cytosol. The direct interaction of the peptides with the bilayer create the fluid domains. The changes caused in the packing of the phospholipids lead to the delocalization of membrane bound proteins, thus contributing to the cell's destruction. The changes made to the membrane appeared to be not dependent on the compn. of the phospholipid bilayer. The distortion caused to the fluidity of the membrane by the AMPs is sufficient to facilitate the entering of the peptides and leakage of the cytosol. Here we show in vivo that cationic AMPs cause "membrane leaks" at the site of membrane insertion by altering the organization and fluidity of the membrane. Our findings thus contribute to the understanding of the membrane perturbation characteristic of cationic AMPs.
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  38. 38
    Parasassi, T.; Gratton, E. Membrane lipid domains and dynamics as detected by Laurdan fluorescence. J. Fluoresc 1995, 5, 59– 69,  DOI: 10.1007/BF00718783
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    38
    Membrane lipid domains and dynamics as detected by Laurdan fluorescence
    Parasassi, Tiziana; Gratton, Enrico
    Journal of Fluorescence (1995), 5 (1), 59-69CODEN: JOFLEN; ISSN:1053-0509.
    A review with 58 refs. 2-Dimethylamino-6-lauroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different max. and shape in the 2 phospholipid phases, due to differences in the polarity and in the amt. of dipolar relaxation. In bilayers composed of a mixt. of gel and liq.-cryst. phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using generalized polarization (GP). The wavelength dependence of the GP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liq.-cryst. phases was detd. The influence of cholesterol on the phase properties of the 2 phospholipid phases is proposed to be the cause of the phase behavior obsd. in natural membranes.
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  39. 39
    Wenzel, M.; Vischer, N. O. E.; Strahl, H.; Hamoen, L. W. Assessing membrane fluidity and visualizing fluid membrane domains in bacteria using fluorescent membrane dyes. Bio-protocol 2018, 8, e3063  DOI: 10.21769/BioProtoc.3063
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    39
    Assessing membrane fluidity and visualizing fluid membrane domains in bacteria using fluorescent membrane dyes
    Wenzel, Michaela; Vischer, Norbert O. E.; Strahl, Henrik; Hamoen, Leendert W.
    Bio-Protocol (2018), 8 (20), e3063CODEN: BIOPCG; ISSN:2331-8325. (Bio-Protocol LLC)
    Membrane fluidity is a key parameter of bacterial membranes that undergoes quick adaptation in response to environmental challenges and has recently emerged as an important factor in the antibacterial mechanism of membrane-targeting antibiotics. The specific level of membrane fluidity is not uniform across the bacterial cell membrane. Rather, specialized microdomains assocd. with different cellular functions can exhibit fluidity values that significantly deviate from the av. Assessing changes in the overall membrane fluidity and formation of membrane microdomains is therefore pivotal to understand both the functional organization of the bacterial cell membrane as well as antibiotic mechanisms. Here we describe how two fluorescent membrane dyes, laurdan and DiIC12, can be employed to assess membrane fluidity in living bacteria. We focus on Bacillus subtilis, since this organism has been relatively well-studied with respect to membrane domains. However, we also describe how these assays can be adapted for other bacteria such as Staphylococcus aureus and Streptococcus pneumoniae.
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  40. 40
    Strahl, H.; Hamoen, L. W. Membrane potential is important for bacterial cell division. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 12281– 12286,  DOI: 10.1073/pnas.1005485107
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    40
    Membrane potential is important for bacterial cell division
    Strahl, Henrik; Hamoen, Leendert W.
    Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (27), 12281-12286, S12281/1-S12281/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Many cell division-related proteins are located at specific positions in the bacterial cell, and this organized distribution of proteins requires energy. Here, the authors report that the proton motive force, or more specifically the (trans)membrane potential, is directly involved in protein localization. It emerged that the membrane potential modulates the distribution of several conserved cell division proteins such as MinD, FtsA, and the bacterial cytoskeletal protein MreB. The authors show for MinD that this is based on the membrane potential stimulated binding of its C-terminal amphipathic helix. This function of the membrane potential has implications for how these morphogenetic proteins work and provide an explanation for the effects obsd. with certain antimicrobial compds.
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  41. 41
    Omardien, S.; Drijfhout, J. W.; Zaat, S. A.; Brul, S. Cationic amphipathic antimicrobial peptides perturb the inner membrane of germinated ppores thus inhibiting their outgrowth. Front. Microbiol. 2018, 9, 2277,  DOI: 10.3389/fmicb.2018.02277
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    41
    Cationic Amphipathic Antimicrobial Peptides Perturb the Inner Membrane of Germinated Spores Thus Inhibiting Their Outgrowth
    Omardien Soraya; Brul Stanley; Drijfhout Jan Wouter; Zaat Sebastian A
    Frontiers in microbiology (2018), 9 (), 2277 ISSN:1664-302X.
    The mode of action of four cationic amphipathic antimicrobial peptides (AMPs) was evaluated against the non-pathogenic, Gram-positive, spore-forming bacterium, Bacillus subtilis. The AMPs were TC19, TC84, BP2, and the lantibiotic Nisin A. TC19 and TC84 were derived from the human thrombocidin-1. Bactericidal peptide 2 (BP2) was derived from the human bactericidal permeability increasing protein (BPI). We employed structured illumination microscopy (SIM), fluorescence microscopy, Alexa 488-labeled TC84, B. subtilis mutants producing proteins fused to the green fluorescent protein (GFP) and single-cell live imaging to determine the effects of the peptides against spores. TC19, TC84, BP2, and Nisin A showed to be bactericidal against germinated spores by perturbing the inner membrane, thus preventing outgrowth to vegetative cells. Single cell live imaging showed that the AMPs do not affect the germination process, but the burst time and subsequent generation time of vegetative cells. Alexa 488-labeled TC84 suggested that the TC84 might be binding to the dormant spore-coat. Therefore, dormant spores were also pre-coated with the AMPs and cultured on AMP-free culture medium during single-cell live imaging. Pre-coating of the spores with TC19, TC84, and BP2 had no effect on the germination process, and variably affected the burst time and generation time. However, the percentage of spores that burst and grew out into vegetative cells was drastically lower when pre-coated with Nisin A, suggesting a novel application potential of this lantibiotic peptide against spores. Our findings contribute to the understanding of AMPs and show the potential of AMPs as eventual therapeutic agents against spore-forming bacteria.
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  42. 42
    Skoczinski, P.; Volkenborn, K.; Fulton, A.; Bhadauriya, A.; Nutschel, C.; Gohlke, H.; Knapp, A.; Jaeger, K.-E. Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis. Microb. Cell Fact. 2017, 16, 160,  DOI: 10.1186/s12934-017-0772-z
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    42
    Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis
    Skoczinski Pia; Volkenborn Kristina; Fulton Alexander; Knapp Andreas; Jaeger Karl-Erich; Skoczinski Pia; Fulton Alexander; Bhadauriya Anuseema; Nutschel Christina; Gohlke Holger; Gohlke Holger; Jaeger Karl-Erich
    Microbial cell factories (2017), 16 (1), 160 ISSN:.
    BACKGROUND: Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. RESULTS: In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. CONCLUSIONS: Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.
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  43. 43
    Jones, S. E.; Lloyd, L. J.; Tan, K. K.; Buck, M. Secretion defects that activate the phage shock response of Escherichia coli. J. Bacteriol. 2003, 185, 6707– 6711,  DOI: 10.1128/JB.185.22.6707-6711.2003
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    43
    Secretion defects that activate the phage-shock response of Escherichia coli
    Jones, Susan E.; Lloyd, Louise J.; Tan, Kum K.; Buck, Martin
    Journal of Bacteriology (2003), 185 (22), 6707-6711CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    The phage shock protein (psp) operon of Escherichia coli is induced by membrane-damaging cues. Earlier studies linked defects in secretion across the inner membrane to induction of the psp response. Here we show that defects in yidC and sec secretion induce psp but that defects in tat and srp have no effect. We have also detd. the cellular location of PspB and PspD proteins.
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  44. 44
    te Winkel, J. D.; Gray, D. A.; Seistrup, K. H.; Hamoen, L. W.; Strahl, H. Analysis of antimicrobial-triggered membrane depolarization using voltage sensitive dyes. Front. Cell Dev. Biol. 2016, 4, 29,  DOI: 10.3389/fcell.2016.00029
    Google Scholar
    44
    Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes
    Te Winkel J Derk; Gray Declan A; Seistrup Kenneth H; Strahl Henrik; Hamoen Leendert W
    Frontiers in cell and developmental biology (2016), 4 (), 29 ISSN:2296-634X.
    The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.
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  45. 45
    Rowland, S. L.; Fu, X.; Sayed, M. A.; Zhang, Y.; Cook, W. R.; Rothfield, L. I. Membrane redistribution of the Escherichia coli MinD protein induced by MinE. J. Bacteriol. 2000, 182, 613– 619,  DOI: 10.1128/JB.182.3.613-619.2000
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    45
    Membrane redistribution of the Escherichia coli MinD protein induced by MinE
    Rowland, S. L.; Fu, X.; Sayed, M. A.; Zhang, Y.; Cook, W. R.; Rothfield, L. I.
    Journal of Bacteriology (2000), 182 (3), 613-619CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    Escherichia coli cells contain potential division sites at midcell and adjacent to the cell poles. Selection of the correct division site at midcell is controlled by three proteins: MinC, MinD, and MinE. It has previously been shown that a MinE-green fluorescent protein chimera (MinE-Gfp) localizes to the midcell site in an MinD-dependent manner. We use here Gfp-MinD to show that MinD assocs. with the membrane around the entire periphery of the cell in the absence of the other Min proteins and that MinE is capable of altering the membrane distribution pattern of Gfp-MinD. Studies with the isolated N-terminal and C-terminal MinE domains indicated different roles for the two MinE domains in the redistribution of membrane-assocd. MinD.
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  46. 46
    Munch, D.; Muller, A.; Schneider, T.; Kohl, B.; Wenzel, M.; Bandow, J. E.; Maffioli, S.; Sosio, M.; Donadio, S.; Wimmer, R.; Sahl, H.-G. The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functions. J. Biol. Chem. 2014, 289, 12063– 12076,  DOI: 10.1074/jbc.M113.537449
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    46
    The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functions
    Munch Daniela; Sahl Hans-Georg; Muller Anna; Schneider Tanja; Kohl Bastian; Wenzel Michaela; Bandow Julia Elisabeth; Maffioli Sonia; Sosio Margherita; Donadio Stefano; Wimmer Reinhard
    The Journal of biological chemistry (2014), 289 (17), 12063-12076 ISSN:.
    The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.
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  47. 47
    Errington, J.; Daniel, R. A.; Scheffers, D.-J. Cytokinesis in bacteria. Microbiol Mol. Biol. Rev. 2003, 67, 52– 65,  DOI: 10.1128/MMBR.67.1.52-65.2003
    Google Scholar
    47
    Cytokinesis in bacteria
    Errington, Jeffery; Daniel, Richard A.; Scheffers, Dirk-Jan
    Microbiology and Molecular Biology Reviews (2003), 67 (1), 52-65CODEN: MMBRF7; ISSN:1092-2172. (American Society for Microbiology)
    A review. Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two neg. regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid free spaces near the cell poles in a manner that is beginning to be understood in cytol. and biochem. terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.
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  48. 48
    Omardien, S.; Drijfhout, J. W.; van Veen, H.; Schachtschabel, S.; Riool, M.; Hamoen, L. W.; Brul, S.; Zaat, S. A. J. Synthetic antimicrobial peptides delocalize membrane bound proteins thereby inducing a cell envelope stress response. Biochim Biophys acta Biomembr 2018, 1860, 2416– 2427,  DOI: 10.1016/j.bbamem.2018.06.005
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    48
    Synthetic antimicrobial peptides delocalize membrane bound proteins thereby inducing a cell envelope stress response
    Omardien, Soraya; Drijfhout, Jan W.; van Veen, Henk; Schachtschabel, Soraya; Riool, Martijn; Hamoen, Leendert W.; Brul, Stanley; Zaat, Sebastian A. J.
    Biochimica et Biophysica Acta, Biomembranes (2018), 1860 (11), 2416-2427CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    Three amphipathic cationic antimicrobial peptides (AMPs) were characterized by detg. their effect on Gram-pos. bacteria using Bacillus subtilis strain 168 as a model organism. These peptides were TC19 and TC84, derivs. of thrombocidin-1 (TC-1), the major AMPs of human blood platelets, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide based on human bactericidal permeability increasing protein (BPI). To elucidate the possible mode of action of the AMPs we performed a transcriptomic anal. using microarrays. Physiol. analyses were performed using transmission electron microscopy (TEM), fluorescence microscopy and various B. subtilis mutants that produce essential membrane bound proteins fused to green fluorescent protein (GFP). The transcriptome anal. showed that the AMPs induced a cell envelope stress response (cell membrane and cell wall). The cell membrane stress response was confirmed with the physiol. observations that TC19, TC84 and BP2 perturb the membrane of B. subtilis. Using B. subtilis mutants, we established that the cell wall stress response is due to the delocalization of essential membrane bound proteins involved in cell wall synthesis. Other essential membrane proteins, involved in cell membrane synthesis and metab., were also delocalized due to alterations caused by the AMPs. We showed that peptides TC19, TC84 and BP2 perturb the membrane causing essential proteins to delocalize, thus preventing the possible repair of the cell envelope after the initial interference with the membrane. These AMPs show potential for eventual clin. application against Gram-pos. bacterial cells and merit further application-oriented investigation.
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  49. 49
    Jim, K. K.; Engelen-Lee, J.; van der Sar, A. M.; Bitter, W.; Brouwer, M. C.; van der Ende, A.; Veening, J.-W.; van de Beek, D.; Vandenbroucke-Grauls, C. M. J. E. Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model. J. Neuroinflammation 2016, 13, 188,  DOI: 10.1186/s12974-016-0655-y
    Google Scholar
    49
    Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model
    Jim, Kin Ki; Engelen-Lee, JooYeon; van der Sar, Astrid M.; Bitter, Wilbert; Brouwer, Matthijs C.; der Ende, Arie van; Veening, Jan-Willem; vandeBeek, Diederik; Vandenbroucke-Grauls, Christina M. J. E.
    Journal of Neuroinflammation (2016), 13 (), 188/1-188/13CODEN: JNOEB3; ISSN:1742-2094. (BioMed Central Ltd.)
    Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavorable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction. Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy. After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked redn. of phagocytes. This new meningitis model permits detailed anal. and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis.
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  50. 50
    Saeloh, D.; Wenzel, M.; Rungrotmongkol, T.; Hamoen, L. W.; Tipmanee, V.; Voravuthikunchai, S. P. Effects of rhodomyrtone on Gram-positive bacterial tubulin homologue FtsZ. PeerJ. 2017, 5, e2962  DOI: 10.7717/peerj.2962
    Google Scholar
    There is no corresponding record for this reference.
  51. 51
    Stepanek, J. J.; Lukezic, T.; Teichert, I.; Petkovic, H.; Bandow, J. E. Dual mechanism of action of the atypical tetracycline chelocardin. Biochim. Biophys. Acta 2016, 1864, 645– 654,  DOI: 10.1016/j.bbapap.2016.03.004
    Google Scholar
    51
    Dual mechanism of action of the atypical tetracycline chelocardin
    Stepanek, Jennifer J.; Lukezic, Tadeja; Teichert, Ines; Petkovic, Hrvoje; Bandow, Julia E.
    Biochimica et Biophysica Acta, Proteins and Proteomics (2016), 1864 (6), 645-654CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
    FR OLClassical tetracyclines targeting the protein biosynthesis machinery are commonly applied in human and veterinary medicine. OLtThe development and spread of resistance seriously compromise the successful treatment of bacterial infections. The atypical tetracycline chelocardin holds promise as it retains activity against tetracycline-resistant strains. It has been suggested that chelocardin targets the bacterial membrane, thus differing in mode of action from that of classical tetracyclines. The authors investigated the mechanism of action of chelocardin using global proteome anal. The proteome profiles after sublethal chelocardin stress were compared to a ref. compendium contg. antibiotic response profiles of Bacillus subtilis. This approach revealed a concn.-dependent dual mechanism of action. At low concns., like classical tetracyclines, chelocardin induces the proteomic signature for peptidyltransferase inhibition, demonstrating that protein biosynthesis inhibition is the dominant physiol. challenge. At higher concns., B. subtilis mainly responds to membrane stress indicating that at clin. relevant concns. the membrane is the main antibiotic target of chelocardin. Studying the effects on the membrane in more detail, it was found that chelocardin causes membrane depolarization but does not lead to formation of large pores. It is concluded that at growth inhibiting doses chelocardin not only targets protein biosynthesis but also corrupts the integrity of the bacterial membrane. This dual mechanism of action might prove beneficial in slowing the development of new resistance mechanisms against this atypical tetracycline.
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  52. 52
