Dual Action of Eeyarestatin 24 on Sec-Dependent Protein Secretion and Bacterial DNAClick to copy article linkArticle link copied!
- Ann-Britt SchäferAnn-Britt SchäferDivision of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, SwedenMore by Ann-Britt Schäfer
- Maurice SteenhuisMaurice SteenhuisMolecular Microbiology, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The NetherlandsMore by Maurice Steenhuis
- Kin Ki JimKin Ki JimDepartment of Medical Microbiology and Infection Prevention, Amsterdam University Medical Centers - Location Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The NetherlandsAmsterdam Institute for Infection and Immunity, Amsterdam University Medical Centers, 1081 HZ Amsterdam, The NetherlandsMore by Kin Ki Jim
- Jolanda NeefJolanda NeefDepartment of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, The NetherlandsMore by Jolanda Neef
- Sarah O’KeefeSarah O’KeefeSchool of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, United KingdomMore by Sarah O’Keefe
- Roger C. WhiteheadRoger C. WhiteheadSchool of Chemistry, Faculty of Science and Engineering, University of Manchester, Manchester M13 9PL, United KingdomMore by Roger C. Whitehead
- Eileithyia SwantonEileithyia SwantonSchool of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, United KingdomMore by Eileithyia Swanton
- Biwen WangBiwen WangBacterial Cell Biology and Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH Amsterdam, The NetherlandsMore by Biwen Wang
- Sven HalbedelSven HalbedelFG11 Division of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, 38855 Wernigerode, GermanyInstitute for Medical Microbiology and Hospital Hygiene, Otto von Guericke University Magdeburg, 39120 Magdeburg, GermanyMore by Sven Halbedel
- Stephen HighStephen HighSchool of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, United KingdomMore by Stephen High
- Jan Maarten van DijlJan Maarten van DijlDepartment of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, The NetherlandsMore by Jan Maarten van Dijl
- Joen Luirink*Joen Luirink*Email: [email protected]Molecular Microbiology, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The NetherlandsMore by Joen Luirink
- Michaela Wenzel*Michaela Wenzel*Email: [email protected]Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, SwedenMore by Michaela Wenzel
Abstract
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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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
*Disclaimer
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
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Results
Transcriptomic Stress Response Profiling
Antibiotic Concentrations for Phenotypic Analyses
Oxidative Stress
DNA Damage
reporter for | protein | localization pattern | ||||
---|---|---|---|---|---|---|
untreated | 0.53 μM Grab | 15 μM NFTc | 2 μM ES24 | 4 μM ES24 | ||
DNA damage | RecA | cytosolic: dispersed | cytosolic: dispersed | nucleoid-associated focib | nucleoid-associated focib | nucleoid-associated focib |
replication | DnaN | cytosolic: nucleoid-associated | cytosolic: dispersedb | cytosolic: dispersedc | cytosolic: nucleoid-associated | cytosolic: nucleoid-associated |
transcription | RpoC | cytosolic: nucleoid-associated | cytosolic: dispersedb | cytosolic: dispersedb | cytosolic: nucleoid-associated | cytosolic: nucleoid-associated |
translation | RpsB | cytosolic: excluded from nucleoid | cytosolic: dispersedb | cytosolic: dispersedc | cytosolic: excluded from nucleoid | cytosolic: excluded from nucleoid |
membrane potential, cell division | DivIVA | membrane-associated: septal/polar | cytosolic: dispersedb | cytosolic: dispersed, membrane: clustersb | membrane-associated: septal/polar | membrane-associated: septal/polar |
membrane potential, cell division | MinD | membrane-associated: septal/polar | cytosolic: dispersedb | cytosolic: dispersed, membrane: clustersc | membrane-associated: septal/polar | membrane-associated: septal/polar |
ATP synthesis | AtpA | membrane-associated: foci | cytosolic: dispersedb | cytosolic: dispersed, membrane: clustersc | membrane-associated: foci | membrane-associated: foci |
protein secretion | SecA | membrane-associated: foci | cytosolic: dispersedb | cytosolic: dispersed, membrane clustersc | membrane: clustersc | membrane: clustersc |
Fusions showing an effect are in bold.
Immediate effects.
Effects visible after 30–120 min.
Membrane Stress
Inhibition of Protein Secretion
Mechanistic Differences in E. coli
Efficacy in a Zebrafish Embryo Infection Model
Discussion
Differences in Effects on Gram-Negative and Gram-Positive bacteria
Differences between ES24 and NFT
Conclusion
Methods
Antimicrobial Compounds
Strains and Growth Conditions
Strain Construction
Minimal Inhibitory Concentration and Growth Experiments
Transcriptomics
Fluorescence Light Microscopy
Oxyburst Green Assay
ROS Scavenger Assay
Bacterial Cytological Profiling
DiSC(3)5 Spectroscopy
DiSC(3)5 Microscopy
MinD Oscillation Microscopy
Laurdan Spectroscopy
Protein Localization
AmyM Secretion
LipA Secretion
Resazurin Assay
Zebrafish Embryo Experiments
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)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
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
This article references 65 other publications.
