Aggregation of Vibrio cholerae by Cationic Polymers Enhances Quorum Sensing but Overrides Biofilm Dissipation in Response to Autoinduction
- Nicolas Perez-SotoNicolas Perez-SotoSchool of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, United KingdomMore by Nicolas Perez-Soto
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- Oliver CreeseOliver CreeseInstitute of Microbiology and Infection and School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United KingdomMore by Oliver Creese
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- Francisco Fernandez-Trillo*Francisco Fernandez-Trillo*E-mail: [email protected]Institute of Microbiology and Infection and School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United KingdomMore by Francisco Fernandez-Trillo
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- Anne-Marie Krachler*Anne-Marie Krachler*E-mail: [email protected]University of Texas Health Science Center at Houston, McGovern Medical School, Department of Microbiology and Molecular Genetics, 6431 Fannin Street, Houston, Texas 77030, United StatesMore by Anne-Marie Krachler
Copyright © 2018 American Chemical Society. This publication is licensed under CC-BY.
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Abstract
Vibrio cholerae is a Gram-negative bacterium found in aquatic environments and a human pathogen of global significance. Its transition between host-associated and environmental lifestyles involves the tight regulation of niche-specific phenotypes such as motility, biofilm formation, and virulence. V. cholerae’s transition from the host to environmental dispersal usually involves suppression of virulence and dispersion of biofilm communities. In contrast to this naturally occurring transition, bacterial aggregation by cationic polymers triggers a unique response, which is to suppress virulence gene expression while also triggering biofilm formation by V. cholerae, an artificial combination of traits that is potentially very useful to bind and neutralize the pathogen from contaminated water. Here, we set out to uncover the mechanistic basis of this polymer-triggered bacterial behavior. We found that bacteria–polymer aggregates undergo rapid autoinduction and achieve quorum sensing at bacterial densities far below those required for autoinduction in the absence of polymers. We demonstrate this induction of quorum sensing is due both to a rapid formation of autoinducer gradients and local enhancement of autoinducer concentrations within bacterial clusters as well as the stimulation of CAI-1 and AI-2 production by aggregated bacteria. We further found that polymers cause an induction of the biofilm-specific regulator VpsR and the biofilm structural protein RbmA, bypassing the usual suppression of biofilm during autoinduction. Overall, this study highlights that synthetic materials can be used to cross-wire natural bacterial responses to achieve a combination of phenotypes with potentially useful applications.
Both natural and synthetic cationic macromolecules, such as cationic antimicrobial peptides, cationic polymers, and dendrimers, have been extensively reported as antimicrobial. (1−3) Because of their positive charge, these polymers can efficiently bind the negatively charged envelope of Gram-negative and Gram-positive bacteria. (4−6) At high concentrations and charge densities, these molecules have the potential to interfere with membrane integrity and decrease bacterial viability. (1−3) However, antimicrobial activity is heavily dependent on the length and nature of the polymer and, more importantly, on the nature of the targeted microbe. At low concentrations, cationic polymers are still capable of causing bacterial aggregation by mediating electrostatic interactions but do so without significantly affecting bacterial membrane integrity and growth. (7−12)
We and others have previously reported that bacteria clustered by subinhibitory concentrations of cationic polymers display interesting phenotypes resembling those of biofilm communities. (7−12) For instance, we have recently demonstrated that clustering of the diarrheal pathogen Vibrio cholerae with methacrylamides containing primary or tertiary amines leads to accumulation of biomass and extracellular DNA and represses ToxT-regulated virulence factors including cholera toxin. (12) However, what drives these phenotypic changes in response to polymer exposure remains unclear. Similarly, the marine bacterium Vibrio harveyi shows enhanced bioluminescence in response to polymer-mediated clustering. (7,8,11) The bacterial components necessary to produce luminescence are encoded by the luxCDABE genes and subject to complex regulatory mechanisms. A major regulatory cascade controlling luminescence is quorum sensing. Because the regulators controlling luminescence are functionally conserved between Vibrio species, expression of V. harveyi luxCDABE genes can be used as a tool to probe quorum sensing in heterologous hosts. (13)