    Senges, C. H. R.; Stepanek, J. J.; Wenzel, M.; Raatschen, N.; Ay, Ü.; Märtens, Y.; Prochnow, P.; Vázquez Hernández, M.; Yayci, A.; Schubert, B.; Janzing, N. B. M.; Warmuth, H. L.; Kozik, M.; Bongard, J.; Alumasa, J. N.; Albada, B.; Penkova, M.; Lukežič, T.; Sorto, N. A.; Lorenz, N.; Miller, R.; Zhu, B.; Benda, M.; Stülke, J.; Schäkermann, S.; Leichert, L. I.; Scheinpflug, K.; Brötz-Oesterhelt, H.; Hertweck, C.; Shaw, J. T.; Petković, H.; Brunel, J. M.; Keiler, K. C.; Metzler-Nolte, N.; Bandow, J. E. Comparison of proteomic responses as global approach to antibiotic mechanism of action elucidation. Antimicrob. Agents Chemother. 2020, 65, e01373-20  DOI: 10.1128/AAC.01373-20
    Google Scholar
    There is no corresponding record for this reference.
  53. 53
    Rice, A. M.; Long, Y.; King, S. B. Nitroaromatic antibiotics as nitrogen oxide sources. Biomolecules 2021, 11, 267,  DOI: 10.3390/biom11020267
    Google Scholar
    53
    Nitroaromatic antibiotics as nitrogen oxide sources
    Rice, Allison M.; Long, Yueming; King, S. Bruce
    Biomolecules (2021), 11 (2), 267CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)
    A review. Nitroarom. antibiotics show activity against anaerobic bacteria and parasites, finding use in the treatment of Heliobacter pylori infections, tuberculosis, trichomoniasis, human African trypanosomiasis, Chagas disease and leishmaniasis. Despite this activity and a clear need for the development of new treatments for these conditions, the assocd. toxicity and lack of clear mechanisms of action have limited their therapeutic development. Nitroarom. antibiotics require reductive bioactivation for activity and this reductive metab. can convert the nitro group to nitric oxide (NO) or a related reactive nitrogen species (RNS). As nitric oxide plays important roles in the defensive immune response to bacterial infection through both signaling and redox-mediated pathways, defining controlled NO generation pathways from these antibiotics would allow the design of new therapeutics. This review focuses on the release of nitrogen oxide species from various nitroarom. antibiotics to portend the increased ability for these compds. to pos. impact infectious disease treatment.
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  54. 54
    Martin, B.; García, P.; Castanié, M. P.; Claverys, J. P. The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction. Mol. Microbiol. 1995, 15, 367– 379,  DOI: 10.1111/j.1365-2958.1995.tb02250.x
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    54
    The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction
    Martin, Bernard; Garcia, Pedro; Castanie, Marie-Pierre; Claverys, Jean-Pierre
    Molecular Microbiology (1995), 15 (2), 367-79CODEN: MOMIEE; ISSN:0950-382X. (Blackwell)
    The recently identified recA gene of the naturally transformable bacterium Streptococcus pneumoniae was further characterized by constructing a recA null mutation and by investigating its regulation. The recA mutation confers both DNA repair (as judged from sensitivity to UV and Me methane sulfonate) and recombination deficiencies. Plasmid transformation into the recA mutant was also drastically reduced. Western blotting established that recA gene expression is increased several-fold at the onset of competence for genetic transformation. Increased expression was assocd. with the appearance of a recA-specific transcript, ∼5.7 kb long. This transcript indicated that recA is part of a competence-inducible (cin) operon. The major (∼4.3 kb) transcript detected from noncompetent cells did not include cinA, the first gene in the operon, suggesting that this gene could be specifically required at some stage in the transformation process. Detection of small amts. of the 5.7-kb polycistronic mRNA in cells treated with mitomycin C suggested that the operon could also be damage inducible. In addn., mitomycin C treatment of a recA lysogenic strain did not lead to prophage induction and cell lysis. This is unlike the situation of a recA+ lysogen. Together, these results demonstrate that RecA controls lysogenic induction and suggest the existence of a SOS repair system in S. pneumoniae.
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  55. 55
    Lewis, P. J.; Marston, A. L. GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene 1999, 227, 101– 110,  DOI: 10.1016/S0378-1119(98)00580-0
    Google Scholar
    55
    GFP vectors for controlled expression and dual labeling of protein fusions in Bacillus subtilis
    Lewis, Peter J.; Marston, Adele L.
    Gene (1999), 227 (1), 101-109CODEN: GENED6; ISSN:0378-1119. (Elsevier Science B.V.)
    We report the development of a series of plasmid vectors for the construction of fusions to mutants of the intrinsically fluorescent green fluorescent protein, GFPmut1. Both N- and C-terminal fusions can be produced, and their expression can be finely controlled from the inducible Pxyl promoter following double crossover integration into the amyE locus of the Bacillus subtilis chromosome. Other vectors designed for single crossover insertion into the chromosome allow downstream genes to be placed under inducible control. We also show that fusions to GFPmutI and GFPuv can be co-localized within the cell by virtue of their different excitation spectra.
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  56. 56
    Hauser, P. M.; Karamata, D. A rapid and simple method for Bacillus subtilis transformation on solid media. Microbiology 1994, 140, 1613– 1617,  DOI: 10.1099/13500872-140-7-1613
    Google Scholar
    56
    A rapid and simple method for Bacillus subtilis transformation on solid media
    Hauser, Philippe M.; Karamata, Dimitri
    Microbiology (Reading, United Kingdom) (1994), 140 (7), 1613-17CODEN: MROBEO; ISSN:1350-0872.
    Cells of Bacillus subtilis strains 168 and W23 deprived of an amino acid or a base on a given solid medium were found to develop competence. We describe a rapid and simple method of genetic transformation of this organism consisting in spreading a sample contg. 1 μg DNA and 107 exponentially growing cells of an auxotrophic mutant onto plates devoid of the required amino acid or base. After overnight incubation, about 100-200 prototrophic transformants per plate were obtained, i.e. a frequency of about 10-5, as compared to 10-4 routinely obtained by the method of transformation in liq. medium with frozen competent cells. Plasmids and other chromosomal or plasmid-borne markers, which cannot be directly selected for, were transferred by congression. The dependence of the transformation efficiency on cell d., medium richness, incubation time and the nature of transforming DNA was investigated. We conclude that the development of competence accompanies amino acid or base starvation of cells under appropriate physiol. conditions.
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  57. 57
    Nicolas, P.; Mäder, U.; Dervyn, E.; Rochat, T.; Leduc, A.; Pigeonneau, N.; Bidnenko, E.; Marchadier, E.; Hoebeke, M.; Aymerich, S.; Becher, D.; Bisicchia, P.; Botella, E.; Delumeau, O.; Doherty, G.; Denham, E. L.; Fogg, M. J.; Fromion, V.; Goelzer, A.; Hansen, A.; Härtig, E.; Harwood, C. R.; Homuth, G.; Jarmer, H.; Jules, M.; Klipp, E.; Le Chat, L.; Lecointe, F.; Lewis, P.; Liebermeister, W.; March, A.; Mars, R. A. T.; Nannapaneni, P.; Noone, D.; Pohl, S.; Rinn, B.; Rügheimer, F.; Sappa, P. K.; Samson, F.; Schaffer, M.; Schwikowski, B.; Steil, L.; Stülke, J.; Wiegert, T.; Devine, K. M.; Wilkinson, A. J.; van Dijl, J. M.; Hecker, M.; Völker, U.; Bessières, P.; Noirot, P. Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science 2012, 335, 1103– 1106,  DOI: 10.1126/science.1206848
    Google Scholar
    57
    Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis
    Nicolas, Pierre; Maeder, Ulrike; Dervyn, Etienne; Rochat, Tatiana; Leduc, Aurelie; Pigeonneau, Nathalie; Bidnenko, Elena; Marchadier, Elodie; Hoebeke, Mark; Aymerich, Stephane; Becher, Doerte; Bisicchia, Paola; Botella, Eric; Delumeau, Olivier; Doherty, Geoff; Denham, Emma L.; Fogg, Mark J.; Fromion, Vincent; Goelzer, Anne; Hansen, Annette; Haertig, Elisabeth; Harwood, Colin R.; Homuth, Georg; Jarmer, Hanne; Jules, Matthieu; Klipp, Edda; Le Chat, Ludovic; Lecointe, Francois; Lewis, Peter; Liebermeister, Wolfram; March, Anika; Mars, Ruben A. T.; Nannapaneni, Priyanka; Noone, David; Pohl, Susanne; Rinn, Bernd; Ruegheimer, Frank; Sappa, Praveen K.; Samson, Franck; Schaffer, Marc; Schwikowski, Benno; Steil, Leif; Stuelke, Joerg; Wiegert, Thomas; Devine, Kevin M.; Wilkinson, Anthony J.; Maarten van Dijl, Jan; Hecker, Michael; Voelker, Uwe; Bessieres, Philippe; Noirot, Philippe
    Science (Washington, DC, United States) (2012), 335 (6072), 1103-1106CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the obsd. variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.
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  58. 58
    Morimoto, T.; Ara, K.; Ozaki, K.; Ogasawara, N. A new simple method to introduce marker-free deletions in the Bacillus subtilis genome. Genes Genet Syst 2009, 84, 315– 318,  DOI: 10.1266/ggs.84.315
    Google Scholar
    58
    A new simple method to introduce marker-free deletions in the Bacillus subtilis genome
    Morimoto, Takuya; Ara, Katsutoshi; Ozaki, Katsuya; Ogasawara, Naotake
    Genes & Genetic Systems (2009), 84 (4), 315-318CODEN: GGSYF5; ISSN:1341-7568. (Genetics Society of Japan)
    A genetic tool to introduce marker-free deletions is essential for multiple manipulations of genomes. We report a simple and efficient method to create marker-free deletion mutants of Bacillus subtilis through transformation with recombinant PCR products, using the Escherichia coli mazF gene encoding an endoribonuclease that cleaves free mRNAs as a counter-selection tool. Our method will be applicable to any bacterium in which introduction of the mazF cassette into the genome by double crossover homologous recombination is possible.
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  59. 59
    Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J. Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Fiji: An open-source platform for biological-image analysis. Nat. Methods 2012, 9, 676– 682,  DOI: 10.1038/nmeth.2019
    Google Scholar
    59
    Fiji: an open-source platform for biological-image analysis
    Schindelin, Johannes; Arganda-Carreras, Ignacio; Frise, Erwin; Kaynig, Verena; Longair, Mark; Pietzsch, Tobias; Preibisch, Stephan; Rueden, Curtis; Saalfeld, Stephan; Schmid, Benjamin; Tinevez, Jean-Yves; White, Daniel James; Hartenstein, Volker; Eliceiri, Kevin; Tomancak, Pavel; Cardona, Albert
    Nature Methods (2012), 9 (7_part1), 676-682CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Fiji is a distribution of the popular open-source software ImageJ focused on biol.-image anal. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biol. research communities.
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  60. 60
    Syvertsson, S.; Vischer, N. O. E.; Gao, Y.; Hamoen, L. W. When phase contrast fails: ChainTracer and NucTracer, two ImageJ methods for semi-automated single cell analysis using membrane or DNA staining. PLoS One 2016, 11, e0151267,  DOI: 10.1371/journal.pone.0151267
    Google Scholar
    60
    When phase contrast fails: chaintracer and nuctracer, two imageJ methods for semi- automated single cell analysis using membrane or DNA staining
    Syvertsson, Simon; Vischer, Norbert O. E.; Gao, Yongqiang; Hamoen, Leendert W.
    PLoS One (2016), 11 (3), e0151267/1-e0151267/11CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Within bacterial populations, genetically identical cells often behave differently. Single-cell measurement methods are required to observe this heterogeneity. Flow cytometry and fluorescence light microscopy are the primary methods to do this. However, flow cytometry requires reasonably strong fluorescence signals and is impractical when bacteria grow in cell chains. Therefore fluorescence light microscopy is often used to measure population heterogeneity in bacteria. Automatic microscopy image anal. programs typically use phase contrast images to identify cells. However, many bacteria divide by forming a crosswall that is not detectable by phase contrast.We have developed 'ChainTracer', a method based on the ImageJ plugin ObjectJ. It can automatically identify individual cells stained by fluorescent membrane dyes, and measure fluorescence intensity, chain length, cell length, and cell diam. As a complementary anal. method we developed 'NucTracer', which uses DAPI stained nucleoids as a proxy for single cells. The latter method is esp. useful when dealing with crowded images. The methods were tested with Bacillus subtilis and Lactococcus lactis cells expressing a GFP-reporter. In conclusion, ChainTracer and Nuc- Tracer are useful single cell measurement methods when bacterial cells are difficult to distinguish with phase contrast.
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  61. 61
    Ducret, A.; Quardokus, E. M.; Brun, Y. V. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat. Microbiol 2016, 1, 16077,  DOI: 10.1038/nmicrobiol.2016.77
    Google Scholar
    61
    MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis
    Ducret, Adrien; Quardokus, Ellen M.; Brun, Yves V.
    Nature Microbiology (2016), 1 (7), 16077CODEN: NMAICH; ISSN:2058-5276. (Nature Publishing Group)
    Single-cell anal. of bacteria and subcellular protein localization dynamics has shown that bacteria have elaborate life cycles, cytoskeletal protein networks and complex signal transduction pathways driven by localized proteins. The vol. of multidimensional images generated in such expts. and the computation time required to detect, assoc. and track cells and subcellular features pose considerable challenges, esp. for high-throughput expts. There is therefore a need for a versatile, computationally efficient image anal. tool capable of extg. the desired relationships from images in a meaningful and unbiased way. Here, we present MicrobeJ, a plug-in for the open-source platform ImageJ1. MicrobeJ provides a comprehensive framework to process images derived from a wide variety of microscopy expts. with special emphasis on large image sets. It performs the most common intensity and morphol. measurements as well as customized detection of poles, septa, fluorescent foci and organelles, dets. their subcellular localization with subpixel resoln., and tracks them over time. Because a dynamic link is maintained between the images, measurements and all data representations derived from them, the editor and suite of advanced data presentation tools facilitates the image anal. process and provides a robust way to verify the accuracy and veracity of the data.
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  62. 62
    Steenhuis, M.; Ten Hagen-Jongman, C. M.; van Ulsen, P.; Luirink, J. Stress-based high-throughput screening assays to identify inhibitors of cell envelope biogenesis. Antibiot (Basel, Switzerland) 2020, 9, 808,  DOI: 10.3390/antibiotics9110808
    Google Scholar
    62
    Stress-based high-throughput screening assays to identify inhibitors of cell envelope biogenesis
    Steenhuis, Maurice; ten Hagen-Jongman, Corinne M.; van Ulsen, Peter; Luirink, Joen
    Antibiotics (Basel, Switzerland) (2020), 9 (11), 808CODEN: ABSNC4; ISSN:2079-6382. (MDPI AG)
    The structural integrity of the Gram-neg. cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addn. of antibacterial compds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compd. screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.
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  63. 63
    Shih, Y.-L.; Le, T.; Rothfield, L. Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 7865– 7870,  DOI: 10.1073/pnas.1232225100
    Google Scholar
    63
    Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles
    Shih, Yu-Ling; Le, Trung; Rothfield, Lawrence
    Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (13), 7865-7870CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The MinCDE proteins of E. coli are required for proper placement of the division septum at mid-cell. The site selection process requires the rapid oscillatory redistribution of the proteins from pole to pole. We report that the 3 Min proteins are organized into extended membrane-assocd. coiled structures that wind around the cell between the 2 poles. The pole-to-pole oscillation of the proteins reflects oscillatory changes in their distribution within the coiled structure. We also report that the E. coli MreB protein, which is required for maintaining the rod shape of the cell, also forms extended coiled structures, which are similar to the MreB structures that have previously been reported in Bacillus subtilis. The MreB and MinCDE coiled arrays do not appear identical. The results suggest that ≥2 functionally distinct cytoskeletal-like elements are present in E. coli and that structures of this type can undergo dynamic changes that play important roles in division site placement and possibly other aspects of the life of the cell.
    >> More from SciFinder ®
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  64. 64
    Cruz, R. A. L. A comparative study of native and heterologous enzyme production in Bacillus subtilis. Ph.D. Thesis, Newcastle University, Newcastle upon Tyne, England, 2016.
    Google Scholar
    There is no corresponding record for this reference.
  65. 65
    Jonkers, T. J. H.; Steenhuis, M.; Schalkwijk, L.; Luirink, J.; Bald, D.; Houtman, C. J.; Kool, J.; Lamoree, M. H.; Hamers, T. Development of a high-throughput bioassay for screening of antibiotics in aquatic environmental samples. Sci. Total Environ. 2020, 729, 139028,  DOI: 10.1016/j.scitotenv.2020.139028
    Google Scholar
    65
    Development of a high-throughput bioassay for screening of antibiotics in aquatic environmental samples
    Jonkers, Tim J. H.; Steenhuis, Maurice; Schalkwijk, Louis; Luirink, Joen; Bald, Dirk; Houtman, Corine J.; Kool, Jeroen; Lamoree, Marja H.; Hamers, Timo
    Science of the Total Environment (2020), 729 (), 139028CODEN: STENDL; ISSN:0048-9697. (Elsevier B.V.)
    The goal of the present study was to select a Gram-pos. (Gram+) and Gram-neg. (Gram-) strain to measure antimicrobial activity in environmental samples, allowing high-throughput environmental screening. The sensitivity of eight pre-selected bacterial strains were tested to a training set of ten antibiotics, i.e. three Gram+ Bacillus subtilis strains with different read-outs, and five Gram- strains. The latter group consisted of a bioluminescent Allivibrio fischeri strain and four Escherichia coli strains, i.e. a wild type (WT) and three strains with a modified cell envelope to increase their sensitivity. The WT B. subtilis and an E. coli strain newly developed in this study, were most sensitive to the training set. This E. coli strain carries an open variant of an outer membrane protein combined with an inactivated multidrug efflux transport system. The assay conditions of these two strains were optimized and validated by exposure to a validation set of thirteen antibiotics with clin. and environmental relevance. The assay sensitivity ranged from the ng/mL to μg/mL range. The applicability of the assays for toxicol. characterization of aquatic environmental samples was demonstrated for hospital effluent ext. A future application includes effect-directed anal. to identify yet unknown antibiotic contaminants or their transformation products.
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  • Abstract