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- 3Butler, 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.2016.72Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s7ks1amtA%253D%253D&md5=4baaeaed43677d32f5c68170c732a72bAntibiotics in the clinical pipeline at the end of 2015Butler Mark S; Blaskovich Mark At; Cooper Matthew AThe 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.
- 4Theuretzbacher, U.; Outterson, K.; Engel, A.; Karlén, A. The global preclinical antibacterial pipeline. Nat. Rev. Microbiol 2020, 18, 275– 285, DOI: 10.1038/s41579-019-0288-0Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MfgvFyksA%253D%253D&md5=23ae61239f8792a0fa6ed5b48ae11dcfThe global preclinical antibacterial pipelineTheuretzbacher Ursula; Outterson Kevin; Outterson Kevin; Engel Aleks; Karlen AndersNature 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.
- 5Fiebiger, 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.e03-07-0506Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXivFalsrs%253D&md5=c6952152891fc81f5b18cd98030c32e6Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatinFiebiger, Edda; Hirsch, Christian; Vyas, Jatin M.; Gordon, Eva; Ploegh, Hidde L.; Tortorella, DomenicoMolecular 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.
- 6Sannino, S.; Brodsky, J. L. Targeting protein quality control pathways in breast cancer. BMC Biol. 2017, 15, 109, DOI: 10.1186/s12915-017-0449-4Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2isbnK&md5=44a741298b27e72f86d1dc537fb7faf6Targeting protein quality control pathways in breast cancerSannino, 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.
- 7Aletrari, 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.0022713Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVKgtL7E&md5=4c81e54bee25c38f435995b57890967fEeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathwaysAletrari, 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.
- 8McKibbin, 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/BJ20111220Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivV2iu74%253D&md5=ddc55c09736e307c81963bbc027a91f6Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein responseMcKibbin, 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, EileithyiaBiochemical 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.
- 9Wang, 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.0015479Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3M%252Fhs1KmtA%253D%253D&md5=617613b70b215171b10227ce6458f65fThe ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory groupWang Qiuyan; Shinkre Bidhan A; Lee Jin-gu; Weniger Marc A; Liu Yanfen; Chen Weiping; Wiestner Adrian; Trenkle William C; Ye YihongPloS 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.
- 10Wang, 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.M708347200Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjt1emurs%253D&md5=07da868005f68246bc48501c2aae0c6dInhibition of p97-dependent Protein Degradation by Eeyarestatin IWang, Qiuyan; Li, Lianyun; Ye, YihongJournal 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.
- 11Gamayun, 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.2019.01.010Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjs1SitrY%253D&md5=671c645abe9b552b4108e69578364176Eeyarestatin Compounds Selectively Enhance Sec61-Mediated Ca2+ Leakage from the Endoplasmic ReticulumGamayun, 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, AdolfoCell 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.
- 12McKenna, 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.182352Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVCksL3M&md5=3d17571c5f62703f1e37a78ff57218efMechanistic insights into the inhibition of Sec61-dependent co- and post-translational translocation by mycolactoneMcKenna, Michael; Simmonds, Rachel E.; High, StephenJournal 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.
- 13Cross, 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.054494Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmtVKrsg%253D%253D&md5=9c681f79f5af809d7c48a1ce0c749e22Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulumCross, 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, EileithyiaJournal 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.
- 14Itskanov, 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 ScholarThere is no corresponding record for this reference.
- 15Denks, 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: 10.3109/09687688.2014.907455Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmsl2jsb4%253D&md5=8d3be9ef509ca27671ce4a5b14aa871bThe Sec translocon mediated protein transport in prokaryotes and eukaryotesDenks, Kaert; Vogt, Andreas; Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-GeorgMolecular 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.
- 16Steenhuis, 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.14589Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslelurfF&md5=c41d960ac1d5a9c13323cc0233b4c713Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogensSteenhuis, Maurice; Koningstein, Gregory M.; Oswald, Julia; Pick, Tillman; O'Keefe, Sarah; Koch, Hans-Georg; Cavalie, Adolfo; Whitehead, Roger C.; Swanton, Eileithyia; High, Stephen; Luirink, JoenMolecular 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.
- 17du 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.2010.08.016Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvFentLk%253D&md5=25625dba0ed9cfd169df9af572bd5949The Sec translocasedu 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.
- 18Yuan, 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-xGoogle Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXis1ejuw%253D%253D&md5=006f598125d0851f4b30c6a198a9d960Protein transport across and into cell membranes in bacteria and archaeaYuan, 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.
- 19Neef, 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/EBC20200171Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVOjtb7N&md5=75e6d01af4cc62f4b3b9ceecacc6baf2Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potentialNeef, Jolanda; van Dijl, Jan Maarten; Buist, GirbeEssays 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.
- 20Gardiner, 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.2019.002Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cfjs1Cgtg%253D%253D&md5=98e7fe77525c4badb651f0576a5ab5f9Nitrofurantoin and fosfomycin for resistant urinary tract infections: old drugs for emerging problemsGardiner 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 YAustralian 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:
- 21Wenzel, 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/s42003-021-01809-8Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtV2hsb3M&md5=0b7319a9dd8a7f3d917b8399ae5dc9d5A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracyclineWenzel, 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.