In V. cholerae, four parallel quorum sensing pathways operate, (14) each governed by an autoinducer synthase, which produces a small, freely diffusible molecule that is released in the environment and can be sensed by a corresponding sensor/kinase component that controls the activity of the LuxU/LuxO phosphorelay. Of the four pathways, the LuxS/LuxPQ system, which produces and detects the interspecies autoinducer AI-2 (S-TMHF-borate) and the CqsA/CqsS system, which produces and senses the Vibrio specific autoinducer S-3-hydroxytridecan-4-one (CAI-1), are the best characterized. CqsA and LuxS synthesize the autoinducers, and the hybrid sensor/kinases CqsS and LuxQ sense and respond to their cognate autoinducers by dephosphorylating and inactivating the regulator LuxO. When extracellular autoinducer concentrations are low, LuxO is phosphorylated and activates the transcription of the small RNAs Qrr1–4, which in turn inhibits the expression of HapR, a master regulator controlling diverse cellular functions including luminescence, biofilm formation, and virulence. (15) When the extracellular autoinducer concentration increases above a threshold, the sensor/kinases instead act as phosphatases, leading to dephosphorylation and inactivation of LuxO, ultimately allowing the expression of HapR. Consequently, when autoinducer concentration is high, HapR activates luminescence but suppresses virulence and biofilm genes. This regulatory mechanism is thought to mediate the dissipation of host-associated biofilms and enable transmission of V. cholerae from the intestine to the environment. (16) Because in V. harveyi exposure to subinhibitory concentrations of cationic polymers leads to enhanced luminescence, we set out here to study whether V. cholerae would also activate quorum sensing in response to polymer-mediated clustering and how quorum sensing could be related to the observed phenotypic changes, including lowered virulence and enhanced biofilm formation.
Results
ARTICLE SECTIONS
Polymers Enhance Quorum Sensing in V. cholerae
First, we set out to test if clustering by the cationic polymers poly(N-[3-aminopropyl] methacrylamide) (P1, Figure 1A) and poly(N-[3-(dimethylamino)propyl] methacrylamide) (P2, Figure 1B) induced quorum sensing in V. cholerae. As previously shown and detailed here by N-SIM super-resolution microscopy, even low concentrations of polymers induced rapid clustering of bacteria without affecting bacterial viability (Figure 1C). For creating a fast and direct read-out for quorum sensing, the cosmid pBB1, which contains the luxCDABE luminescence genes from V. harveyi, (17) was used to transform V. cholerae serogroup O1 biovar El Tor strain A1552 (serotype Inaba). The transformed bacteria were then grown in LB for 20 h, washed with artificial marine water (AMW), and adjusted to an OD of 0.2 in AMW alone or AMW containing polymers at concentrations ranging from 0.005 to 0.5 mg mL–1. In the absence of polymer, luminescence as a read-out of quorum sensing first decreased due to back-dilution of the culture from a high density overnight culture to OD 0.2 and then gradually increased due to accumulation of autoinducers, peaking at 4.5 h (Figure 1D and E, black traces). Interestingly, the quorum induction kinetics were significantly different in the presence of either P1 (Figure 1D) or P2 (Figure 1E) with initial luminescence being sustained and reaching a higher second peak at ∼2.5 h as opposed to 4.5 h in the absence of polymers. The magnitude of induction was higher in polymer-treated cultures (∼5-fold at peak quorum induction) compared to untreated cultures. Results were similar for the Ogawa serotype strain E7646 in that quorum sensing was initially sustained and then further enhanced (∼4-fold at peak quorum induction) by polymer-mediated bacterial aggregation (Figure 1F, G). In both strains, the magnitude of luminescence induction was independent of the polymer concentration used, suggesting a threshold response.