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    Figure 1

    Figure 1. Summary of B. subtilis transcriptomic stress profiling results. (A) Chemical structures of ES24 and NFT. The nitrofuran ring is highlighted in red. (B) Number of regulated transcripts per regulon. Sec components and proteases potentially indicating secretion stress are highlighted. NfrAB is a nitro/flavin reductase under transcriptional control of the Spx regulator and among the most strongly activated transcripts for both ES24 and NFT treatment. (C) Number of induced transcripts per functional category of the respective gene products. Functional categories were assigned according to function, regulation, interaction, and expression data available on http://subtiwiki.uni-goettingen.de/. Transcripts that showed a log2-fold change of ≥3 (p < 0.05) were considered as differentially regulated.

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    Figure 2

    Figure 2. Oxidative stress assays. (A) Oxyburst Green assay for detecting ROS in B. subtilis 168CA. The fluorescent probe shows a marked increase in fluorescence when ROS are present. It should be noted that Oxyburst Green has been reported to be specific for the detection of superoxide, yet we have found that it reacts to different sources of ROS (see Figure S2). (B) Influence of ROS scavengers on antibiotic activity against B. subtilis 168CA. MICs were determined in the absence or presence of the superoxide scavenger tiron or the hydroxyl radical scavenger thiourea. If one of these ROS species contributes to the activity of the compound, the MIC should increase in the presence of the respective scavenger.

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    Figure 3

    Figure 3. Bacterial cytological profiling of B. subtilis 168CA treated with ES24 or NFT. Red arrows indicate membrane aberrations, and blue arrows indicate nucleoid aberrations. See Figure S4 for corresponding images of cells treated with hydrogen peroxide and paraquat. Scale bar, 2 μM.

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    Figure 4

    Figure 4. Localization of RecA-GFP (B. subtilis UG10) after treatment with ES24 or NFT. Gramicidin (Gra) was used as additional control. Yellow arrows indicate clustered protein. Scale bar, 2 μM.

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    Figure 5

    Figure 5. Effects of ES24 and NFT on B. subtilis membrane potential and fluidity. (A) Kinetic DiSC(3)5 spectroscopy measurements of B. subtilis 168CA treated with ES24, NFT, or positive control gramicidin (Gra). An increase in fluorescence intensity indicates membrane depolarization. The arrow indicates the time point of antibiotic addition. (B) End point measurements under the same conditions as in panel A up to 90 min. (C) Kinetic laurdan generalized polarization (GP) spectroscopy measurements of B. subtilis 168CA treated with ES24, NFT, or positive control benzyl alcohol (BA). A decrease in GP indicates membrane fluidization. The arrow indicates the time point of antibiotic addition. (D) End point measurements under the same conditions as in panel C up to 120 min. The p-values were calculated using a paired two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001.

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    Figure 6

    Figure 6. Localization of SecA-GFP after treatment with ES24 or NFT. Gramicidin (Gra) was used as additional control. Yellow arrows: clustered protein. Green arrows: dispersed protein. Scale bar, 2 μM.

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    Figure 7

    Figure 7. Effect on AmyM secretion. B. subtilis BWB09 (ΔxynA ΔamyE) carrying pCS73 (PamyQ–amyM) to express amyM from the constitutive PamyQ promoter was grown until mid-log phase and treated with different concentrations of ES24 or NFT for 3 h prior to SDS-PAGE and Western blotting. (A) Western blot analysis of culture supernatant using an α-AmyM antibody (top) and of cell lysate using an α-streptavidin antibody detecting the cytosolic protein PycA (bottom). (B) Quantification of pixel intensity in AmyM bands. **p < 0.01.

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    Figure 8

    Figure 8. Effects of ES24 on cell membrane function in E. coli. (A) DiSC(3)5 measurements of E. coli MC4100 carrying pABCON2-fhuA ΔC/Δ4L treated with ES24 or NFT. Polymyxin B (PolB) was used as positive control. p-Values were calculated using a heteroscedastic, two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001. (B) MinD oscillation in E. coli RC1 carrying pFX9 treated with ES24 or NFT. The proton ionophore CCCP, which is known to abolish MinD oscillation due to membrane depolarization, (40) was used as positive control. (C) Quantification of FtsZ delocalization. Cells displaying an entirely cytosolic GFP signal, GFP clusters in the cell membrane, double bands, or spirals of FtsZ were counted as delocalized. Cells displaying single FtsZ bands, two opposing foci, or one focus at midcell (indicative of a closing septum) were counted as normal.

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    Figure 9

    Figure 9. In vivo efficacy of ES24 in an S. pneumoniae zebrafish embryo infection model. (A) Viability of S. pneumoniae D39 after overnight incubation with a concentrations series of ES24 or NFT measured with resazurin. (B) Survival of 2 days postfertilization zebrafish embryos infected with S. pneumoniae D39. Embryos were injected with 150 CFU of bacteria in the caudal vein and subsequently treated with 5 μM ES24 or 5 μM NFT by adding the compounds directly to the water 1 h post infection (hpi). ES24 treatment improved the survival rate significantly in zebrafish embryos infected with S. pneumoniae D39 compared with NFT-treated embryos (p < 0.0001) or nontreated infected embryos (p < 0.0001). NFT also improved the survival rate compared with nontreated infected embryos, albeit not significantly (p = 0.4139). Data represents the mean ± SEM. Experiments were performed in biological triplicates with 10 fish per condition in each replicate. Survival rates were compared using log-rank statistics.

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    Figure 10

    Figure 10. Current model of ES24 mechanism of action in Gram-positive and Gram-negative bacteria in comparison with NFT. NFT affects different cellular processes, including DNA packing; localization of DNA-binding proteins; and membrane integrity, membrane fluidity, and membrane protein binding. No clear differences were observed in its mechanism against E. coli and B. subtilis. ES24 affects Sec-mediated protein secretion and causes DNA damage in both organisms, but only affects cell division in E. coli. Reporter protein functions: MinD and DivIVA, cell division site regulation; FtsZ, cell division; SecA, Sec-dependent protein secretion; AtpA, ATP synthesis; RecA, DNA damage repair; DnaN, replication; RpoC, transcription; RpsB: translation.

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  • References

    This article references 65 other publications.