- 22Tu, Y.; McCalla, D. R. Effect of activated nitrofurans on DNA. Biochim. Biophys. Acta 1975, 402, 142– 149, DOI: 10.1016/0005-2787(75)90032-5Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2MXlslanu7w%253D&md5=e69f1e0da349a0204a9dae12a82f89f6Effect of activated nitrofurans on DNATu, 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.
- 23Whiteway, 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.180.21.5529-5539.1998Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXnt1WqtLo%253D&md5=bae1ba43c6a9ec3f320799c06c00c994Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coliWhiteway, 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.
- 24Valle, 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/s00253-011-3787-0Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjvFKku7k%253D&md5=66189cb7ef6ecb75e15703e3a9823a24Study 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 propertiesValle, Antonio; Borgne, Sylvie; Bolivar, Jorge; Cabrera, Gema; Cantero, DomingoApplied 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.).
- 25Pedreira, 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/gkab943Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1ChsbY%253D&md5=fe91d9a47d44a20acdb75099e95cae76The current state of SubtiWiki, the database for the model organism Bacillus subtilisPedreira, Tiago; Elfmann, Christoph; Stuelke, JoergNucleic 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.
- 26Wenzel, 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.00802-18Google ScholarThere is no corresponding record for this reference.
- 27Saeloh, 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.1006876Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsleltrnO&md5=189cefeee0d335a11855ebc5f44f17d8The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluiditySaeloh, 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.
- 28Müller, 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.1611173113Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslalsLrL&md5=900e3e7b23f443b37e0a0a1ba6ec590fDaptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomainsMueller, 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.
- 29Garcia 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.1995.1076Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2M3jt1OktA%253D%253D&md5=33276d7c0ce660a7861782a5fd6755e5Nitrofurantoin-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 RToxicology 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.
- 30Mols, M.; Abee, T. Primary and secondary oxidative stress in Bacillus. Environ. Microbiol 2011, 13, 1387– 1394, DOI: 10.1111/j.1462-2920.2011.02433.xGoogle Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptVyiur0%253D&md5=823939ad7e0e57c142e775d1380ed964Primary and secondary oxidative stress in BacillusMols, Maarten; Abee, TjakkoEnvironmental 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.
- 31Ameziane, El.; Hassani, R.; Dupuy, C. Detection of intracellular reactive oxygen species (CM-H2DCFDA). Bio-protocol 2013, 3, e313 DOI: 10.21769/BioProtoc.313Google ScholarThere is no corresponding record for this reference.
- 32Taiwo, F. A. Mechanism of tiron as scavenger of superoxide ions and free electrons. Spectroscopy 2008, 22, 491– 498, DOI: 10.1155/2008/953692Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlOmtr7J&md5=0aefadc1d21feac4af48b436527579c3Mechanism of tiron as scavenger of superoxide ions and free electronsTaiwo, 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.
- 33Chueca, 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.0094072Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1ejs73I&md5=1fd7d01e100d0cbe960771d2c1224fcbDifferential mechanism of Escherichia coli inactivation by (+)-limonene as a function of cell physiological state and drug concentrationChueca, Beatriz; Pagan, Rafael; Garcia-Gonzalo, DiegoPLoS 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.
- 34Lenhart, 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.01494-14Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFens7nI&md5=0fbf8aeadd48115b22f977e4f13e2d08RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stressLenhart, 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.
- 35Wenzel, 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.01380-12Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsF2iurjI&md5=e6a8a0185973060a7c5cda069fe8db9fProteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membraneWenzel, 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.
- 36Scheinpflug, 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/srep44332Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1czksFSluw%253D%253D&md5=72ccc162c8def53e16ae72f4c082befcAntimicrobial peptide cWFW kills by combining lipid phase separation with autolysisScheinpflug Kathi; Krylova Oxana; Dathe Margitta; Wenzel Michaela; Bandow Julia E; Strahl HenrikScientific 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.
- 37Omardien, 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.2018.06.004Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFKlt7fF&md5=ffbcd8c558e53bac162adfd63092eaf2Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayerOmardien, Soraya; Drijfhout, Jan W.; Vaz, Frederic M.; Wenzel, Michaela; Hamoen, Leendert W.; Zaat, Sebastian A. J.; Brul, StanleyBiochimica 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.
- 38Parasassi, T.; Gratton, E. Membrane lipid domains and dynamics as detected by Laurdan fluorescence. J. Fluoresc 1995, 5, 59– 69, DOI: 10.1007/BF00718783Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXlsFyitb4%253D&md5=114aea7168301cb5cb2da6bc2b36005cMembrane lipid domains and dynamics as detected by Laurdan fluorescenceParasassi, Tiziana; Gratton, EnricoJournal 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.
- 39Wenzel, 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.3063Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1GrurnJ&md5=7af68bde241d07915045565f399d7c56Assessing membrane fluidity and visualizing fluid membrane domains in bacteria using fluorescent membrane dyesWenzel, 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.