Figure 1
Figure 1. Polymers enhance quorum sensing in Vibrio cholerae. Chemical structures of P1 (A) and P2 (B). N-SIM super-resolution image of LIVE/DEAD-stained V. cholerae A1552 clustered by P1 (C). V. cholerae El Tor strains A1552 (D, E, H) or E7646 (F, G, I) containing the luminescence reporter pBB1 were adjusted to an OD600 of 0.2 following 16 h of growth and incubated in AMW alone (black) or AMW-containing polymer P1 (D, F) or P2 (E, G) at concentrations of 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL–1. Luminescence was recorded every 30 min for 10 h and plotted as means ± SEM from at least three biological replicates. For the effect of polymers on bacterial viability to be tested, samples were removed after 10 h, serially diluted, and plated on LB (H, I).
For assessing viable bacterial counts at the experimental end point, samples were serially diluted in high-salt buffer to disrupt clusters as previously described (12) and plated. Similar numbers of colony-forming units were recovered from untreated or polymer-treated samples (Figure 1H, I), suggesting that the presence of polymers did not affect bacterial viability or proliferation in agreement with our previous data. (12) Taken together, our data demonstrate that these cationic polymers that cluster V. cholerae modulate the community behavior to give an accelerated and more robust autoinduction.
Bacterial Density Shapes the Kinetics of Quorum Induction in Response to Polymer
Because quorum sensing is usually tightly linked to bacterial density, we set out to explore how initial culture density affects quorum induction in the presence of polymers. V. cholerae carrying pBB1 as a quorum sensing reporter was adjusted to optical densities of 0.005, 0.05, and 0.5 in AMW alone or AMW containing 0.005–0.5 mg mL–1 P1 or P2, and luminescence was monitored (Figure 2). The onset of autoinduction was not significantly modulated by the addition of polymers P1 or P2 to higher density cultures (OD600 of 0.5), but an approximate 6- (P1) to 9-fold (P2) increase in peak luminescence was observed (Figure 2A, D). At lower culture densities, autoinduction of the untreated cultures was less pronounced (0.05) and eventually ceased (OD 0.005) because not enough autoinducer accumulated to reach the threshold concentration. In the presence of polymers, the initial quorum present in the culture was sustained, even in very dilute cultures, and the enhancement in peak luminescence was much more pronounced compared to that of untreated cultures (Figure 2B–F). Interestingly, clustering of very dilute cultures led to two peaks in luminescence with a gap of ∼4 h (Figure 2C, F). Of note, the luminescence was not a result of bacterial growth, which was negligible under the observed conditions (AMW) and over the time frame described, both at high and low initial densities (Figure 2G, H).
Figure 2
Figure 2. Bacterial density shapes the kinetics of quorum induction in response to polymer. Cultures of V. cholerae A1552 containing pBB1 were grown for 16 h and diluted into AMW alone (black) or AMW containing 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL–1 of P1 (A–C) or P2 (D–F). Bacterial densities were adjusted to result in OD600 values of 0.5 (A, D), 0.05 (B, E), and 0.005 (C, F), respectively. Luminescence and OD600 were recorded every 30 min for 10 h, and values are means ± SEM from at least three biological replicates. No significant growth was detected over this time frame at either initial density of 0.5 (G) or 0.005 (H).
For the process of bacterial clustering and luminescence induction to be visualized, V. cholerae A1552 containing pBB1 was incubated in the presence of P1, P2, or AMW alone in glass-bottom plates. Bacteria were imaged every 30 min to simultaneously visualize clustering and luminescence induction. Both P1 and P2 bacterial clusters were observed within minutes and remained stable over the duration of the experiment (Figure 3). Luminescence appears to originate from and be restricted to bacterial clusters. Quantification of luminescence based on integrated pixel intensities showed a polymer-mediated enhancement of autoinduction (Figure 3H) in agreement with spectroscopic data.
Figure 3
Figure 3. Polymers enhance quorum sensing in V. cholerae. V. cholerae A1552 containing pBB1 was grown for 16 h and then adjusted to an OD600 of 0.2 in the presence of P1 (A–C), P2 (D–F), or AMW alone (G). Polymers were adjusted to final concentrations of 0.005 (A, D), 0.05 (B, E), or 0.5 (C, F) mg mL–1 in AMW. Luminescence of samples was imaged every 30 min for 15 h, and representative images for each time point are shown from 0 h (top left) to 15 h (bottom right) of each panel. Luminescence intensities over time were analyzed by quantifying pixel intensities, and means ± SEM from at least three biological replicates are shown for P1 (left) and P2 (right panel) (H).