    1. 1
      Agnew, E.; Dolecek, C.; Hasan, R.; Lahra, M.; Merk, H.; Perovic, O.; Sievert, D.; Smith, R.; Taylor, A.; Turner, P. Global antimicrobial resistance and use surveillance system (GLASS) report; World Health Organization, 2021.
      There is no corresponding record for this reference.
    2. 2
      Tacconelli, E.; Carrara, E.; Savoldi, A.; Harbarth, S. Discovery, Research, and Development of New Antibiotics: The WHO Priority List of Antibiotic-Resistant Bacteria and Tuberculosis. Lancet Infect. Dis. 2018, 18 (3), 318– 327,  DOI: 10.1016/S1473-3099(17)30753-3
      2
      Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis
      Tacconelli Evelina; Carrara Elena; Savoldi Alessia; Harbarth Stephan; Mendelson Marc; Monnet Dominique L; Pulcini Celine; Kahlmeter Gunnar; Kluytmans Jan; Carmeli Yehuda; Ouellette Marc; Outterson Kevin; Patel Jean; Cavaleri Marco; Cox Edward M; Houchens Chris R; Grayson M Lindsay; Hansen Paul; Singh Nalini; Theuretzbacher Ursula; Magrini Nicola
      The Lancet. Infectious diseases (2018), 18 (3), 318-327 ISSN:.
      BACKGROUND: The spread of antibiotic-resistant bacteria poses a substantial threat to morbidity and mortality worldwide. Due to its large public health and societal implications, multidrug-resistant tuberculosis has been long regarded by WHO as a global priority for investment in new drugs. In 2016, WHO was requested by member states to create a priority list of other antibiotic-resistant bacteria to support research and development of effective drugs. METHODS: We used a multicriteria decision analysis method to prioritise antibiotic-resistant bacteria; this method involved the identification of relevant criteria to assess priority against which each antibiotic-resistant bacterium was rated. The final priority ranking of the antibiotic-resistant bacteria was established after a preference-based survey was used to obtain expert weighting of criteria. FINDINGS: We selected 20 bacterial species with 25 patterns of acquired resistance and ten criteria to assess priority: mortality, health-care burden, community burden, prevalence of resistance, 10-year trend of resistance, transmissibility, preventability in the community setting, preventability in the health-care setting, treatability, and pipeline. We stratified the priority list into three tiers (critical, high, and medium priority), using the 33rd percentile of the bacterium's total scores as the cutoff. Critical-priority bacteria included carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, and carbapenem-resistant and third-generation cephalosporin-resistant Enterobacteriaceae. The highest ranked Gram-positive bacteria (high priority) were vancomycin-resistant Enterococcus faecium and meticillin-resistant Staphylococcus aureus. Of the bacteria typically responsible for community-acquired infections, clarithromycin-resistant Helicobacter pylori, and fluoroquinolone-resistant Campylobacter spp, Neisseria gonorrhoeae, and Salmonella typhi were included in the high-priority tier. INTERPRETATION: Future development strategies should focus on antibiotics that are active against multidrug-resistant tuberculosis and Gram-negative bacteria. The global strategy should include antibiotic-resistant bacteria responsible for community-acquired infections such as Salmonella spp, Campylobacter spp, N gonorrhoeae, and H pylori. FUNDING: World Health Organization.
      >> More from SciFinder ®
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    3. 3
      Butler, M. S.; Blaskovich, M. A.; Cooper, M. A. Antibiotics in the clinical pipeline at the end of 2015. J. Antibiot (Tokyo) 2017, 70, 3– 24,  DOI: 10.1038/ja.2016.72
      3
      Antibiotics in the clinical pipeline at the end of 2015
      Butler Mark S; Blaskovich Mark At; Cooper Matthew A
      The Journal of antibiotics (2017), 70 (1), 3-24 ISSN:.
      There is growing global recognition that the continued emergence of multidrug-resistant bacteria poses a serious threat to human health. Action plans released by the World Health Organization and governments of the UK and USA in particular recognize that discovering new antibiotics, particularly those with new modes of action, is one essential element required to avert future catastrophic pandemics. This review lists the 30 antibiotics and two β-lactamase/β-lactam combinations first launched since 2000, and analyzes in depth seven new antibiotics and two new β-lactam/β-lactamase inhibitor combinations launched since 2013. The development status, mode of action, spectra of activity and genesis (natural product, natural product-derived, synthetic or protein/mammalian peptide) of the 37 compounds and six β-lactamase/β-lactam combinations being evaluated in clinical trials between 2013 and 2015 are discussed. Compounds discontinued from clinical development since 2013 and new antibacterial pharmacophores are also reviewed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s7ks1amtA%253D%253D&md5=4baaeaed43677d32f5c68170c732a72b
    4. 4
      Theuretzbacher, U.; Outterson, K.; Engel, A.; Karlén, A. The global preclinical antibacterial pipeline. Nat. Rev. Microbiol 2020, 18, 275– 285,  DOI: 10.1038/s41579-019-0288-0
      4
      The global preclinical antibacterial pipeline
      Theuretzbacher Ursula; Outterson Kevin; Outterson Kevin; Engel Aleks; Karlen Anders
      Nature reviews. Microbiology (2020), 18 (5), 275-285 ISSN:.
      Antibacterial resistance is a great concern and requires global action. A critical question is whether enough new antibacterial drugs are being discovered and developed. A review of the clinical antibacterial drug pipeline was recently published, but comprehensive information about the global preclinical pipeline is unavailable. This Review focuses on discovery and preclinical development projects and has found, as of 1 May 2019, 407 antibacterial projects from 314 institutions. The focus is on Gram-negative pathogens, particularly bacteria on the WHO priority bacteria list. The preclinical pipeline is characterized by high levels of diversity and interesting scientific concepts, with 135 projects on direct-acting small molecules that represent new classes, new targets or new mechanisms of action. There is also a strong trend towards non-traditional approaches, including diverse antivirulence approaches, microbiome-modifying strategies, and engineered phages and probiotics. The high number of pathogen-specific and adjunctive approaches is unprecedented in antibiotic history. Translational hurdles are not adequately addressed yet, especially development pathways to show clinical impact of non-traditional approaches. The innovative potential of the preclinical pipeline compared with the clinical pipeline is encouraging but fragile. Much more work, focus and funding are needed for the novel approaches to result in effective antibacterial therapies to sustainably combat antibacterial resistance.
      >> More from SciFinder ®
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    5. 5
      Fiebiger, E.; Hirsch, C.; Vyas, J. M.; Gordon, E.; Ploegh, H. L.; Tortorella, D. Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin. Mol. Biol. Cell 2004, 15, 1635– 1646,  DOI: 10.1091/mbc.e03-07-0506
      5
      Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin
      Fiebiger, Edda; Hirsch, Christian; Vyas, Jatin M.; Gordon, Eva; Ploegh, Hidde L.; Tortorella, Domenico
      Molecular Biology of the Cell (2004), 15 (4), 1635-1646CODEN: MBCEEV; ISSN:1059-1524. (American Society for Cell Biology)
      The mammalian endoplasmic reticulum (ER)-to-cytosol degrdn. pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degrdn. The ability to do so is hampered by the small no. of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I mols. We used this change in signal intensity as a readout to screen a chem. library of 16,320 compds. and identified two structurally related compds. (eeyarestatin I and II) that interfered with the degrdn. of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degrdn. of a second misfolded type I membrane protein, T-cell receptor α. Both compds. stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degrdn. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation.
      >> More from SciFinder ®
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    6. 6
      Sannino, S.; Brodsky, J. L. Targeting protein quality control pathways in breast cancer. BMC Biol. 2017, 15, 109,  DOI: 10.1186/s12915-017-0449-4
      6
      Targeting protein quality control pathways in breast cancer
      Sannino, Sara; Brodsky, Jeffrey L.
      BMC Biology (2017), 15 (), 109/1-109/20CODEN: BBMIF7; ISSN:1741-7007. (BioMed Central Ltd.)
      The efficient prodn., folding, and secretion of proteins is crit. for cancer cell survival. However, cancer cells thrive under stress conditions that damage proteins, so many cancer cells overexpress mol. chaperones that facilitate protein folding and target misfolded proteins for degrdn. via the ubiquitin-proteasome or autophagy pathway. Stress response pathway induction is also important for cancer cell survival. Indeed, validated targets for anti-cancer treatments include mol. chaperones, components of the unfolded protein response, the ubiquitin-proteasome system, and autophagy. We will focus on links between breast cancer and these processes, as well as the development of drug resistance, relapse, and treatment.
      >> More from SciFinder ®
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    7. 7
      Aletrari, M.-O.; McKibbin, C.; Williams, H.; Pawar, V.; Pietroni, P.; Lord, J. M.; Flitsch, S. L.; Whitehead, R.; Swanton, E.; High, S.; Spooner, R. A. Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways. PLoS One 2011, 6, e22713  DOI: 10.1371/journal.pone.0022713
      7
      Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways
      Aletrari, Mina-Olga; McKibbin, Craig; Williams, Helen; Pawar, Vidya; Pietroni, Paola; Michael Lord, J.; Flitsch, Sabine L.; Whitehead, Roger; Swanton, Eileithyia; High, Stephen; Spooner, Robert A.
      PLoS One (2011), 6 (7), e22713CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Background: The small mol. Eeyarestatin I (ESI) inhibits the endoplasmic reticulum (ER)-cytosol dislocation and subsequent degrdn. of ERAD (ER assocd. protein degrdn.) substrates. Toxins such as ricin and Shiga/Shiga-like toxins (SLTx) are endocytosed and trafficked to the ER. Their catalytic subunits are thought to utilize ERAD-like mechanisms to dislocate from the ER into the cytosol, where a proportion uncouples from the ERAD process, recovers a catalytic conformation and destroys their cellular targets. We therefore investigated ESI as a potential inhibitor of toxin dislocation. Methodol. and Principal Findings: Using cytotoxicity measurements, we found no role for ESI as an inhibitor of toxin dislocation from the ER, but instead found that for SLTx, ESI treatment of cells was protective by reducing the rate of toxin delivery to the ER. Microscopy of the trafficking of labeled SLTx and its B chain (lacking the toxic A chain) showed a delay in its accumulation at a peri-nuclear location, confirmed to be the Golgi by examn. of SLTx B chain metabolically labeled in the trans-Golgi cisternae. The drug also reduced the rate of endosomal trafficking of diphtheria toxin, which enters the cytosol from acidified endosomes, and delayed the Golgi-specific glycan modifications and eventual plasma membrane appearance of tsO45 VSV-G protein, a classical marker for anterograde trafficking. Conclusions and Significance: ESI acts on one or more components that function during vesicular transport, while at least one retrograde trafficking pathway, that of ricin, remains unperturbed.
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    8. 8
      McKibbin, C.; Mares, A.; Piacenti, M.; Williams, H.; Roboti, P.; Puumalainen, M.; Callan, A. C.; Lesiak-Mieczkowska, K.; Linder, S.; Harant, H.; High, S.; Flitsch, S. L.; Whitehead, R. C.; Swanton, E. Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response. Biochem. J. 2012, 442, 639– 648,  DOI: 10.1042/BJ20111220
      8
      Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response
      McKibbin, Craig; Mares, Alina; Piacenti, Michela; Williams, Helen; Roboti, Peristera; Puumalainen, Marjo; Callan, Anna C.; Lesiak-Mieczkowska, Karolina; Linder, Stig; Harant, Hanna; High, Stephen; Flitsch, Sabine L.; Whitehead, Roger C.; Swanton, Eileithyia
      Biochemical Journal (2012), 442 (3), 639-648CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      Selective small-mol. inhibitors represent powerful tools for the dissection of complex biol. processes. ESI (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addn. to acutely inhibiting ERAD (ER-assocd. degrdn.), ESI causes prodn. of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation.
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    9. 9
      Wang, Q.; Shinkre, B. A.; Lee, J.; Weniger, M. A.; Liu, Y.; Chen, W.; Wiestner, A.; Trenkle, W. C.; Ye, Y. The ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group. PLoS One 2010, 5, e15479  DOI: 10.1371/journal.pone.0015479
      9
      The ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group
      Wang Qiuyan; Shinkre Bidhan A; Lee Jin-gu; Weniger Marc A; Liu Yanfen; Chen Weiping; Wiestner Adrian; Trenkle William C; Ye Yihong
      PloS one (2010), 5 (11), e15479 ISSN:.
      BACKGROUND: Protein homeostasis in the endoplasmic reticulum (ER) has recently emerged as a therapeutic target for cancer treatment. Disruption of ER homeostasis results in ER stress, which is a major cause of cell death in cells exposed to the proteasome inhibitor Bortezomib, an anti-cancer drug approved for treatment of multiple myeloma and Mantle cell lymphoma. We recently reported that the ERAD inhibitor Eeyarestatin I (EerI) also disturbs ER homeostasis and has anti-cancer activities resembling that of Bortezomib. METHODOLOGY AND PRINCIPAL FINDINGS: Here we developed in vitro binding and cell-based functional assays to demonstrate that a nitrofuran-containing (NFC) group in EerI is the functional domain responsible for the cytotoxicity. Using both SPR and pull down assays, we show that EerI directly binds the p97 ATPase, an essential component of the ERAD machinery, via the NFC domain. An aromatic domain in EerI, although not required for p97 interaction, can localize EerI to the ER membrane, which improves its target specificity. Substitution of the aromatic module with another benzene-containing domain that maintains membrane localization generates a structurally distinct compound that nonetheless has similar biologic activities as EerI. CONCLUSIONS AND SIGNIFICANCE: Our findings reveal a class of bifunctional chemical agents that can preferentially inhibit membrane-bound p97 to disrupt ER homeostasis and to induce tumor cell death. These results also suggest that the AAA ATPase p97 may be a potential drug target for cancer therapeutics.
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    10. 10
      Wang, Q.; Li, L.; Ye, Y. Inhibition of p97-dependent protein degradation by Eeyarestatin I. J. Biol. Chem. 2008, 283, 7445– 7454,  DOI: 10.1074/jbc.M708347200
      10
      Inhibition of p97-dependent Protein Degradation by Eeyarestatin I
      Wang, Qiuyan; Li, Lianyun; Ye, Yihong
      Journal of Biological Chemistry (2008), 283 (12), 7445-7454CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-assocd. degrdn. involves substrate retrotranslocation from the ER lumen into the cytosol for degrdn. by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which hydrolyzes ATP to dislocate polyubiquitinated substrates into the cytosol. Polypeptides extd. by p97 are eventually transferred to the proteasome for destruction. In mammalian cells, ERAD can be blocked by a chem. inhibitor termed Eeyarestatin I, but the mechanism of EerI action is unclear. Here we report that EerI can assoc. with a p97 complex to inhibit ERAD. The interaction of EerI with the p97 complex appears to neg. influence a deubiquitinating process that is mediated by p97-assocd. deubiquitinating enzymes. We further show that ataxin-3, a p97-assocd. deubiquitinating enzyme previously implicated in ER-assocd. degrdn., is among those affected. Interestingly, p97-assocd. deubiquitination is also involved in degrdn. of a sol. substrate. Our analyses establish a role for a novel deubiquitinating process in proteasome-dependent protein turnover.
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    11. 11
      Gamayun, I.; O’Keefe, S.; Pick, T.; Klein, M. C.; Nguyen, D.; McKibbin, C.; Piacenti, M.; Williams, H. M.; Flitsch, S. L.; Whitehead, R. C.; Swanton, E.; Helms, V.; High, S.; Zimmermann, R.; Cavalié, A. Eeyarestatin compounds selectively enhance Sec61-mediated Ca 2+ leakage from the endoplasmic reticulum. Cell Chem. Biol. 2019, 26, 571– 583, e6  DOI: 10.1016/j.chembiol.2019.01.010
      11
      Eeyarestatin Compounds Selectively Enhance Sec61-Mediated Ca2+ Leakage from the Endoplasmic Reticulum
      Gamayun, Igor; O'Keefe, Sarah; Pick, Tillman; Klein, Marie-Christine; Nguyen, Duy; McKibbin, Craig; Piacenti, Michela; Williams, Helen M.; Flitsch, Sabine L.; Whitehead, Roger C.; Swanton, Eileithyia; Helms, Volkhard; High, Stephen; Zimmermann, Richard; Cavalie, Adolfo
      Cell Chemical Biology (2019), 26 (4), 571-583.e6CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
      Eeyarestatin 1 (ES1) inhibits p97-dependent protein degrdn., Sec61-dependent protein translocation into the endoplasmic reticulum (ER), and vesicular transport within the endomembrane system. Here, we show that ES1 impairs Ca2+ homeostasis by enhancing the Ca2+ leakage from mammalian ER. A comparison of various ES1 analogs suggested that the 5-nitrofuran (5-NF) ring of ES1 is crucial for this effect. Accordingly, the analog ES24, which conserves the 5-NF domain of ES1, selectively inhibited protein translocation into the ER, displayed the highest potency on ER Ca2+ leakage of ES1 analogs studied and induced Ca2+-dependent cell death. Using small interfering RNA-mediated knockdown of Sec61a, we identified Sec61 complexes as the targets that mediate the gain of Ca2+ leakage induced by ES1 and ES24. By interacting with the lateral gate of Sec61a, ES1 and ES24 likely capture Sec61 complexes in a Ca2+-permeable, open state, in which Sec61 complexes allow Ca2+ leakage but are translocation incompetent.
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    12. 12
      McKenna, M.; Simmonds, R. E.; High, S. Mechanistic insights into the inhibition of Sec61-dependent co- and post-translational translocation by mycolactone. J. Cell Sci. 2016, 129, 1404– 1415,  DOI: 10.1242/jcs.182352
      12
      Mechanistic insights into the inhibition of Sec61-dependent co- and post-translational translocation by mycolactone
      McKenna, Michael; Simmonds, Rachel E.; High, Stephen
      Journal of Cell Science (2016), 129 (7), 1404-1415CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