- 40Strahl, 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.1005485107Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXovFyitLk%253D&md5=ec185ef57c2bf7d25a16692ba446186bMembrane potential is important for bacterial cell divisionStrahl, 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.
- 41Omardien, 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.2018.02277Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3czptFylsg%253D%253D&md5=1f0b6a0b99fb19d2bb2668ce9d47b9a7Cationic Amphipathic Antimicrobial Peptides Perturb the Inner Membrane of Germinated Spores Thus Inhibiting Their OutgrowthOmardien Soraya; Brul Stanley; Drijfhout Jan Wouter; Zaat Sebastian AFrontiers 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.
- 42Skoczinski, 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-zGoogle Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M%252FitF2qtw%253D%253D&md5=466831289f4cd00b03b88e182ceedf2dContribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilisSkoczinski Pia; Volkenborn Kristina; Fulton Alexander; Knapp Andreas; Jaeger Karl-Erich; Skoczinski Pia; Fulton Alexander; Bhadauriya Anuseema; Nutschel Christina; Gohlke Holger; Gohlke Holger; Jaeger Karl-ErichMicrobial 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.
- 43Jones, 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.185.22.6707-6711.2003Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovFKhtbY%253D&md5=724f71a70d1da4a942100c3bc51af6b0Secretion defects that activate the phage-shock response of Escherichia coliJones, Susan E.; Lloyd, Louise J.; Tan, Kum K.; Buck, MartinJournal 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.
- 44te 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.2016.00029Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28bms1yntw%253D%253D&md5=83ccf852d3f2697dcaeb9460bc635362Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive DyesTe Winkel J Derk; Gray Declan A; Seistrup Kenneth H; Strahl Henrik; Hamoen Leendert WFrontiers 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.
- 45Rowland, 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.182.3.613-619.2000Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXltFCrsQ%253D%253D&md5=b9ed6977a49a671cfa9e4640c05a3addMembrane redistribution of the Escherichia coli MinD protein induced by MinERowland, 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.
- 46Munch, 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.M113.537449Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2crjsF2jtw%253D%253D&md5=db90837d4999c5855303639f2229e499The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functionsMunch Daniela; Sahl Hans-Georg; Muller Anna; Schneider Tanja; Kohl Bastian; Wenzel Michaela; Bandow Julia Elisabeth; Maffioli Sonia; Sosio Margherita; Donadio Stefano; Wimmer ReinhardThe 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.
- 47Errington, J.; Daniel, R. A.; Scheffers, D.-J. Cytokinesis in bacteria. Microbiol Mol. Biol. Rev. 2003, 67, 52– 65, DOI: 10.1128/MMBR.67.1.52-65.2003Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXis1yjsLo%253D&md5=4b1f6d80d698bd33d16e00b97b6f33abCytokinesis in bacteriaErrington, Jeffery; Daniel, Richard A.; Scheffers, Dirk-JanMicrobiology 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.
- 48Omardien, 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.2018.06.005Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFCqtLzM&md5=ac9a05083a9086b703662a77c4077982Synthetic antimicrobial peptides delocalize membrane bound proteins thereby inducing a cell envelope stress responseOmardien, 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.
- 49Jim, 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-yGoogle Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVaisbbI&md5=c2053b62d4bcc5c9ab1c7267239463e4Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis modelJim, 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.
- 50Saeloh, 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.2962Google ScholarThere is no corresponding record for this reference.
- 51Stepanek, 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.2016.03.004Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktVehsbc%253D&md5=f57282eb31bb35c4a7c0cc7c9afa4396Dual mechanism of action of the atypical tetracycline chelocardinStepanek, 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.
- 52Senges, 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.01373-20Google ScholarThere is no corresponding record for this reference.
- 53Rice, A. M.; Long, Y.; King, S. B. Nitroaromatic antibiotics as nitrogen oxide sources. Biomolecules 2021, 11, 267, DOI: 10.3390/biom11020267Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlsFemu70%253D&md5=a7896d2980877d4ea5a7f746d40e8bbdNitroaromatic antibiotics as nitrogen oxide sourcesRice, Allison M.; Long, Yueming; King, S. BruceBiomolecules (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.
- 54Martin, 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.xGoogle Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjs1Wqtrk%253D&md5=8a77614c02f73dd6032d2406b330e429The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic inductionMartin, Bernard; Garcia, Pedro; Castanie, Marie-Pierre; Claverys, Jean-PierreMolecular 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.
- 55Lewis, 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)00580-0Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXht1aitL8%253D&md5=214646660f7aea5499c2db330a5af39eGFP vectors for controlled expression and dual labeling of protein fusions in Bacillus subtilisLewis, 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.
- 56Hauser, P. M.; Karamata, D. A rapid and simple method for Bacillus subtilis transformation on solid media. Microbiology 1994, 140, 1613– 1617, DOI: 10.1099/13500872-140-7-1613Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjvFensw%253D%253D&md5=b6617c66e447e6b32614402ca47008e8A rapid and simple method for Bacillus subtilis transformation on solid mediaHauser, Philippe M.; Karamata, DimitriMicrobiology (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.