Polymer-Mediated Luminescence Is Not Because of Nutrient Starvation within Clusters
Although autoinduction controls HapR and luminescence via the regulator LuxO, other environmental cues, including nutrient availability, have been reported to feed into the LuxO signaling cascade and thus have the potential to affect luminescence. Nutrient sensing and the LuxO signaling pathway converge at the cyclic AMP (cAMP) receptor protein (CRP). (18) During limitation of PTS sugars such as glucose, cAMP-CRP is capable of modulating LuxO activation by affecting the expression of autoinducer synthases. (19) Because clustering of bacteria by polymers may limit nutrient access and induce starvation, we tested whether a CRP deletion strain would be capable of activating luminescence in the presence of polymers. The V. cholerae Δcrp strain was transformed with pBB1 to monitor luminescence. However, the culture produced very low levels of luminescence both in the presence and absence of polymers (Figure 4A, B), which is in agreement with previous work on V. fischeri CRP. (20) Although this suggests that cross-talk between CRP and LuxO signaling is a dominant cue for luminescence induction in V. cholerae as well, this made it unfeasible to determine whether the presence of polymers induced a state of carbon starvation, leading to luminescence induction via CRP. Instead, we repeated the experiment using the pBB1 containing V. cholerae wild-type strain and supplementing the AMW with additional glucose. If clustering would limit nutrient diffusion, increasing the nutrient concentration should be able to overcome this and revert the bacteria to a nonluminescent phenotype. However, polymers still enhanced and sustained luminescence in the presence of 1% glucose to a similar extent as in AMW alone (Figure 4C), suggesting that the effect was not due to nutrient limitation in the clusters.
Figure 4
Figure 4. Polymer-mediated luminescence is not due to nutrient starvation within clusters. V. cholerae E7646 Δcrp containing the luminescence reporter pBB1 was grown for 16 h and then diluted to an OD600 of 0.2 in AMW alone (black) or AMW containing P1 (A) or P2 (B) at concentrations of 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL–1. Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown. (C) V. cholerae E7646 wild-type containing pBB1 was grown for 16 h and adjusted to an OD600 of 0.2 in AMW containing 1% glucose alone (orange) or in the presence of 0.5 mg mL–1 of P1 (light blue) or P2 (dark blue). Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown.
Polymer-Mediated Enhancement of Quorum Sensing Is Dominated by CAI-1
In V. cholerae, at least four parallel quorum sensing pathways converge to control the activity of the quorum sensing regulator LuxO and thus quorum-regulated phenotypes including biofilm formation and virulence. (14) We set out to test whether the polymer-mediated effect on quorum sensing was specific for any one pathway. V. cholerae wild-type strain A1552 was mixed with equal numbers of cells of quorum sensing mutants transformed with pBB1 either deficient in the production of AI-2 (DH231) or CAI-1 (WN1103) to give a final OD600 of 0.2. Additionally, the mutants were unable to sense the presence of AI-2 or CAI-1. Cultures producing less AI-2 produced a luminescence profile similar in shape and magnitude to quorum sensing proficient cells in response to polymer P1 addition (Figure 5A). In contrast, cultures producing less CAI-1 still had a comparable response profile upon addition of P1, but the magnitude of the luminescence enhancement was decreased (Figure 5B) compared to that of cultures producing less AI-2 (Figure 5A). In cocultures producing less of both autoinducers (containing strain BH1578), the luminescence response to polymer was abolished (Figure 5C). Similarly, cocultures containing a low-density locked mutant of the downstream quorum regulator LuxO (LuxOD47E) showed no luminescence induction upon addition of polymer P1 (Figure 5D). These data pointed at both autoinducers being involved in the quorum sensing enhancement in response to polymer with CAI-1 being the dominant inducer. Additionally, the luminescence response to the polymer seems to proceed through the canonical LuxO-dependent pathway.