      The virulence factor mycolactone is responsible for the immunosuppression and tissue necrosis that characterize Buruli ulcer, a disease caused by infection with Mycobacterium ulcerans. In this study, we confirm that Sec61, the protein-conducting channel that coordinates entry of secretory proteins into the endoplasmic reticulum, is a primary target of mycolactone, and characterize the nature of its inhibitory effect. We conclude that mycolactone constrains the ribosome-nascent-chain-Sec61 complex, consistent with its broad-ranging perturbation of the co-translational translocation of classical secretory proteins. In contrast, the effect of mycolactone on the post-translational ribosome-independent translocation of short secretory proteins through the Sec61 complex is dependent on both signal sequence hydrophobicity and the translocation competence of the mature domain. Changes to protease sensitivity strongly suggest that mycolactone acts by inducing a conformational change in the pore-forming Sec61α subunit. These findings establish that mycolactone inhibits Sec61-mediated protein translocation and highlight differences between the co- and post-translational routes that the Sec61 complex mediates. We propose that mycolactone also provides a useful tool for further delineating the mol. mechanisms of Sec61-dependent protein translocation.
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    13. 13
      Cross, B. C. S.; McKibbin, C.; Callan, A. C.; Roboti, P.; Piacenti, M.; Rabu, C.; Wilson, C. M.; Whitehead, R.; Flitsch, S. L.; Pool, M. R.; High, S.; Swanton, E. Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum. J. Cell Sci. 2009, 122, 4393– 4400,  DOI: 10.1242/jcs.054494
      13
      Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum
      Cross, Benedict C. S.; McKibbin, Craig; Callan, Anna C.; Roboti, Peristera; Piacenti, Michela; Rabu, Catherine; Wilson, Cornelia M.; Whitehead, Roger; Flitsch, Sabine L.; Pool, Martin R.; High, Stephen; Swanton, Eileithyia
      Journal of Cell Science (2009), 122 (23), 4393-4400CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
      Prodn. and trafficking of proteins entering the secretory pathway of eukaryotic cells is coordinated at the endoplasmic reticulum (ER) in a process that begins with protein translocation via the membrane-embedded ER translocon. The same complex is also responsible for the co-translational integration of membrane proteins and orchestrates polypeptide modifications that are often essential for protein function. We now show that the previously identified inhibitor of ER-assocd. degrdn. (ERAD) eeyarestatin 1 (ESI) is a potent inhibitor of protein translocation. We have characterized this inhibition of ER translocation both in vivo and in vitro, and provide evidence that ESI targets a component of the Sec61 complex that forms the membrane pore of the ER translocon. Further analyses show that ESI acts by preventing the transfer of the nascent polypeptide from the co-translational targeting machinery to the Sec61 complex. These results identify a novel effect of ESI, and suggest that the drug can modulate canonical protein transport from the cytosol into the mammalian ER both in vitro and in vivo.
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    14. 14
      Itskanov, S.; Wang, L.; Junne, T.; Sherriff, R.; Xiao, L.; Blanchard, N.; Shi, W. Q.; Forsyth, C.; Hoepfner, D.; Spiess, M.; Park, E. A common mechanism of Sec61 translocon inhibition by small molecules. bioRxiv (Biochemistry) , August 11, 2022, 503542. DOI: 10.1101/2022.08.11.503542 .
      There is no corresponding record for this reference.
    15. 15
      Denks, K.; Vogt, A.; Sachelaru, I.; Petriman, N.-A.; Kudva, R.; Koch, H.-G. The Sec translocon mediated protein transport in prokaryotes and eukaryotes. Mol. Membr. Biol. 2014, 31, 58– 84,  DOI: 10.3109/09687688.2014.907455
      15
      The Sec translocon mediated protein transport in prokaryotes and eukaryotes
      Denks, Kaert; Vogt, Andreas; Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-Georg
      Molecular Membrane Biology (2014), 31 (2-3), 58-84CODEN: MMEBE7; ISSN:0968-7688. (Informa Healthcare)
      A review. Protein transport via the Sec translocon represents an evolutionary conserved mechanism for delivering cytosolically synthesized proteins to extracytosolic compartments. The Sec translocon has a 3-subunit core, termed Sec61 in eukaryotes and SecYEG in bacteria. It is located in the endoplasmic reticulum (ER) of eukaryotes and in the cytoplasmic membrane of bacteria where it constitutes a channel that can be activated by multiple partner proteins. These partner proteins det. the mechanism of polypeptide movement across the channel. During signal recognition particle (SRP)-dependent co-translational targeting, the ribosome threads the nascent protein directly into the Sec channel. This pathway in bacteria is mainly dedicated for membrane proteins, but in eukaryotes it is also employed by secretory proteins. The alternative pathway, leading to post-translational translocation across the Sec translocon engages an ATP-dependent pushing mechanism by motor protein SecA in bacteria and a ratcheting mechanism by lumenal chaperone BiP in eukaryotes. Protein transport and biogenesis is also assisted by addnl. proteins at the lateral gate of SecY/Sec61α and in the lumen of the ER or in the periplasm of bacterial cells. The modular assembly enables the Sec complex to transport a vast array of substrates. Here, the authors summarize recent biochem. and structural information on the prokaryotic and eukaryotic Sec translocons and describe the remarkably complex interaction network of the Sec complexes.
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    16. 16
      Steenhuis, M.; Koningstein, G. M.; Oswald, J.; Pick, T.; O’Keefe, S.; Koch, H.-G.; Cavalié, A.; Whitehead, R. C.; Swanton, E.; High, S.; Luirink, J. Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogens. Mol. Microbiol. 2021, 115, 28– 40,  DOI: 10.1111/mmi.14589
      16
      Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogens
      Steenhuis, Maurice; Koningstein, Gregory M.; Oswald, Julia; Pick, Tillman; O'Keefe, Sarah; Koch, Hans-Georg; Cavalie, Adolfo; Whitehead, Roger C.; Swanton, Eileithyia; High, Stephen; Luirink, Joen
      Molecular Microbiology (2021), 115 (1), 28-40CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
      Eeyarestatin 1 (ES1) is an inhibitor of endoplasmic reticulum (ER) assocd. protein degrdn., Sec61-dependent Ca2+ homeostasis and protein translocation into the ER. Recently, evidence was presented showing that a smaller analog of ES1, ES24, targets the Sec61-translocon, and captures it in an open conformation that is translocation-incompetent. We now show that ES24 impairs protein secretion and membrane protein insertion in Escherichia coli via the homologous SecYEG-translocon. Transcriptomic anal. suggested that ES24 has a complex mode of action, probably involving multiple targets. Interestingly, ES24 shows antibacterial activity toward clin. relevant strains. Furthermore, the antibacterial activity of ES24 is equiv. to or better than that of nitrofurantoin, a known antibiotic that, although structurally similar to ES24, does not interfere with SecYEG-dependent protein trafficking. Like nitrofurantoin, we find that ES24 requires activation by the NfsA and NfsB nitroreductases, suggesting that the formation of highly reactive nitroso intermediates is essential for target inactivation in vivo.
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    17. 17
      du Plessis, D. J. F.; Nouwen, N.; Driessen, A. J. M. The Sec translocase. Biochim. Biophys. Acta 2011, 1808, 851– 865,  DOI: 10.1016/j.bbamem.2010.08.016
      17
      The Sec translocase
      du Plessis, David J. F.; Nouwen, Nico; Driessen, Arnold J. M.
      Biochimica et Biophysica Acta, Biomembranes (2011), 1808 (3), 851-865CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      A review. The vast majority of proteins trafficking across or into the bacterial cytoplasmic membrane occur via the translocon. The translocon consists of the SecYEG complex that forms an evolutionarily conserved heterotrimeric protein-conducting membrane channel that functions in conjunction with a variety of ancillary proteins. For post-translational protein translocation, the translocon interacts with cytosolic motor protein SecA that drives the ATP-dependent stepwise translocation of unfolded polypeptides across the membrane. For the cotranslational integration of membrane proteins, the translocon interacts with ribosome-nascent chain complexes and membrane insertion is coupled to polypeptide chain elongation at the ribosome. These processes are assisted by the YidC and SecDF(yajC) complex that transiently interacts with the translocon. This review summarizes the authors' current understanding of the structure-function relation of the translocon and its interactions with ancillary components during protein translocation and membrane protein insertion.
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    18. 18
      Yuan, J.; Zweers, J. C.; van Dijl, J. M.; Dalbey, R. E. Protein transport across and into cell membranes in bacteria and archaea. Cell. Mol. Life Sci. 2010, 67, 179– 199,  DOI: 10.1007/s00018-009-0160-x
      18
      Protein transport across and into cell membranes in bacteria and archaea
      Yuan, Jijun; Zweers, Jessica C.; van Dijl, Jan Maarten; Dalbey, Ross E.
      Cellular and Molecular Life Sciences (2010), 67 (2), 179-199CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
      A review. In the three domains of life, the Sec, YidC/Oxa1, and Tat translocases play important roles in protein translocation across membranes and membrane protein insertion. While extensive studies have been performed on the endoplasmic reticular and Escherichia coli systems, far fewer studies have been done on archaea, other Gram-neg. bacteria, and Gram-pos. bacteria. Interestingly, work carried out to date has shown that there are differences in the protein transport systems in terms of the no. of translocase components and, in some cases, the translocation mechanisms and energy sources that drive translocation. In this review, we will describe the different systems employed to translocate and insert proteins across or into the cytoplasmic membrane of archaea and bacteria.
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    19. 19
      Neef, J.; van Dijl, J. M.; Buist, G. Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential. Essays Biochem 2021, 65, 187– 195,  DOI: 10.1042/EBC20200171
      19
      Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential
      Neef, Jolanda; van Dijl, Jan Maarten; Buist, Girbe
      Essays in Biochemistry (2021), 65 (2), 187-195CODEN: ESBIAV; ISSN:1744-1358. (Portland Press Ltd.)
      Secreted recombinant proteins are of great significance for industry, healthcare and a sustainable bio-based economy. Consequently, there is an ever-increasing need for efficient prodn. platforms to deliver such proteins in high amts. and high quality. Gram-pos. bacteria, particularly bacilli such as Bacillus subtilis, are favored for the prodn. of secreted industrial enzymes. Nevertheless, recombinant protein prodn. in the B. subtilis cell factory can be very challenging due to bottlenecks in the general (Sec) secretion pathway as well as this bacterium's intrinsic capability to secrete a cocktail of highly potent proteases. This has placed another Gram-pos. bacterium, Lactococcus lactis, in the focus of attention as an alternative, non-proteolytic, cell factory for secreted proteins. Here we review our current understanding of the secretion pathways exploited in B. subtilis and L. lactis to deliver proteins from their site of synthesis, the cytoplasm, into the fermn. broth. An advantage of this cell factory comparison is that it identifies opportunities for protein secretion pathway engineering to remove or bypass current prodn. bottlenecks. Noteworthy new developments in cell factory engineering are the mini-Bacillus concept, highlighting potential advantages of massive genome minimization, and the application of thus far untapped 'non-classical' protein secretion routes. Altogether, it is foreseen that engineered lactococci will find future applications in the prodn. of high-quality proteins at the relatively small pilot scale, while engineered bacilli will remain a favored choice for protein prodn. in bulk.
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    20. 20
      Gardiner, B. J.; Stewardson, A. J.; Abbott, I. J.; Peleg, A. Y. Nitrofurantoin and fosfomycin for resistant urinary tract infections: old drugs for emerging problems. Aust Prescr 2019, 42, 14– 19,  DOI: 10.18773/austprescr.2019.002
      20
      Nitrofurantoin and fosfomycin for resistant urinary tract infections: old drugs for emerging problems
      Gardiner Bradley J; Stewardson Andrew J; Abbott Iain J; Peleg Anton Y; Gardiner Bradley J; Stewardson Andrew J; Abbott Iain J; Peleg Anton Y; Gardiner Bradley J; Stewardson Andrew J; Abbott Iain J; Peleg Anton Y
      Australian prescriber (2019), 42 (1), 14-19 ISSN:0312-8008.
      UNCOMPLICATED URINARY TRACT INFECTION IS ONE OF THE MOST COMMON INDICATIONS FOR ANTIBIOTIC USE IN THE COMMUNITY HOWEVER THE GRAM-NEGATIVE ORGANISMS THAT CAN CAUSE THE INFECTION ARE BECOMING MORE RESISTANT TO ANTIBIOTICS: MANY MULTIDRUG RESISTANT ORGANISMS RETAIN SUSCEPTIBILITY TO TWO OLD ANTIBIOTICS NITROFURANTOIN AND FOSFOMYCIN ADVANTAGES OVER NEWER DRUGS INCLUDE THEIR HIGH URINARY CONCENTRATIONS AND MINIMAL TOXICITY: FOSFOMYCIN IS A POTENTIAL TREATMENT OPTION FOR PATIENTS WITH UNCOMPLICATED URINARY TRACT INFECTION DUE TO RESISTANT ORGANISMS NITROFURANTOIN MAY BE MORE EFFECTIVE AND CAN BE USED FOR URINARY INFECTIONS IN PREGNANT WOMEN:
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    21. 21
      Wenzel, M.; Dekker, M. P.; Wang, B.; Burggraaf, M. J.; Bitter, W.; van Weering, J. R. T.; Hamoen, L. W. A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline. Commun. Biol. 2021, 4, 306,  DOI: 10.1038/s42003-021-01809-8
      21
      A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline
      Wenzel, Michaela; Dekker, Marien P.; Wang, Biwen; Burggraaf, Maroeska J.; Bitter, Wilbert; van Weering, Jan R. T.; Hamoen, Leendert W.
      Communications Biology (2021), 4 (1), 306CODEN: CBOIDQ; ISSN:2399-3642. (Nature Research)
      Transmission electron microscopy of cell sample sections is a popular technique in microbiol. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphol. phenotypes due to the lack of sufficient nos. of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture vols. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphol. phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.
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    22. 22
      Tu, Y.; McCalla, D. R. Effect of activated nitrofurans on DNA. Biochim. Biophys. Acta 1975, 402, 142– 149,  DOI: 10.1016/0005-2787(75)90032-5
      22
      Effect of activated nitrofurans on DNA
      Tu, Yu; McCalla, D. R.
      Biochimica et Biophysica Acta, Nucleic Acids and Protein Synthesis (1975), 402 (2), 142-9CODEN: BBNPAS; ISSN:0005-2787.
      Enzymically activated nitrofurazone (I) [59-87-0] reacted with covalently closed circular DNA (derived from Escherichia coli minicells carrying λdv) to give ≥2 kinds of damage: breaks which were detected on neutral sucrose gradients and alkali-labile lesions in DNA which were converted to breaks when the DNA was subsequently treated with alkali. DNA, isolated from minicells exposed to the drug, also contained lesions which were converted to breaks upon treatment with endonuclease prepns. obtained from Micrococcus luteus. Minicells repaired both breaks and nuclease-susceptible lesions within 2 hr but did not repair alkali labile lesions within that time. Expts. with 3 other nitrofurans showed that there were considerable differences in the degree to which DNA was damaged by activated metabolites of various derivs. and that the potency of the compds. as mutagens and carcinogens was correlated with the amt. of damage caused to minicell DNA.
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    23. 23
      Whiteway, J.; Koziarz, P.; Veall, J.; Sandhu, N.; Kumar, P.; Hoecher, B.; Lambert, I. B. Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli. J. Bacteriol. 1998, 180, 5529– 5539,  DOI: 10.1128/JB.180.21.5529-5539.1998
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      Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli
      Whiteway, J.; Koziarz, P.; Veall, J.; Sandhu, N.; Kumar, P.; Hoecher, B.; Lambert, I. B.
      Journal of Bacteriology (1998), 180 (21), 5529-5539CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Nitroheterocyclic and nitroarom. compds. constitute an enormous range of chems. whose potent biol. activity has significant human health and environmental implications. The biol. activity of nitro-substituted compds. is derived from reductive metab. of the nitro moiety, a process catalyzed by a variety of nitroreductase activities. Resistance of bacteria to nitro-substituted compds. is believed to result primarily from mutations in genes encoding oxygen-insensitive nitroreductases. We have characterized the nfsA and nfsB genes of a large no. of nitrofuran-resistant mutants of Escherichia coli and have correlated mutation with cell ext. nitroreductase activity. Our studies demonstrate that first-step resistance to furazolidone or nitrofurazone results from an nfsA mutation, while the increased resistance assocd. with second-step mutants is a consequence of an nfsB mutation. Inferences made from mutation about the structure-function relationships of NfsA and NfsB are discussed, esp. with regard to the identification of FMN binding sites. We show that expression of plasmid-carried nfsA and nfsB genes in resistant mutants restores sensitivity to nitrofurans. Among the 20 first-step and 53 second-step mutants isolated in this study, 65% and 49%, resp., contained insertion sequence elements in nfsA and nfsB. IS1 integrated in both genes, while IS30 and IS186 were found only in nfsA and IS2 and IS5 were obsd. only in nfsB. Insertion hot spots for IS30 and IS186 are indicated in nfsA, and a hot spot for IS5 insertion is evident in nfsB. We discuss potential regional and sequence-specific determinants for insertion sequence element integration in nfsA and nfsB.
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    24. 24
      Valle, A.; Le Borgne, S.; Bolívar, J.; Cabrera, G.; Cantero, D. Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2’-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biol. Appl. Microbiol. Biotechnol. 2012, 94, 163– 171,  DOI: 10.1007/s00253-011-3787-0
      24
      Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2'-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties
      Valle, Antonio; Borgne, Sylvie; Bolivar, Jorge; Cabrera, Gema; Cantero, Domingo