- 57Nicolas, 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.1206848Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivVGhtro%253D&md5=847103bb32e621c62f3f54472d0c216eCondition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilisNicolas, 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, PhilippeScience (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.
- 58Morimoto, 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.84.315Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXivVamur8%253D&md5=3a081f38e979074ae600fcfc4044ea12A new simple method to introduce marker-free deletions in the Bacillus subtilis genomeMorimoto, Takuya; Ara, Katsutoshi; Ozaki, Katsuya; Ogasawara, NaotakeGenes & 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.
- 59Schindelin, 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.2019Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVKnurbJ&md5=ad150521a33367d37a800bee853dd9dbFiji: an open-source platform for biological-image analysisSchindelin, 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, AlbertNature 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.
- 60Syvertsson, 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.0151267Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFehtb7L&md5=0eb8d1c2c0c64a0c263c63426775f1eaWhen phase contrast fails: chaintracer and nuctracer, two imageJ methods for semi- automated single cell analysis using membrane or DNA stainingSyvertsson, 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.
- 61Ducret, 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.2016.77Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXkvFyrsrY%253D&md5=863ef625fc4c31448d1cabeddb975544MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysisDucret, 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.
- 62Steenhuis, 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/antibiotics9110808Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXkt1amur8%253D&md5=1767b810f54f1327badf7369162a62baStress-based high-throughput screening assays to identify inhibitors of cell envelope biogenesisSteenhuis, Maurice; ten Hagen-Jongman, Corinne M.; van Ulsen, Peter; Luirink, JoenAntibiotics (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.
- 63Shih, 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.1232225100Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXlt1WqsbY%253D&md5=8824af893612b45c3b1463fc3d48ef3fDivision site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell polesShih, Yu-Ling; Le, Trung; Rothfield, LawrenceProceedings 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.
- 64Cruz, 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 ScholarThere is no corresponding record for this reference.
- 65Jonkers, 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.2020.139028Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptVChsb4%253D&md5=fa7f0719c71aa6782a3e37dc02e1fe5bDevelopment of a high-throughput bioassay for screening of antibiotics in aquatic environmental samplesJonkers, Tim J. H.; Steenhuis, Maurice; Schalkwijk, Louis; Luirink, Joen; Bald, Dirk; Houtman, Corine J.; Kool, Jeroen; Lamoree, Marja H.; Hamers, TimoScience 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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- 1Agnew, 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.
- 2Tacconelli, 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)30753-32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MzkvFOgsA%253D%253D&md5=14021ec57670b29fec6a88226a6a1fddDiscovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosisTacconelli 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 NicolaThe 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.
- 3Butler, 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.2016.723https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s7ks1amtA%253D%253D&md5=4baaeaed43677d32f5c68170c732a72bAntibiotics in the clinical pipeline at the end of 2015Butler Mark S; Blaskovich Mark At; Cooper Matthew AThe 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.
- 4Theuretzbacher, U.; Outterson, K.; Engel, A.; Karlén, A. The global preclinical antibacterial pipeline. Nat. Rev. Microbiol 2020, 18, 275– 285, DOI: 10.1038/s41579-019-0288-04https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MfgvFyksA%253D%253D&md5=23ae61239f8792a0fa6ed5b48ae11dcfThe global preclinical antibacterial pipelineTheuretzbacher Ursula; Outterson Kevin; Outterson Kevin; Engel Aleks; Karlen AndersNature 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.
- 5Fiebiger, 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.e03-07-05065https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXivFalsrs%253D&md5=c6952152891fc81f5b18cd98030c32e6Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatinFiebiger, Edda; Hirsch, Christian; Vyas, Jatin M.; Gordon, Eva; Ploegh, Hidde L.; Tortorella, DomenicoMolecular 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.
- 6Sannino, S.; Brodsky, J. L. Targeting protein quality control pathways in breast cancer. BMC Biol. 2017, 15, 109, DOI: 10.1186/s12915-017-0449-46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2isbnK&md5=44a741298b27e72f86d1dc537fb7faf6Targeting protein quality control pathways in breast cancerSannino, 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.
- 7Aletrari, 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.00227137https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVKgtL7E&md5=4c81e54bee25c38f435995b57890967fEeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathwaysAletrari, 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.
- 8McKibbin, 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/BJ201112208https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivV2iu74%253D&md5=ddc55c09736e307c81963bbc027a91f6Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein responseMcKibbin, 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, EileithyiaBiochemical 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.
- 9Wang, 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.00154799https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3M%252Fhs1KmtA%253D%253D&md5=617613b70b215171b10227ce6458f65fThe ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory groupWang Qiuyan; Shinkre Bidhan A; Lee Jin-gu; Weniger Marc A; Liu Yanfen; Chen Weiping; Wiestner Adrian; Trenkle William C; Ye YihongPloS 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.
- 10Wang, 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.M70834720010https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjt1emurs%253D&md5=07da868005f68246bc48501c2aae0c6dInhibition of p97-dependent Protein Degradation by Eeyarestatin IWang, Qiuyan; Li, Lianyun; Ye, YihongJournal 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.