Figure 5
Figure 5. Polymer-mediated enhancement of quorum sensing is mainly driven by CAI-1. V. cholerae A1552 wild-type (dark) and quorum sensing mutants containing pBB1 were grown for 16 h and then diluted into AMW to give equal cell densities and a total OD600 of 0.2. Strains were grown together in AMW alone (black) or AMW containing P1 at 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL–1. Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown. Mutants grown in coculture with the wild-type were (A) DH231 (ΔluxSΔcqsS) producing no AI-2, (B) WN1103 (ΔluxQΔcqsA) producing no CAI-1, (C) BH1578 (ΔluxSΔcqsA) producing no AI-2 or CAI-1, and (D) BH1651 (luxOD47E).
Enhanced Quorum Sensing Is Driven by Enhanced Production of Autoinducers in Response to Polymers
It has been described that, at least under some conditions, quorum sensing may be subject to positive feedback, where quorum induction leads to increased production of one of the autoinducer synthases. (21) Therefore, the enhancement in luminescence in response to polymers could be due to positive feedback as a result of the polymers increasing the local concentration of autoinducers above the threshold. Alternatively, the polymers could have a direct effect on the production of autoinducers. We set out to test this by establishing a reporter assay that allowed us to decouple quorum sensing from the production of autoinducers. For this assay, we used as a luminescence reporter a V. cholerae strain transformed with pBB1 that could sense both CAI-1 and AI-2 but could not produce either molecule (BH1578). This reporter strain was exposed to supernatants from producer strains grown under different conditions to evaluate the effect of the polymer. Initially, we evaluated the assay by growing the reporter in the presence of supernatants harvested from wild-type V. cholerae or strains incapable of producing AI-2, CAI-1, or both autoinducers. Supernatant harvested from the quorum-proficient wild-type strain grown to high cell density triggered the highest level of luminescence in BH1578 (Figure 6A). The luminescence triggered by the AI-2-deficient strain was slightly decreased (Figure 6B), whereas luminescence was significantly decreased in response to supernatant from the CAI-1-deficient strain (Figure 6C) and was abolished in response to the strain deficient in both CAI-1 and AI-2 production (Figure 6D). Hence, the assay was capable of detecting different levels of autoinducers produced by a second strain.
Figure 6
Figure 6. Enhanced quorum sensing is driven by enhanced production of autoinducers in response to polymers. Cultures of V. cholerae were adjusted to an OD of 0.2 and grown for 16 h in LB medium, and the supernatants were harvested, filtered, and incubated with V. cholerae BH1578 containing pBB1. Strains used to harvest supernatants were (A) wild-type A1552, (B) DH231 (ΔluxSΔcqs), (C) WN1103 (ΔluxQΔcqsA), and (D) BH1578 ((ΔluxSΔcqsA). Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown. V. cholerae wild-type was adjusted to an OD600 of 0.2 in AMW alone or AMW containing 0.005–0.5 mg mL–1 P1 (E) or P2 (F), and the supernatants were harvested and filtered 16 h later. For their autoinducer content to be determined, the supernatants were incubated with the reporter strain V. cholerae BH1578 containing pBB1. Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown.
We took this assay forward and harvested supernatants from wild-type cells exposed to AMW alone or AMW containing 0.005–0.5 mg mL–1 polymers P1 or P2, and exposed the reporter strain to filtered supernatants to test if the levels of autoinducers produced by the wild-type strain in the presence of polymers were different. The reporter strain was not clustered under the assay conditions. Although wild-type cells exposed to AMW alone did not produce a detectable amount of autoinducer and thus no significant luminescence reading in the reporter strain, both polymers P1 and P2 enhanced the production of autoinducers by wild-type V. cholerae, leading to an increase in luminescence upon exposure of the reporter to the supernatants (Figure 6E, F). These data demonstrate that polymer exposure leads to enhanced production of autoinducers by the bacteria.