      Applied Microbiology and Biotechnology (2012), 94 (1), 163-171CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Benzohydroxamic acids, such as 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chem. synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, Et 2-(2'-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroarom. compds., which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analyzed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, resp. In both cases, the biotransformation capacity of the strains was recovered (6.09 ± 0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33% ± 9.42% and 59.68% ± 2.0% resp.).
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    25. 25
      Pedreira, T.; Elfmann, C.; Stülke, J. The current state of SubtiWiki, the database for the model organism Bacillus subtilis. Nucleic Acids Res. 2022, 50, D875– D882,  DOI: 10.1093/nar/gkab943
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      The current state of SubtiWiki, the database for the model organism Bacillus subtilis
      Pedreira, Tiago; Elfmann, Christoph; Stuelke, Joerg
      Nucleic Acids Research (2022), 50 (D1), D875-D882CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      Bacillus subtilis is a Gram-pos. model bacterium with extensive documented annotation. However, with the rise of high-throughput techniques, the amt. of complex data being generated every year has been increasing at a fast pace. Thus, having platforms ready to integrate and give a representation to these data becomes a priority. To address it, SubtiWiki was created in 2008 and has been growing in data and viewership ever since. With millions of requests every year, it is the most visited B. subtilis database, providing scientists all over the world with curated information about its genes and proteins, as well as intricate protein-protein interactions, regulatory elements, expression data and metabolic pathways. However, there is still a large portion of annotation to be unveiled for some biol. elements. Thus, to facilitate the development of new hypotheses for research, we have added a Homol. section covering potential protein homologs in other organisms. Here, we present the recent developments of SubtiWiki and give a guided tour of our database and the current state of the data for this organism.
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    26. 26
      Wenzel, M.; Rautenbach, M.; Vosloo, J. A.; Siersma, T.; Aisenbrey, C. H. M.; Zaitseva, E.; Laubscher, W. E.; van Rensburg, W.; Behrends, J.; Bechinger, B.; Hamoen, L. W. The multifaceted antibacterial mechanisms of the pioneering peptide antibiotics tyrocidine and gramicidin S. mBio 2018, 9, e00802-18  DOI: 10.1128/mBio.00802-18
      There is no corresponding record for this reference.
    27. 27
      Saeloh, D.; Tipmanee, V.; Jim, K. K.; Dekker, M. P.; Bitter, W.; Voravuthikunchai, S. P.; Wenzel, M.; Hamoen, L. W. The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluidity. PLoS Pathog 2018, 14, e1006876  DOI: 10.1371/journal.ppat.1006876
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      The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluidity
      Saeloh, Dennapa; Tipmanee, Varomyalin; Jim, Kin Ki; Dekker, Marien P.; Bitter, Wilbert; Voravuthikunchai, Supayang P.; Wenzel, Michaela; Hamoen, Leendert W.
      PLoS Pathogens (2018), 14 (2), e1006876/1-e1006876/35CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
      The acylphloroglucinol rhodomyrtone is a promising new antibiotic isolated from the rose myrtle Rhodomyrtus tomentosa, a plant used in Asian traditional medicine. While many studies have demonstrated its antibacterial potential in a variety of clin. applications, very little is known about the mechanism of action of rhodomyrtone. Preceding studies have been focused on intracellular targets, but no specific intracellular protein could be confirmed as main target. Using live cell, high-resoln., and electron microscopy we demonstrate that rhodomyrtone causes large membrane invaginations with a dramatic increase in fluidity, which attract a broad range of membrane proteins. Invaginations then form intracellular vesicles, thereby trapping these proteins. Aberrant protein localization impairs several cellular functions, including the respiratory chain and the ATP synthase complex. Being uncharged and devoid of a particular amphipathic structure, rhodomyrtone did not seem to be a typical membrane-inserting mol. In fact, mol. dynamics simulations showed that instead of inserting into the bilayer, rhodomyrtone transiently binds to phospholipid head groups and causes distortion of lipid packing, providing explanations for membrane fluidization and induction of membrane curvature. Both its transient binding mode and its ability to form protein- trapping membrane vesicles are unique, making it an attractive new antibiotic candidate with a novel mechanism of action.
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    28. 28
      Müller, A.; Wenzel, M.; Strahl, H.; Grein, F.; Saaki, TN V; Kohl, B.; Siersma, T.; Bandow, J. E.; Sahl, H.-G.; Schneider, T.; Hamoen, L. W. Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E7077– E7086,  DOI: 10.1073/pnas.1611173113
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      Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains
      Mueller, Anna; Wenzel, Michaela; Strahl, Henrik; Grein, Fabian; Saaki, Terrens N. V.; Kohl, Bastian; Siersma, Tjalling; Bandow, Julia E.; Sahl, Hans-Georg; Schneider, Tanja; Hamoen, Leendert W.
      Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (45), E7077-E7086CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clin. importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different expts. have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To det. which model is correct, the authors carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. The authors found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although the authors found no evidence for altered membrane curvature, the authors confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, the authors showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-assocd. lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization obsd. with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to the authors' understanding of membrane-active antibiotics.
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    29. 29
      Garcia Martinez, P.; Winston, G. W.; Metash-Dickey, C.; O’Hara, S. C.; Livingstone, D. R. Nitrofurantoin-stimulated reactive oxygen species production and genotoxicity in digestive gland microsomes and cytosol of the common mussel (Mytilus edulis L.). Toxicol. Appl. Pharmacol. 1995, 131, 332– 341,  DOI: 10.1006/taap.1995.1076
      29
      Nitrofurantoin-stimulated reactive oxygen species production and genotoxicity in digestive gland microsomes and cytosol of the common mussel (Mytilus edulis L.)
      Garcia Martinez P; Winston G W; Metash-Dickey C; O'Hara S C; Livingstone D R
      Toxicology and applied pharmacology (1995), 131 (2), 332-41 ISSN:0041-008X.
      The ability of nitrofurantoin (NF) to produce reactive oxygen species (ROS) was investigated in subcellular fractions of digestive gland of the mussel Mytilus edulis in terms of oxygen consumption and the formation of superoxide anion radical (O2-) (measured as SOD-sensitive cytochrome c reduction or SOD-sensitive sensitive .OH production), H2O2 (effect of catalase), and hydroxyl radical (.OH) (iron/EDTA-mediated oxidation of KMBA to ethylene). Additionally, the genotoxic effects of NF were examined using the Salmonella typhimurium umu mutagenicity assay. Microsomal NAD(P)H-dependent oxygen consumption was stimulated by NF, leading to the formation of H2O2. Stimulation of microsomal O2- production by NF was evident for NADH but not NADPH, confirming redox cycling at least with the former coenzyme. No stimulation of O2- production was obvious for cytosolic fraction with either coenzyme. NF stimulated microsomal NAD(P)H-dependent .OH production; the rates of .OH production were greater for NADH than NADPH; and the .OH was indicated to be formed, at least in part, by an iron-catalyzed Haber-Weiss reaction. A role was indicated for a free radical driven Fenton reaction in the NF-stimulated microsomal production of .OH from NADPH. The production of mutagenic species from NF was observed for cytosol but not for microsomes, and the former effects were greater for NADH than NADPH. Overall, the NAD(P)H-dependent microsomal generation of ROS, and the lack of correlation of ROS production with mutagenicity, are considered indicative of the potential of digestive gland to metabolize NF by both one-electron and two-electron reductive pathways. From this and other studies, enhanced ROS production by NF and other redox cycling xenobiotics is indicated to be a widespread phenomenon in aquatic organisms and a potential mechanism of pollutant-mediated toxicity.
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    30. 30
      Mols, M.; Abee, T. Primary and secondary oxidative stress in Bacillus. Environ. Microbiol 2011, 13, 1387– 1394,  DOI: 10.1111/j.1462-2920.2011.02433.x
      30
      Primary and secondary oxidative stress in Bacillus
      Mols, Maarten; Abee, Tjakko
      Environmental Microbiology (2011), 13 (6), 1387-1394CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)
      A review. Coping with oxidative stress originating from oxidizing compds. or reactive oxygen species (ROS), assocd. with the exposure to agents that cause environmental stresses, is one of the prerequisites for an aerobic lifestyle of Bacillus spp. such as B. subtilis, B. cereus and B. anthracis. This minireview highlights novel insights in the primary oxidative stress response caused by oxidizing compds. including hydrogen peroxide and the secondary oxidative stress responses apparent upon exposure to a range of agents and conditions leading to environmental stresses such as antibiotics, heat and acid. Insights in the pathways and damaging radicals involved have been compiled based among others on transcriptome studies, network analyses and fluorescence techniques for detection of ROS at single cell level. Exploitation of the current knowledge for the control of spoilage and pathogenic bacteria is discussed.
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    31. 31
      Ameziane, El.; Hassani, R.; Dupuy, C. Detection of intracellular reactive oxygen species (CM-H2DCFDA). Bio-protocol 2013, 3, e313  DOI: 10.21769/BioProtoc.313
      There is no corresponding record for this reference.
    32. 32
      Taiwo, F. A. Mechanism of tiron as scavenger of superoxide ions and free electrons. Spectroscopy 2008, 22, 491– 498,  DOI: 10.1155/2008/953692
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      Mechanism of tiron as scavenger of superoxide ions and free electrons
      Taiwo, Fatai A.
      Spectroscopy (Amsterdam, Netherlands) (2008), 22 (6), 491-498CODEN: SPIJDZ; ISSN:0712-4813. (IOS Press)
      Sodium 4,5-dihydroxybenzene-1,3-disulfonate (tiron) has been reported to be an efficient chelator of certain metal ions, and a substrate in several enzyme reactions. Its small size facilitates cell entry and therefore modulates intracellular electron transfer reactions as an antioxidant by scavenging free radicals. Its redn. by electrochem. and enzymic methods gives identical products; a semiquinone detectable by EPR spectroscopy. In a test of its use as a spin trap, in comparison with DMPO, tiron does not form a mol. spin-adduct but proves more functional as an electron trap. Electron addn. to tiron is more facile than redn. of dioxygen as obsd. by the non-formation of DMPO-OOH spin-adduct in the system XO/HPX/O2/DMPO/tiron. Rather, it is the tiron semiquinone radical which is formed quant. with increasing concn. of hypoxanthine independent of oxygen concn. These results offer explanation for the action of tiron and its suitability for measuring electron release in hypoxic conditions, and also for mitigating redox-induced toxicity in drug regimes by acting as an electron scavenger.
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    33. 33
      Chueca, B.; Pagán, R.; García-Gonzalo, D. Differential mechanism of Escherichia coli inactivation by (+)-limonene as a function of cell physiological state and drug’s concentration. PLoS One 2014, 9, e94072  DOI: 10.1371/journal.pone.0094072
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      Differential mechanism of Escherichia coli inactivation by (+)-limonene as a function of cell physiological state and drug concentration
      Chueca, Beatriz; Pagan, Rafael; Garcia-Gonzalo, Diego
      PLoS One (2014), 9 (4), e94072/1-e94072/7, 7 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      (+)-Limonene is a lipophilic antimicrobial compd., extd. from citrus fruits' essential oils, that is used as a flavouring agent and org. solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. The objective here was to det. whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 h. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2'-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was obsd. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as obsd. for bactericidal drugs. However, several differences have been obsd. between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, the results demonstrated that E. coli inactivation was influenced by its physiol. state and the drug's concn. e. Expts. with stationary-phase cells or 4000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. This study has also shown that drug's concn. is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compd., and in clarifying the controversy about the mechanism of inactivation by bactericidal antibiotics.
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    34. 34
      Lenhart, J. S.; Brandes, E. R.; Schroeder, J. W.; Sorenson, R. J.; Showalter, H. D.; Simmons, L. A. RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stress. J. Bacteriol. 2014, 196, 2851– 2860,  DOI: 10.1128/JB.01494-14
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      RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stress
      Lenhart, Justin S.; Brandes, Eileen R.; Schroeder, Jeremy W.; Sorenson, Roderick J.; Showalter, Hollis D.; Simmons, Lyle A.
      Journal of Bacteriology (2014), 196 (15), 2851-2860, 11 pp.CODEN: JOBAAY; ISSN:1098-5530. (American Society for Microbiology)
      RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these 2 pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examd., including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an addnl. component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.
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    35. 35
      Wenzel, M.; Kohl, B.; Münch, D.; Raatschen, N.; Albada, H. B.; Hamoen, L.; Metzler-Nolte, N.; Sahl, H. G.; Bandow, J. E. Proteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membrane. Antimicrob. Agents Chemother. 2012, 56, 5749– 5757,  DOI: 10.1128/AAC.01380-12
      35
      Proteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membrane
      Wenzel, Michaela; Kohl, Bastian; Muench, Daniela; Raatschen, Nadja; Albada, H. Bauke; Hamoen, Leendert; Metzler-Nolte, Nils; Sahl, Hans-Georg; Bandow, Julia E.
      Antimicrobial Agents and Chemotherapy (2012), 56 (11), 5749-5757CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
      Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides contg. lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane compn. The authors studied the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane.
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    36. 36
      Scheinpflug, K.; Wenzel, M.; Krylova, O.; Bandow, E. J.; Dathe, M.; Strahl, H. Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis. Sci. Rep 2017, 7, 44332,  DOI: 10.1038/srep44332
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      Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis
      Scheinpflug Kathi; Krylova Oxana; Dathe Margitta; Wenzel Michaela; Bandow Julia E; Strahl Henrik
      Scientific reports (2017), 7 (), 44332 ISSN:.
      The synthetic cyclic hexapeptide cWFW (cyclo(RRRWFW)) has a rapid bactericidal activity against both Gram-positive and Gram-negative bacteria. Its detailed mode of action has, however, remained elusive. In contrast to most antimicrobial peptides, cWFW neither permeabilizes the membrane nor translocates to the cytoplasm. Using a combination of proteome analysis, fluorescence microscopy, and membrane analysis we show that cWFW instead triggers a rapid reduction of membrane fluidity both in live Bacillus subtilis cells and in model membranes. This immediate activity is accompanied by formation of distinct membrane domains which differ in local membrane fluidity, and which severely disrupts membrane protein organisation by segregating peripheral and integral proteins into domains of different rigidity. These major membrane disturbances cause specific inhibition of cell wall synthesis, and trigger autolysis. This novel antibacterial mode of action holds a low risk to induce bacterial resistance, and provides valuable information for the design of new synthetic antimicrobial peptides.
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    37. 37
      Omardien, S.; Drijfhout, J. W.; Vaz, F. M.; Wenzel, M.; Hamoen, L. W.; Zaat, S. A. J.; Brul, S. Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer. Biochim. Biophys. Acta 2018, 1860, 2404– 2415,  DOI: 10.1016/j.bbamem.2018.06.004
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      Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer
      Omardien, Soraya; Drijfhout, Jan W.; Vaz, Frederic M.; Wenzel, Michaela; Hamoen, Leendert W.; Zaat, Sebastian A. J.; Brul, Stanley
      Biochimica et Biophysica Acta, Biomembranes (2018), 1860 (11), 2404-2415CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      Amphipathic cationic antimicrobial peptides (AMPs) TC19 and TC84, derived from the major AMPs of human blood platelets, thrombocidins, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide showed to perturb the membrane of Bacillus subtilis. We aimed to det. the means by which the three AMPs cause membrane perturbation in vivo using B. subtilis and to evaluate whether the membrane alterations are dependent on the phospholipid compn. of the membrane. Physiol. anal. was employed using Alexa Fluor 488 labeled TC84, various fluorescence dyes, fluorescent microscopy techniques and structured illumination microscopy. TC19, TC84 and BP2 created extensive fluidity domains in the membrane that are permeable, thus facilitating the entering of the peptides and the leakage of the cytosol. The direct interaction of the peptides with the bilayer create the fluid domains. The changes caused in the packing of the phospholipids lead to the delocalization of membrane bound proteins, thus contributing to the cell's destruction. The changes made to the membrane appeared to be not dependent on the compn. of the phospholipid bilayer. The distortion caused to the fluidity of the membrane by the AMPs is sufficient to facilitate the entering of the peptides and leakage of the cytosol. Here we show in vivo that cationic AMPs cause "membrane leaks" at the site of membrane insertion by altering the organization and fluidity of the membrane. Our findings thus contribute to the understanding of the membrane perturbation characteristic of cationic AMPs.