- 11Gamayun, 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.2019.01.01011https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjs1SitrY%253D&md5=671c645abe9b552b4108e69578364176Eeyarestatin Compounds Selectively Enhance Sec61-Mediated Ca2+ Leakage from the Endoplasmic ReticulumGamayun, 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, AdolfoCell 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.
- 12McKenna, 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.18235212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVCksL3M&md5=3d17571c5f62703f1e37a78ff57218efMechanistic insights into the inhibition of Sec61-dependent co- and post-translational translocation by mycolactoneMcKenna, Michael; Simmonds, Rachel E.; High, StephenJournal 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.
- 13Cross, 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.05449413https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmtVKrsg%253D%253D&md5=9c681f79f5af809d7c48a1ce0c749e22Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulumCross, 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, EileithyiaJournal 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.
- 14Itskanov, 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.
- 15Denks, 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: 10.3109/09687688.2014.90745515https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmsl2jsb4%253D&md5=8d3be9ef509ca27671ce4a5b14aa871bThe Sec translocon mediated protein transport in prokaryotes and eukaryotesDenks, Kaert; Vogt, Andreas; Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-GeorgMolecular 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.
- 16Steenhuis, 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.1458916https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslelurfF&md5=c41d960ac1d5a9c13323cc0233b4c713Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogensSteenhuis, Maurice; Koningstein, Gregory M.; Oswald, Julia; Pick, Tillman; O'Keefe, Sarah; Koch, Hans-Georg; Cavalie, Adolfo; Whitehead, Roger C.; Swanton, Eileithyia; High, Stephen; Luirink, JoenMolecular 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.
- 17du 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.2010.08.01617https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvFentLk%253D&md5=25625dba0ed9cfd169df9af572bd5949The Sec translocasedu 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.
- 18Yuan, 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-x18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXis1ejuw%253D%253D&md5=006f598125d0851f4b30c6a198a9d960Protein transport across and into cell membranes in bacteria and archaeaYuan, 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.
- 19Neef, 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/EBC2020017119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVOjtb7N&md5=75e6d01af4cc62f4b3b9ceecacc6baf2Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potentialNeef, Jolanda; van Dijl, Jan Maarten; Buist, GirbeEssays 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.
- 20Gardiner, 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.2019.00220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cfjs1Cgtg%253D%253D&md5=98e7fe77525c4badb651f0576a5ab5f9Nitrofurantoin and fosfomycin for resistant urinary tract infections: old drugs for emerging problemsGardiner 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 YAustralian 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:
- 21Wenzel, 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/s42003-021-01809-821https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtV2hsb3M&md5=0b7319a9dd8a7f3d917b8399ae5dc9d5A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracyclineWenzel, 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.
- 22Tu, Y.; McCalla, D. R. Effect of activated nitrofurans on DNA. Biochim. Biophys. Acta 1975, 402, 142– 149, DOI: 10.1016/0005-2787(75)90032-522https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2MXlslanu7w%253D&md5=e69f1e0da349a0204a9dae12a82f89f6Effect of activated nitrofurans on DNATu, 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.
- 23Whiteway, 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.180.21.5529-5539.199823https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXnt1WqtLo%253D&md5=bae1ba43c6a9ec3f320799c06c00c994Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coliWhiteway, 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.
- 24Valle, 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/s00253-011-3787-024https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjvFKku7k%253D&md5=66189cb7ef6ecb75e15703e3a9823a24Study 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 propertiesValle, Antonio; Borgne, Sylvie; Bolivar, Jorge; Cabrera, Gema; Cantero, DomingoApplied 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.).
- 25Pedreira, 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/gkab94325https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1ChsbY%253D&md5=fe91d9a47d44a20acdb75099e95cae76The current state of SubtiWiki, the database for the model organism Bacillus subtilisPedreira, Tiago; Elfmann, Christoph; Stuelke, JoergNucleic 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.
- 26Wenzel, 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.00802-18There is no corresponding record for this reference.
- 27Saeloh, 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.100687627https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsleltrnO&md5=189cefeee0d335a11855ebc5f44f17d8The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluiditySaeloh, 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.
- 28Müller, 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.161117311328https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslalsLrL&md5=900e3e7b23f443b37e0a0a1ba6ec590fDaptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomainsMueller, 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.
- 29Garcia 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.1995.107629https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2M3jt1OktA%253D%253D&md5=33276d7c0ce660a7861782a5fd6755e5Nitrofurantoin-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 RToxicology 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.
- 30Mols, M.; Abee, T. Primary and secondary oxidative stress in Bacillus. Environ. Microbiol 2011, 13, 1387– 1394, DOI: 10.1111/j.1462-2920.2011.02433.x30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptVyiur0%253D&md5=823939ad7e0e57c142e775d1380ed964Primary and secondary oxidative stress in BacillusMols, Maarten; Abee, TjakkoEnvironmental 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.