Polymer-Mediated Quorum Induction Overrides the Canonical Biofilm Dissipation Program in V. cholerae
Contrary to many bacteria that use quorum signaling as a means to induce biofilm formation, in V. cholerae autoinduction promotes repression of biofilm production and dissemination via the regulator HapR. (22) However, we had previously observed enhanced accumulation of V. cholerae upon prolonged exposure to cationic polymers, but whether this was accompanied by transcriptional changes at the level of biofilm production was not known. Thus, we grew V. cholerae containing transcriptional fusions to promoters regulating key biofilm components in the presence or absence of P1 and P2 (Figure 7). V. cholerae biofilms contain the structural protein RbmA and require the regulator VpsR, which controls the expression of the vps polysaccharide biosynthesis genes. (23−25) Upon exposure to either P1 or P2, vpsR and rbmA were both significantly induced (Figure 7A, B), suggesting that upon polymer-mediated clustering, quorum sensing does not, as usual, suppress genes involved in biofilm production, but instead their transcription is enhanced. AphA, which is another direct target usually induced by VpsR, is suppressed in the presence of polymers (Figure 7C).
Figure 7
Figure 7. Polymer-mediated quorum induction overrides the canonical biofilm dissipation program in V. cholerae. V. cholerae wild-type A1552 containing pRW50T lacZ reporters for the promoters of rbmA (A) or vpsR (B) were grown for 16 h and then diluted into AMW alone or containing 0.005 or 0.05 mg mL–1 P1 or P2 as indicated to give an OD600 of 0.2. Following 16 h of incubation, clustered bacteria were removed and either processed for β-galactosidase assays or treated with high-salt PBS to disperse the cultures for OD600 measurements. Transcriptional activities were calculated and normalized to untreated cultures. Shown are means ± SEM and individual measurements for three biological replicates. Statistical significance was determined by ANOVA and a Dunnett’s multiple comparison test and is depicted as ****p-values ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, and ns for not significant (p ≥ 0.05). (C) V. cholerae wild-type A1552 containing pRW50T lacZ reporter for the aphA promoter was grown in AMW alone (orange) or containing P1 (light blue) at 0.05 mg mL–1 or P2 (dark blue) at 0.5 mg mL–1 for 18 h. Clustered bacteria were removed at indicated times and either processed for β-galactosidase assays or treated with high-salt PBS to disperse the cultures for OD600 measurements. Transcriptional activities were calculated and normalized to the activities of untreated cultures at 2 h. Shown are means ± SEM and individual measurements for three biological replicates.
Discussion
ARTICLE SECTIONS
Traditionally, work on cationic polymers has been carried out with the development of antimicrobial materials as a main goal. (1−3) However, recent work by our groups and others has demonstrated that such cationic polymers can be titrated against bacteria to achieve a charge balance that allows for the rapid and efficient clustering of bacteria but avoids membrane disruption and bacterial cell death. (7−12)
The use of cationic polymers to induce rapid bacterial clustering in this way has proven as an interesting path to study effects of cell aggregation and crowding on bacterial physiology. Although such behaviors are often studied in batch cultures, by incubating bacterial cultures over a prolonged time, this means aggregation is accompanied by bacterial growth and eventually nutrient limitation, which makes it difficult to establish the primary cause of the observed phenotypes. In contrast, cationic polymers induce cell aggregation rapidly, within minutes, which allows us to study these phenomena independent of cellular proliferation and nutrient limitation.
We and others have previously observed that cationic polymers and dendrimers can, under certain conditions, trigger bioluminescence in the marine bacterium V. harveyi, suggesting they may induce or enhance quorum sensing. (7,8,10,11) In a more recent study where we extended this work to the human pathogen V. cholerae, we observed that polymer-mediated clustering led to enhanced deposition of biomass and extracellular DNA, whereas it interfered with the induction of virulence genes in an infection model. (12) Because virulence and biofilm production are both regulated by quorum sensing but are usually both regulated concurrently, the goal of this study was to test whether cationic polymers would trigger quorum sensing in V. cholerae and how this would affect downstream transcription of biofilm genes.