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    38. 38
      Parasassi, T.; Gratton, E. Membrane lipid domains and dynamics as detected by Laurdan fluorescence. J. Fluoresc 1995, 5, 59– 69,  DOI: 10.1007/BF00718783
      38
      Membrane lipid domains and dynamics as detected by Laurdan fluorescence
      Parasassi, Tiziana; Gratton, Enrico
      Journal of Fluorescence (1995), 5 (1), 59-69CODEN: JOFLEN; ISSN:1053-0509.
      A review with 58 refs. 2-Dimethylamino-6-lauroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different max. and shape in the 2 phospholipid phases, due to differences in the polarity and in the amt. of dipolar relaxation. In bilayers composed of a mixt. of gel and liq.-cryst. phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using generalized polarization (GP). The wavelength dependence of the GP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liq.-cryst. phases was detd. The influence of cholesterol on the phase properties of the 2 phospholipid phases is proposed to be the cause of the phase behavior obsd. in natural membranes.
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    39. 39
      Wenzel, M.; Vischer, N. O. E.; Strahl, H.; Hamoen, L. W. Assessing membrane fluidity and visualizing fluid membrane domains in bacteria using fluorescent membrane dyes. Bio-protocol 2018, 8, e3063  DOI: 10.21769/BioProtoc.3063
      39
      Assessing membrane fluidity and visualizing fluid membrane domains in bacteria using fluorescent membrane dyes
      Wenzel, Michaela; Vischer, Norbert O. E.; Strahl, Henrik; Hamoen, Leendert W.
      Bio-Protocol (2018), 8 (20), e3063CODEN: BIOPCG; ISSN:2331-8325. (Bio-Protocol LLC)
      Membrane fluidity is a key parameter of bacterial membranes that undergoes quick adaptation in response to environmental challenges and has recently emerged as an important factor in the antibacterial mechanism of membrane-targeting antibiotics. The specific level of membrane fluidity is not uniform across the bacterial cell membrane. Rather, specialized microdomains assocd. with different cellular functions can exhibit fluidity values that significantly deviate from the av. Assessing changes in the overall membrane fluidity and formation of membrane microdomains is therefore pivotal to understand both the functional organization of the bacterial cell membrane as well as antibiotic mechanisms. Here we describe how two fluorescent membrane dyes, laurdan and DiIC12, can be employed to assess membrane fluidity in living bacteria. We focus on Bacillus subtilis, since this organism has been relatively well-studied with respect to membrane domains. However, we also describe how these assays can be adapted for other bacteria such as Staphylococcus aureus and Streptococcus pneumoniae.
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    40. 40
      Strahl, H.; Hamoen, L. W. Membrane potential is important for bacterial cell division. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 12281– 12286,  DOI: 10.1073/pnas.1005485107
      40
      Membrane potential is important for bacterial cell division
      Strahl, Henrik; Hamoen, Leendert W.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (27), 12281-12286, S12281/1-S12281/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Many cell division-related proteins are located at specific positions in the bacterial cell, and this organized distribution of proteins requires energy. Here, the authors report that the proton motive force, or more specifically the (trans)membrane potential, is directly involved in protein localization. It emerged that the membrane potential modulates the distribution of several conserved cell division proteins such as MinD, FtsA, and the bacterial cytoskeletal protein MreB. The authors show for MinD that this is based on the membrane potential stimulated binding of its C-terminal amphipathic helix. This function of the membrane potential has implications for how these morphogenetic proteins work and provide an explanation for the effects obsd. with certain antimicrobial compds.
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    41. 41
      Omardien, S.; Drijfhout, J. W.; Zaat, S. A.; Brul, S. Cationic amphipathic antimicrobial peptides perturb the inner membrane of germinated ppores thus inhibiting their outgrowth. Front. Microbiol. 2018, 9, 2277,  DOI: 10.3389/fmicb.2018.02277
      41
      Cationic Amphipathic Antimicrobial Peptides Perturb the Inner Membrane of Germinated Spores Thus Inhibiting Their Outgrowth
      Omardien Soraya; Brul Stanley; Drijfhout Jan Wouter; Zaat Sebastian A
      Frontiers in microbiology (2018), 9 (), 2277 ISSN:1664-302X.
      The mode of action of four cationic amphipathic antimicrobial peptides (AMPs) was evaluated against the non-pathogenic, Gram-positive, spore-forming bacterium, Bacillus subtilis. The AMPs were TC19, TC84, BP2, and the lantibiotic Nisin A. TC19 and TC84 were derived from the human thrombocidin-1. Bactericidal peptide 2 (BP2) was derived from the human bactericidal permeability increasing protein (BPI). We employed structured illumination microscopy (SIM), fluorescence microscopy, Alexa 488-labeled TC84, B. subtilis mutants producing proteins fused to the green fluorescent protein (GFP) and single-cell live imaging to determine the effects of the peptides against spores. TC19, TC84, BP2, and Nisin A showed to be bactericidal against germinated spores by perturbing the inner membrane, thus preventing outgrowth to vegetative cells. Single cell live imaging showed that the AMPs do not affect the germination process, but the burst time and subsequent generation time of vegetative cells. Alexa 488-labeled TC84 suggested that the TC84 might be binding to the dormant spore-coat. Therefore, dormant spores were also pre-coated with the AMPs and cultured on AMP-free culture medium during single-cell live imaging. Pre-coating of the spores with TC19, TC84, and BP2 had no effect on the germination process, and variably affected the burst time and generation time. However, the percentage of spores that burst and grew out into vegetative cells was drastically lower when pre-coated with Nisin A, suggesting a novel application potential of this lantibiotic peptide against spores. Our findings contribute to the understanding of AMPs and show the potential of AMPs as eventual therapeutic agents against spore-forming bacteria.
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    42. 42
      Skoczinski, P.; Volkenborn, K.; Fulton, A.; Bhadauriya, A.; Nutschel, C.; Gohlke, H.; Knapp, A.; Jaeger, K.-E. Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis. Microb. Cell Fact. 2017, 16, 160,  DOI: 10.1186/s12934-017-0772-z
      42
      Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis
      Skoczinski Pia; Volkenborn Kristina; Fulton Alexander; Knapp Andreas; Jaeger Karl-Erich; Skoczinski Pia; Fulton Alexander; Bhadauriya Anuseema; Nutschel Christina; Gohlke Holger; Gohlke Holger; Jaeger Karl-Erich
      Microbial cell factories (2017), 16 (1), 160 ISSN:.
      BACKGROUND: Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. RESULTS: In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. CONCLUSIONS: Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.
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    43. 43
      Jones, S. E.; Lloyd, L. J.; Tan, K. K.; Buck, M. Secretion defects that activate the phage shock response of Escherichia coli. J. Bacteriol. 2003, 185, 6707– 6711,  DOI: 10.1128/JB.185.22.6707-6711.2003
      43
      Secretion defects that activate the phage-shock response of Escherichia coli
      Jones, Susan E.; Lloyd, Louise J.; Tan, Kum K.; Buck, Martin
      Journal of Bacteriology (2003), 185 (22), 6707-6711CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      The phage shock protein (psp) operon of Escherichia coli is induced by membrane-damaging cues. Earlier studies linked defects in secretion across the inner membrane to induction of the psp response. Here we show that defects in yidC and sec secretion induce psp but that defects in tat and srp have no effect. We have also detd. the cellular location of PspB and PspD proteins.
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    44. 44
      te Winkel, J. D.; Gray, D. A.; Seistrup, K. H.; Hamoen, L. W.; Strahl, H. Analysis of antimicrobial-triggered membrane depolarization using voltage sensitive dyes. Front. Cell Dev. Biol. 2016, 4, 29,  DOI: 10.3389/fcell.2016.00029
      44
      Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes
      Te Winkel J Derk; Gray Declan A; Seistrup Kenneth H; Strahl Henrik; Hamoen Leendert W
      Frontiers in cell and developmental biology (2016), 4 (), 29 ISSN:2296-634X.
      The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.
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    45. 45
      Rowland, S. L.; Fu, X.; Sayed, M. A.; Zhang, Y.; Cook, W. R.; Rothfield, L. I. Membrane redistribution of the Escherichia coli MinD protein induced by MinE. J. Bacteriol. 2000, 182, 613– 619,  DOI: 10.1128/JB.182.3.613-619.2000
      45
      Membrane redistribution of the Escherichia coli MinD protein induced by MinE
      Rowland, S. L.; Fu, X.; Sayed, M. A.; Zhang, Y.; Cook, W. R.; Rothfield, L. I.
      Journal of Bacteriology (2000), 182 (3), 613-619CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Escherichia coli cells contain potential division sites at midcell and adjacent to the cell poles. Selection of the correct division site at midcell is controlled by three proteins: MinC, MinD, and MinE. It has previously been shown that a MinE-green fluorescent protein chimera (MinE-Gfp) localizes to the midcell site in an MinD-dependent manner. We use here Gfp-MinD to show that MinD assocs. with the membrane around the entire periphery of the cell in the absence of the other Min proteins and that MinE is capable of altering the membrane distribution pattern of Gfp-MinD. Studies with the isolated N-terminal and C-terminal MinE domains indicated different roles for the two MinE domains in the redistribution of membrane-assocd. MinD.
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    46. 46
      Munch, D.; Muller, A.; Schneider, T.; Kohl, B.; Wenzel, M.; Bandow, J. E.; Maffioli, S.; Sosio, M.; Donadio, S.; Wimmer, R.; Sahl, H.-G. The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functions. J. Biol. Chem. 2014, 289, 12063– 12076,  DOI: 10.1074/jbc.M113.537449
      46
      The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functions
      Munch Daniela; Sahl Hans-Georg; Muller Anna; Schneider Tanja; Kohl Bastian; Wenzel Michaela; Bandow Julia Elisabeth; Maffioli Sonia; Sosio Margherita; Donadio Stefano; Wimmer Reinhard
      The Journal of biological chemistry (2014), 289 (17), 12063-12076 ISSN:.
      The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.
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    47. 47
      Errington, J.; Daniel, R. A.; Scheffers, D.-J. Cytokinesis in bacteria. Microbiol Mol. Biol. Rev. 2003, 67, 52– 65,  DOI: 10.1128/MMBR.67.1.52-65.2003
      47
      Cytokinesis in bacteria
      Errington, Jeffery; Daniel, Richard A.; Scheffers, Dirk-Jan
      Microbiology and Molecular Biology Reviews (2003), 67 (1), 52-65CODEN: MMBRF7; ISSN:1092-2172. (American Society for Microbiology)
      A review. Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two neg. regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid free spaces near the cell poles in a manner that is beginning to be understood in cytol. and biochem. terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.
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    48. 48
      Omardien, S.; Drijfhout, J. W.; van Veen, H.; Schachtschabel, S.; Riool, M.; Hamoen, L. W.; Brul, S.; Zaat, S. A. J. Synthetic antimicrobial peptides delocalize membrane bound proteins thereby inducing a cell envelope stress response. Biochim Biophys acta Biomembr 2018, 1860, 2416– 2427,  DOI: 10.1016/j.bbamem.2018.06.005
      48
      Synthetic antimicrobial peptides delocalize membrane bound proteins thereby inducing a cell envelope stress response
      Omardien, Soraya; Drijfhout, Jan W.; van Veen, Henk; Schachtschabel, Soraya; Riool, Martijn; Hamoen, Leendert W.; Brul, Stanley; Zaat, Sebastian A. J.
      Biochimica et Biophysica Acta, Biomembranes (2018), 1860 (11), 2416-2427CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      Three amphipathic cationic antimicrobial peptides (AMPs) were characterized by detg. their effect on Gram-pos. bacteria using Bacillus subtilis strain 168 as a model organism. These peptides were TC19 and TC84, derivs. of thrombocidin-1 (TC-1), the major AMPs of human blood platelets, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide based on human bactericidal permeability increasing protein (BPI). To elucidate the possible mode of action of the AMPs we performed a transcriptomic anal. using microarrays. Physiol. analyses were performed using transmission electron microscopy (TEM), fluorescence microscopy and various B. subtilis mutants that produce essential membrane bound proteins fused to green fluorescent protein (GFP). The transcriptome anal. showed that the AMPs induced a cell envelope stress response (cell membrane and cell wall). The cell membrane stress response was confirmed with the physiol. observations that TC19, TC84 and BP2 perturb the membrane of B. subtilis. Using B. subtilis mutants, we established that the cell wall stress response is due to the delocalization of essential membrane bound proteins involved in cell wall synthesis. Other essential membrane proteins, involved in cell membrane synthesis and metab., were also delocalized due to alterations caused by the AMPs. We showed that peptides TC19, TC84 and BP2 perturb the membrane causing essential proteins to delocalize, thus preventing the possible repair of the cell envelope after the initial interference with the membrane. These AMPs show potential for eventual clin. application against Gram-pos. bacterial cells and merit further application-oriented investigation.
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    49. 49
      Jim, K. K.; Engelen-Lee, J.; van der Sar, A. M.; Bitter, W.; Brouwer, M. C.; van der Ende, A.; Veening, J.-W.; van de Beek, D.; Vandenbroucke-Grauls, C. M. J. E. Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model. J. Neuroinflammation 2016, 13, 188,  DOI: 10.1186/s12974-016-0655-y
      49
      Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model
      Jim, Kin Ki; Engelen-Lee, JooYeon; van der Sar, Astrid M.; Bitter, Wilbert; Brouwer, Matthijs C.; der Ende, Arie van; Veening, Jan-Willem; vandeBeek, Diederik; Vandenbroucke-Grauls, Christina M. J. E.
      Journal of Neuroinflammation (2016), 13 (), 188/1-188/13CODEN: JNOEB3; ISSN:1742-2094. (BioMed Central Ltd.)
      Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavorable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction. Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy. After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked redn. of phagocytes. This new meningitis model permits detailed anal. and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis.
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    50. 50
      Saeloh, D.; Wenzel, M.; Rungrotmongkol, T.; Hamoen, L. W.; Tipmanee, V.; Voravuthikunchai, S. P. Effects of rhodomyrtone on Gram-positive bacterial tubulin homologue FtsZ. PeerJ. 2017, 5, e2962  DOI: 10.7717/peerj.2962
      There is no corresponding record for this reference.
    51. 51
      Stepanek, J. J.; Lukezic, T.; Teichert, I.; Petkovic, H.; Bandow, J. E. Dual mechanism of action of the atypical tetracycline chelocardin. Biochim. Biophys. Acta 2016, 1864, 645– 654,  DOI: 10.1016/j.bbapap.2016.03.004
      51
      Dual mechanism of action of the atypical tetracycline chelocardin
      Stepanek, Jennifer J.; Lukezic, Tadeja; Teichert, Ines; Petkovic, Hrvoje; Bandow, Julia E.
      Biochimica et Biophysica Acta, Proteins and Proteomics (2016), 1864 (6), 645-654CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
      FR OLClassical tetracyclines targeting the protein biosynthesis machinery are commonly applied in human and veterinary medicine. OLtThe development and spread of resistance seriously compromise the successful treatment of bacterial infections. The atypical tetracycline chelocardin holds promise as it retains activity against tetracycline-resistant strains. It has been suggested that chelocardin targets the bacterial membrane, thus differing in mode of action from that of classical tetracyclines. The authors investigated the mechanism of action of chelocardin using global proteome anal. The proteome profiles after sublethal chelocardin stress were compared to a ref. compendium contg. antibiotic response profiles of Bacillus subtilis. This approach revealed a concn.-dependent dual mechanism of action. At low concns., like classical tetracyclines, chelocardin induces the proteomic signature for peptidyltransferase inhibition, demonstrating that protein biosynthesis inhibition is the dominant physiol. challenge. At higher concns., B. subtilis mainly responds to membrane stress indicating that at clin. relevant concns. the membrane is the main antibiotic target of chelocardin. Studying the effects on the membrane in more detail, it was found that chelocardin causes membrane depolarization but does not lead to formation of large pores. It is concluded that at growth inhibiting doses chelocardin not only targets protein biosynthesis but also corrupts the integrity of the bacterial membrane. This dual mechanism of action might prove beneficial in slowing the development of new resistance mechanisms against this atypical tetracycline.
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    52. 52
      Senges, C. H. R.; Stepanek, J. J.; Wenzel, M.; Raatschen, N.; Ay, Ü.; Märtens, Y.; Prochnow, P.; Vázquez Hernández, M.; Yayci, A.; Schubert, B.; Janzing, N. B. M.; Warmuth, H. L.; Kozik, M.; Bongard, J.; Alumasa, J. N.; Albada, B.; Penkova, M.; Lukežič, T.; Sorto, N. A.; Lorenz, N.; Miller, R.; Zhu, B.; Benda, M.; Stülke, J.; Schäkermann, S.; Leichert, L. I.; Scheinpflug, K.; Brötz-Oesterhelt, H.; Hertweck, C.; Shaw, J. T.; Petković, H.; Brunel, J. M.; Keiler, K. C.; Metzler-Nolte, N.; Bandow, J. E. Comparison of proteomic responses as global approach to antibiotic mechanism of action elucidation. Antimicrob. Agents Chemother. 2020, 65, e01373-20  DOI: 10.1128/AAC.01373-20
      There is no corresponding record for this reference.
    53. 53
      Rice, A. M.; Long, Y.; King, S. B. Nitroaromatic antibiotics as nitrogen oxide sources. Biomolecules 2021, 11, 267,  DOI: 10.3390/biom11020267