- 31Ameziane, El.; Hassani, R.; Dupuy, C. Detection of intracellular reactive oxygen species (CM-H2DCFDA). Bio-protocol 2013, 3, e313 DOI: 10.21769/BioProtoc.313There is no corresponding record for this reference.
- 32Taiwo, F. A. Mechanism of tiron as scavenger of superoxide ions and free electrons. Spectroscopy 2008, 22, 491– 498, DOI: 10.1155/2008/95369232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlOmtr7J&md5=0aefadc1d21feac4af48b436527579c3Mechanism of tiron as scavenger of superoxide ions and free electronsTaiwo, 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.
- 33Chueca, 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.009407233https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1ejs73I&md5=1fd7d01e100d0cbe960771d2c1224fcbDifferential mechanism of Escherichia coli inactivation by (+)-limonene as a function of cell physiological state and drug concentrationChueca, Beatriz; Pagan, Rafael; Garcia-Gonzalo, DiegoPLoS 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.
- 34Lenhart, 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.01494-1434https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFens7nI&md5=0fbf8aeadd48115b22f977e4f13e2d08RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stressLenhart, 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.
- 35Wenzel, 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.01380-1235https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsF2iurjI&md5=e6a8a0185973060a7c5cda069fe8db9fProteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membraneWenzel, 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.
- 36Scheinpflug, 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/srep4433236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1czksFSluw%253D%253D&md5=72ccc162c8def53e16ae72f4c082befcAntimicrobial peptide cWFW kills by combining lipid phase separation with autolysisScheinpflug Kathi; Krylova Oxana; Dathe Margitta; Wenzel Michaela; Bandow Julia E; Strahl HenrikScientific 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.
- 37Omardien, 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.2018.06.00437https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFKlt7fF&md5=ffbcd8c558e53bac162adfd63092eaf2Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayerOmardien, Soraya; Drijfhout, Jan W.; Vaz, Frederic M.; Wenzel, Michaela; Hamoen, Leendert W.; Zaat, Sebastian A. J.; Brul, StanleyBiochimica 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.
- 38Parasassi, T.; Gratton, E. Membrane lipid domains and dynamics as detected by Laurdan fluorescence. J. Fluoresc 1995, 5, 59– 69, DOI: 10.1007/BF0071878338https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXlsFyitb4%253D&md5=114aea7168301cb5cb2da6bc2b36005cMembrane lipid domains and dynamics as detected by Laurdan fluorescenceParasassi, Tiziana; Gratton, EnricoJournal 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.
- 39Wenzel, 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.306339https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1GrurnJ&md5=7af68bde241d07915045565f399d7c56Assessing membrane fluidity and visualizing fluid membrane domains in bacteria using fluorescent membrane dyesWenzel, 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.
- 40Strahl, 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.100548510740https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXovFyitLk%253D&md5=ec185ef57c2bf7d25a16692ba446186bMembrane potential is important for bacterial cell divisionStrahl, 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.
- 41Omardien, 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.2018.0227741https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3czptFylsg%253D%253D&md5=1f0b6a0b99fb19d2bb2668ce9d47b9a7Cationic Amphipathic Antimicrobial Peptides Perturb the Inner Membrane of Germinated Spores Thus Inhibiting Their OutgrowthOmardien Soraya; Brul Stanley; Drijfhout Jan Wouter; Zaat Sebastian AFrontiers 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.
- 42Skoczinski, 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-z42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M%252FitF2qtw%253D%253D&md5=466831289f4cd00b03b88e182ceedf2dContribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilisSkoczinski Pia; Volkenborn Kristina; Fulton Alexander; Knapp Andreas; Jaeger Karl-Erich; Skoczinski Pia; Fulton Alexander; Bhadauriya Anuseema; Nutschel Christina; Gohlke Holger; Gohlke Holger; Jaeger Karl-ErichMicrobial 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.
- 43Jones, 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.185.22.6707-6711.200343https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovFKhtbY%253D&md5=724f71a70d1da4a942100c3bc51af6b0Secretion defects that activate the phage-shock response of Escherichia coliJones, Susan E.; Lloyd, Louise J.; Tan, Kum K.; Buck, MartinJournal 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.
- 44te 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.2016.0002944https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28bms1yntw%253D%253D&md5=83ccf852d3f2697dcaeb9460bc635362Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive DyesTe Winkel J Derk; Gray Declan A; Seistrup Kenneth H; Strahl Henrik; Hamoen Leendert WFrontiers 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.
- 45Rowland, 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.182.3.613-619.200045https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXltFCrsQ%253D%253D&md5=b9ed6977a49a671cfa9e4640c05a3addMembrane redistribution of the Escherichia coli MinD protein induced by MinERowland, 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.
- 46Munch, 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.M113.53744946https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2crjsF2jtw%253D%253D&md5=db90837d4999c5855303639f2229e499The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functionsMunch Daniela; Sahl Hans-Georg; Muller Anna; Schneider Tanja; Kohl Bastian; Wenzel Michaela; Bandow Julia Elisabeth; Maffioli Sonia; Sosio Margherita; Donadio Stefano; Wimmer ReinhardThe 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.
- 47Errington, J.; Daniel, R. A.; Scheffers, D.-J. Cytokinesis in bacteria. Microbiol Mol. Biol. Rev. 2003, 67, 52– 65, DOI: 10.1128/MMBR.67.1.52-65.200347https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXis1yjsLo%253D&md5=4b1f6d80d698bd33d16e00b97b6f33abCytokinesis in bacteriaErrington, Jeffery; Daniel, Richard A.; Scheffers, Dirk-JanMicrobiology 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.
- 48Omardien, 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.2018.06.00548https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFCqtLzM&md5=ac9a05083a9086b703662a77c4077982Synthetic antimicrobial peptides delocalize membrane bound proteins thereby inducing a cell envelope stress responseOmardien, 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.
- 49Jim, 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-y49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVaisbbI&md5=c2053b62d4bcc5c9ab1c7267239463e4Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis modelJim, 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.
- 50Saeloh, 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.2962There is no corresponding record for this reference.
- 51Stepanek, 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.2016.03.00451https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktVehsbc%253D&md5=f57282eb31bb35c4a7c0cc7c9afa4396Dual mechanism of action of the atypical tetracycline chelocardinStepanek, 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.
- 52Senges, 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.01373-20There is no corresponding record for this reference.
- 53Rice, A. M.; Long, Y.; King, S. B. Nitroaromatic antibiotics as nitrogen oxide sources. Biomolecules 2021, 11, 267, DOI: 10.3390/biom1102026753https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlsFemu70%253D&md5=a7896d2980877d4ea5a7f746d40e8bbdNitroaromatic antibiotics as nitrogen oxide sourcesRice, Allison M.; Long, Yueming; King, S. BruceBiomolecules (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.
- 54Martin, 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.x54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjs1Wqtrk%253D&md5=8a77614c02f73dd6032d2406b330e429The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic inductionMartin, Bernard; Garcia, Pedro; Castanie, Marie-Pierre; Claverys, Jean-PierreMolecular 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.
- 55Lewis, 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)00580-055https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXht1aitL8%253D&md5=214646660f7aea5499c2db330a5af39eGFP vectors for controlled expression and dual labeling of protein fusions in Bacillus subtilisLewis, 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.
- 56Hauser, P. M.; Karamata, D. A rapid and simple method for Bacillus subtilis transformation on solid media. Microbiology 1994, 140, 1613– 1617, DOI: 10.1099/13500872-140-7-161356https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjvFensw%253D%253D&md5=b6617c66e447e6b32614402ca47008e8A rapid and simple method for Bacillus subtilis transformation on solid mediaHauser, Philippe M.; Karamata, DimitriMicrobiology (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.
- 57Nicolas, 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.120684857https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivVGhtro%253D&md5=847103bb32e621c62f3f54472d0c216eCondition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilisNicolas, 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, PhilippeScience (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.
- 58Morimoto, 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.84.31558https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXivVamur8%253D&md5=3a081f38e979074ae600fcfc4044ea12A new simple method to introduce marker-free deletions in the Bacillus subtilis genomeMorimoto, Takuya; Ara, Katsutoshi; Ozaki, Katsuya; Ogasawara, NaotakeGenes & 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.
- 59Schindelin, 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.201959https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVKnurbJ&md5=ad150521a33367d37a800bee853dd9dbFiji: an open-source platform for biological-image analysisSchindelin, 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, AlbertNature 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.
- 60Syvertsson, 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.015126760https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFehtb7L&md5=0eb8d1c2c0c64a0c263c63426775f1eaWhen phase contrast fails: chaintracer and nuctracer, two imageJ methods for semi- automated single cell analysis using membrane or DNA stainingSyvertsson, 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.
- 61Ducret, 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.2016.7761https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXkvFyrsrY%253D&md5=863ef625fc4c31448d1cabeddb975544MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysisDucret, 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.
- 62Steenhuis, 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/antibiotics911080862https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXkt1amur8%253D&md5=1767b810f54f1327badf7369162a62baStress-based high-throughput screening assays to identify inhibitors of cell envelope biogenesisSteenhuis, Maurice; ten Hagen-Jongman, Corinne M.; van Ulsen, Peter; Luirink, JoenAntibiotics (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.
- 63Shih, 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.123222510063https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXlt1WqsbY%253D&md5=8824af893612b45c3b1463fc3d48ef3fDivision site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell polesShih, Yu-Ling; Le, Trung; Rothfield, LawrenceProceedings 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.
- 64Cruz, 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.
- 65Jonkers, 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.2020.13902865https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptVChsb4%253D&md5=fa7f0719c71aa6782a3e37dc02e1fe5bDevelopment of a high-throughput bioassay for screening of antibiotics in aquatic environmental samplesJonkers, Tim J. H.; Steenhuis, Maurice; Schalkwijk, Louis; Luirink, Joen; Bald, Dirk; Houtman, Corine J.; Kool, Jeroen; Lamoree, Marja H.; Hamers, TimoScience 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.
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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