      53
      Nitroaromatic antibiotics as nitrogen oxide sources
      Rice, Allison M.; Long, Yueming; King, S. Bruce
      Biomolecules (2021), 11 (2), 267CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)
      A review. Nitroarom. antibiotics show activity against anaerobic bacteria and parasites, finding use in the treatment of Heliobacter pylori infections, tuberculosis, trichomoniasis, human African trypanosomiasis, Chagas disease and leishmaniasis. Despite this activity and a clear need for the development of new treatments for these conditions, the assocd. toxicity and lack of clear mechanisms of action have limited their therapeutic development. Nitroarom. antibiotics require reductive bioactivation for activity and this reductive metab. can convert the nitro group to nitric oxide (NO) or a related reactive nitrogen species (RNS). As nitric oxide plays important roles in the defensive immune response to bacterial infection through both signaling and redox-mediated pathways, defining controlled NO generation pathways from these antibiotics would allow the design of new therapeutics. This review focuses on the release of nitrogen oxide species from various nitroarom. antibiotics to portend the increased ability for these compds. to pos. impact infectious disease treatment.
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    54. 54
      Martin, B.; García, P.; Castanié, M. P.; Claverys, J. P. The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction. Mol. Microbiol. 1995, 15, 367– 379,  DOI: 10.1111/j.1365-2958.1995.tb02250.x
      54
      The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction
      Martin, Bernard; Garcia, Pedro; Castanie, Marie-Pierre; Claverys, Jean-Pierre
      Molecular Microbiology (1995), 15 (2), 367-79CODEN: MOMIEE; ISSN:0950-382X. (Blackwell)
      The recently identified recA gene of the naturally transformable bacterium Streptococcus pneumoniae was further characterized by constructing a recA null mutation and by investigating its regulation. The recA mutation confers both DNA repair (as judged from sensitivity to UV and Me methane sulfonate) and recombination deficiencies. Plasmid transformation into the recA mutant was also drastically reduced. Western blotting established that recA gene expression is increased several-fold at the onset of competence for genetic transformation. Increased expression was assocd. with the appearance of a recA-specific transcript, ∼5.7 kb long. This transcript indicated that recA is part of a competence-inducible (cin) operon. The major (∼4.3 kb) transcript detected from noncompetent cells did not include cinA, the first gene in the operon, suggesting that this gene could be specifically required at some stage in the transformation process. Detection of small amts. of the 5.7-kb polycistronic mRNA in cells treated with mitomycin C suggested that the operon could also be damage inducible. In addn., mitomycin C treatment of a recA lysogenic strain did not lead to prophage induction and cell lysis. This is unlike the situation of a recA+ lysogen. Together, these results demonstrate that RecA controls lysogenic induction and suggest the existence of a SOS repair system in S. pneumoniae.
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    55. 55
      Lewis, P. J.; Marston, A. L. GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene 1999, 227, 101– 110,  DOI: 10.1016/S0378-1119(98)00580-0
      55
      GFP vectors for controlled expression and dual labeling of protein fusions in Bacillus subtilis
      Lewis, Peter J.; Marston, Adele L.
      Gene (1999), 227 (1), 101-109CODEN: GENED6; ISSN:0378-1119. (Elsevier Science B.V.)
      We report the development of a series of plasmid vectors for the construction of fusions to mutants of the intrinsically fluorescent green fluorescent protein, GFPmut1. Both N- and C-terminal fusions can be produced, and their expression can be finely controlled from the inducible Pxyl promoter following double crossover integration into the amyE locus of the Bacillus subtilis chromosome. Other vectors designed for single crossover insertion into the chromosome allow downstream genes to be placed under inducible control. We also show that fusions to GFPmutI and GFPuv can be co-localized within the cell by virtue of their different excitation spectra.
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    56. 56
      Hauser, P. M.; Karamata, D. A rapid and simple method for Bacillus subtilis transformation on solid media. Microbiology 1994, 140, 1613– 1617,  DOI: 10.1099/13500872-140-7-1613
      56
      A rapid and simple method for Bacillus subtilis transformation on solid media
      Hauser, Philippe M.; Karamata, Dimitri
      Microbiology (Reading, United Kingdom) (1994), 140 (7), 1613-17CODEN: MROBEO; ISSN:1350-0872.
      Cells of Bacillus subtilis strains 168 and W23 deprived of an amino acid or a base on a given solid medium were found to develop competence. We describe a rapid and simple method of genetic transformation of this organism consisting in spreading a sample contg. 1 μg DNA and 107 exponentially growing cells of an auxotrophic mutant onto plates devoid of the required amino acid or base. After overnight incubation, about 100-200 prototrophic transformants per plate were obtained, i.e. a frequency of about 10-5, as compared to 10-4 routinely obtained by the method of transformation in liq. medium with frozen competent cells. Plasmids and other chromosomal or plasmid-borne markers, which cannot be directly selected for, were transferred by congression. The dependence of the transformation efficiency on cell d., medium richness, incubation time and the nature of transforming DNA was investigated. We conclude that the development of competence accompanies amino acid or base starvation of cells under appropriate physiol. conditions.
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    57. 57
      Nicolas, P.; Mäder, U.; Dervyn, E.; Rochat, T.; Leduc, A.; Pigeonneau, N.; Bidnenko, E.; Marchadier, E.; Hoebeke, M.; Aymerich, S.; Becher, D.; Bisicchia, P.; Botella, E.; Delumeau, O.; Doherty, G.; Denham, E. L.; Fogg, M. J.; Fromion, V.; Goelzer, A.; Hansen, A.; Härtig, E.; Harwood, C. R.; Homuth, G.; Jarmer, H.; Jules, M.; Klipp, E.; Le Chat, L.; Lecointe, F.; Lewis, P.; Liebermeister, W.; March, A.; Mars, R. A. T.; Nannapaneni, P.; Noone, D.; Pohl, S.; Rinn, B.; Rügheimer, F.; Sappa, P. K.; Samson, F.; Schaffer, M.; Schwikowski, B.; Steil, L.; Stülke, J.; Wiegert, T.; Devine, K. M.; Wilkinson, A. J.; van Dijl, J. M.; Hecker, M.; Völker, U.; Bessières, P.; Noirot, P. Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science 2012, 335, 1103– 1106,  DOI: 10.1126/science.1206848
      57
      Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis
      Nicolas, Pierre; Maeder, Ulrike; Dervyn, Etienne; Rochat, Tatiana; Leduc, Aurelie; Pigeonneau, Nathalie; Bidnenko, Elena; Marchadier, Elodie; Hoebeke, Mark; Aymerich, Stephane; Becher, Doerte; Bisicchia, Paola; Botella, Eric; Delumeau, Olivier; Doherty, Geoff; Denham, Emma L.; Fogg, Mark J.; Fromion, Vincent; Goelzer, Anne; Hansen, Annette; Haertig, Elisabeth; Harwood, Colin R.; Homuth, Georg; Jarmer, Hanne; Jules, Matthieu; Klipp, Edda; Le Chat, Ludovic; Lecointe, Francois; Lewis, Peter; Liebermeister, Wolfram; March, Anika; Mars, Ruben A. T.; Nannapaneni, Priyanka; Noone, David; Pohl, Susanne; Rinn, Bernd; Ruegheimer, Frank; Sappa, Praveen K.; Samson, Franck; Schaffer, Marc; Schwikowski, Benno; Steil, Leif; Stuelke, Joerg; Wiegert, Thomas; Devine, Kevin M.; Wilkinson, Anthony J.; Maarten van Dijl, Jan; Hecker, Michael; Voelker, Uwe; Bessieres, Philippe; Noirot, Philippe
      Science (Washington, DC, United States) (2012), 335 (6072), 1103-1106CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the obsd. variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.
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    58. 58
      Morimoto, T.; Ara, K.; Ozaki, K.; Ogasawara, N. A new simple method to introduce marker-free deletions in the Bacillus subtilis genome. Genes Genet Syst 2009, 84, 315– 318,  DOI: 10.1266/ggs.84.315
      58
      A new simple method to introduce marker-free deletions in the Bacillus subtilis genome
      Morimoto, Takuya; Ara, Katsutoshi; Ozaki, Katsuya; Ogasawara, Naotake
      Genes & Genetic Systems (2009), 84 (4), 315-318CODEN: GGSYF5; ISSN:1341-7568. (Genetics Society of Japan)
      A genetic tool to introduce marker-free deletions is essential for multiple manipulations of genomes. We report a simple and efficient method to create marker-free deletion mutants of Bacillus subtilis through transformation with recombinant PCR products, using the Escherichia coli mazF gene encoding an endoribonuclease that cleaves free mRNAs as a counter-selection tool. Our method will be applicable to any bacterium in which introduction of the mazF cassette into the genome by double crossover homologous recombination is possible.
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    59. 59
      Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J. Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Fiji: An open-source platform for biological-image analysis. Nat. Methods 2012, 9, 676– 682,  DOI: 10.1038/nmeth.2019
      59
      Fiji: an open-source platform for biological-image analysis
      Schindelin, Johannes; Arganda-Carreras, Ignacio; Frise, Erwin; Kaynig, Verena; Longair, Mark; Pietzsch, Tobias; Preibisch, Stephan; Rueden, Curtis; Saalfeld, Stephan; Schmid, Benjamin; Tinevez, Jean-Yves; White, Daniel James; Hartenstein, Volker; Eliceiri, Kevin; Tomancak, Pavel; Cardona, Albert
      Nature Methods (2012), 9 (7_part1), 676-682CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Fiji is a distribution of the popular open-source software ImageJ focused on biol.-image anal. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biol. research communities.
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    60. 60
      Syvertsson, S.; Vischer, N. O. E.; Gao, Y.; Hamoen, L. W. When phase contrast fails: ChainTracer and NucTracer, two ImageJ methods for semi-automated single cell analysis using membrane or DNA staining. PLoS One 2016, 11, e0151267,  DOI: 10.1371/journal.pone.0151267
      60
      When phase contrast fails: chaintracer and nuctracer, two imageJ methods for semi- automated single cell analysis using membrane or DNA staining
      Syvertsson, Simon; Vischer, Norbert O. E.; Gao, Yongqiang; Hamoen, Leendert W.
      PLoS One (2016), 11 (3), e0151267/1-e0151267/11CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Within bacterial populations, genetically identical cells often behave differently. Single-cell measurement methods are required to observe this heterogeneity. Flow cytometry and fluorescence light microscopy are the primary methods to do this. However, flow cytometry requires reasonably strong fluorescence signals and is impractical when bacteria grow in cell chains. Therefore fluorescence light microscopy is often used to measure population heterogeneity in bacteria. Automatic microscopy image anal. programs typically use phase contrast images to identify cells. However, many bacteria divide by forming a crosswall that is not detectable by phase contrast.We have developed 'ChainTracer', a method based on the ImageJ plugin ObjectJ. It can automatically identify individual cells stained by fluorescent membrane dyes, and measure fluorescence intensity, chain length, cell length, and cell diam. As a complementary anal. method we developed 'NucTracer', which uses DAPI stained nucleoids as a proxy for single cells. The latter method is esp. useful when dealing with crowded images. The methods were tested with Bacillus subtilis and Lactococcus lactis cells expressing a GFP-reporter. In conclusion, ChainTracer and Nuc- Tracer are useful single cell measurement methods when bacterial cells are difficult to distinguish with phase contrast.
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    61. 61
      Ducret, A.; Quardokus, E. M.; Brun, Y. V. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat. Microbiol 2016, 1, 16077,  DOI: 10.1038/nmicrobiol.2016.77
      61
      MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis
      Ducret, Adrien; Quardokus, Ellen M.; Brun, Yves V.
      Nature Microbiology (2016), 1 (7), 16077CODEN: NMAICH; ISSN:2058-5276. (Nature Publishing Group)
      Single-cell anal. of bacteria and subcellular protein localization dynamics has shown that bacteria have elaborate life cycles, cytoskeletal protein networks and complex signal transduction pathways driven by localized proteins. The vol. of multidimensional images generated in such expts. and the computation time required to detect, assoc. and track cells and subcellular features pose considerable challenges, esp. for high-throughput expts. There is therefore a need for a versatile, computationally efficient image anal. tool capable of extg. the desired relationships from images in a meaningful and unbiased way. Here, we present MicrobeJ, a plug-in for the open-source platform ImageJ1. MicrobeJ provides a comprehensive framework to process images derived from a wide variety of microscopy expts. with special emphasis on large image sets. It performs the most common intensity and morphol. measurements as well as customized detection of poles, septa, fluorescent foci and organelles, dets. their subcellular localization with subpixel resoln., and tracks them over time. Because a dynamic link is maintained between the images, measurements and all data representations derived from them, the editor and suite of advanced data presentation tools facilitates the image anal. process and provides a robust way to verify the accuracy and veracity of the data.
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    62. 62
      Steenhuis, M.; Ten Hagen-Jongman, C. M.; van Ulsen, P.; Luirink, J. Stress-based high-throughput screening assays to identify inhibitors of cell envelope biogenesis. Antibiot (Basel, Switzerland) 2020, 9, 808,  DOI: 10.3390/antibiotics9110808
      62
      Stress-based high-throughput screening assays to identify inhibitors of cell envelope biogenesis
      Steenhuis, Maurice; ten Hagen-Jongman, Corinne M.; van Ulsen, Peter; Luirink, Joen
      Antibiotics (Basel, Switzerland) (2020), 9 (11), 808CODEN: ABSNC4; ISSN:2079-6382. (MDPI AG)
      The structural integrity of the Gram-neg. cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addn. of antibacterial compds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compd. screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.
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    63. 63
      Shih, Y.-L.; Le, T.; Rothfield, L. Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 7865– 7870,  DOI: 10.1073/pnas.1232225100
      63
      Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles
      Shih, Yu-Ling; Le, Trung; Rothfield, Lawrence
      Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (13), 7865-7870CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The MinCDE proteins of E. coli are required for proper placement of the division septum at mid-cell. The site selection process requires the rapid oscillatory redistribution of the proteins from pole to pole. We report that the 3 Min proteins are organized into extended membrane-assocd. coiled structures that wind around the cell between the 2 poles. The pole-to-pole oscillation of the proteins reflects oscillatory changes in their distribution within the coiled structure. We also report that the E. coli MreB protein, which is required for maintaining the rod shape of the cell, also forms extended coiled structures, which are similar to the MreB structures that have previously been reported in Bacillus subtilis. The MreB and MinCDE coiled arrays do not appear identical. The results suggest that ≥2 functionally distinct cytoskeletal-like elements are present in E. coli and that structures of this type can undergo dynamic changes that play important roles in division site placement and possibly other aspects of the life of the cell.
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    64. 64
      Cruz, R. A. L. A comparative study of native and heterologous enzyme production in Bacillus subtilis. Ph.D. Thesis, Newcastle University, Newcastle upon Tyne, England, 2016.
      There is no corresponding record for this reference.
    65. 65
      Jonkers, T. J. H.; Steenhuis, M.; Schalkwijk, L.; Luirink, J.; Bald, D.; Houtman, C. J.; Kool, J.; Lamoree, M. H.; Hamers, T. Development of a high-throughput bioassay for screening of antibiotics in aquatic environmental samples. Sci. Total Environ. 2020, 729, 139028,  DOI: 10.1016/j.scitotenv.2020.139028
      65
      Development of a high-throughput bioassay for screening of antibiotics in aquatic environmental samples
      Jonkers, Tim J. H.; Steenhuis, Maurice; Schalkwijk, Louis; Luirink, Joen; Bald, Dirk; Houtman, Corine J.; Kool, Jeroen; Lamoree, Marja H.; Hamers, Timo
      Science of the Total Environment (2020), 729 (), 139028CODEN: STENDL; ISSN:0048-9697. (Elsevier B.V.)
      The goal of the present study was to select a Gram-pos. (Gram+) and Gram-neg. (Gram-) strain to measure antimicrobial activity in environmental samples, allowing high-throughput environmental screening. The sensitivity of eight pre-selected bacterial strains were tested to a training set of ten antibiotics, i.e. three Gram+ Bacillus subtilis strains with different read-outs, and five Gram- strains. The latter group consisted of a bioluminescent Allivibrio fischeri strain and four Escherichia coli strains, i.e. a wild type (WT) and three strains with a modified cell envelope to increase their sensitivity. The WT B. subtilis and an E. coli strain newly developed in this study, were most sensitive to the training set. This E. coli strain carries an open variant of an outer membrane protein combined with an inactivated multidrug efflux transport system. The assay conditions of these two strains were optimized and validated by exposure to a validation set of thirteen antibiotics with clin. and environmental relevance. The assay sensitivity ranged from the ng/mL to μg/mL range. The applicability of the assays for toxicol. characterization of aquatic environmental samples was demonstrated for hospital effluent ext. A future application includes effect-directed anal. to identify yet unknown antibiotic contaminants or their transformation products.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.2c00404.

    • Growth experiments, additional ROS assay controls, quantification and overview images of microscopy, peroxide and paraquat controls, protein localization assays, additional protein secretion experiments and controls, microscopy images of membrane potential and cell division assays in E. coli, cell length measurements, transcriptomics data tables, list of strains, plasmids, and primers, supplementary references (PDF)

    • Transcriptomics raw data (XLSX)


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