We used V. cholerae strains heterologously expressing the luxCDABE luminescence genes (on cosmid pBB1) from V. harveyi to be able to use luminescence as a direct readout for autoinduction. Over 16 h, V. cholerae would grow to high cell densities and as a result was strongly luminescent. Upon dilution into artificial marine water, cell density and autoinducer concentration would rapidly decrease, resulting in a decline in luminescence. After several hours, cells would eventually accumulate sufficient autoinducer to reach the quorum threshold and induce luminescence again. This behavior was observed in AMW alone (Figure 1) and is in agreement with commonly observed results from such experiments. (14,21) In contrast, when cells were diluted into media containing polymers, they would undergo extensive clustering almost instantaneously, and luminescence readouts never dropped but instead further increased immediately (Figure 1), suggesting that clustering not only countered the dilution effect but further increased autoinducer concentration within the clusters. Interestingly, this behavior was observed over a broad space of cell densities (at least 2 orders of magnitude), including in dilute cultures that did not by themselves experience autoinduction (Figure 2C, F), suggesting that during clustering polymers create pockets containing strongly increased concentrations of autoinducers around bacterial aggregates.
We further demonstrated that both CAI-1- and AI-2-dependent quorum sensing cascades are activated in response to polymers and that clustering leads to an enhanced production of both autoinducers (Figures 5 and 6). The effect of the Vibrio-specific autoinducer CAI-1 dominated the clustering-driven luminescence phenotype (Figure 5) in line with previous results obtained for batch cultures of V. cholerae in rich medium. (21)
Some studies have hypothesized that luminescence could be a result of limited diffusion of nutrients in the polymer-mediated bacterial aggregates. (10) Catabolite repression of luminescence has been reported for V. fischeri, where cAMP-CRP stimulates luxCDABE expression. (26) However, this effect is alleviated by high concentrations of autoinducer. (20) In our hands, CRP was essential for luminescence, both triggered by high cell density in the absence of polymers, in line with previous findings for an E. coli Δcrp mutant, (20) as well as in response to polymer-induced clustering. Additionally, supplementation of the media with excess glucose did not quench luminescence even in the absence of autoinduction. This suggests that nutrient limitation within the clusters is not a major cue for luminescence induction but further underpins that cross-talk between nutrient sensing and quorum sensing pathways exists.
Finally, we followed up on our earlier observation that exposure to cationic polymers causes deposition of V. cholerae on inorganic surfaces and release of extracellular DNA, both hallmarks of biofilm formation. (12) Here, we showed that this phenotype is the result of transcriptional activation of genes involved in biofilm production in response to polymer exposure. Biofilm induction may explain the enhanced resistance toward antimicrobials of bacteria that have been exposed to cationic polymers, as previously described by others. (10) The expression of the biofilm regulator VpsR and the biofilm structural protein RbmA were both induced upon exposure to the polymers (Figure 7). This upregulation is in contrast to the canonical biofilm regulation where biofilm genes are repressed during autoinduction. VpsR is a master regulator of biofilm formation and a two component system response regulator. Although no cognate histidine kinase has been identified, VpsR is epistatic to the intracellular hybrid sensor histidine kinase VpsS. (27) Induction of vpsR likely leads to the downstream induction of rbmA we observed here because rbmA is a direct target of VpsR regulation. (28) However, vpsR induction in the presence of polymers seems to happen despite autoinduction, which should normally lead to suppression of vpsR. What is also different from a regular biofilm response is that VpsR in the presence of polymer fails to upregulate one of its other direct targets, aphA. We showed that, in contrast to this canonical response, aphA is strongly suppressed by the presence of polymers (Figure 7C). When Shikuma et al. identified VpsS as a regulator of VpsR, they established the existence of a pathway that proceeds from VpsS through the quorum regulators LuxU and LuxO and results in the VpsR-dependent activation of biofilm production independent of HapR. (27) It may be that, in the presence of polymers, this pathway is active and dominates the effects of the CAI-1 and AI-2 pathways on biofilm. Unfortunately, the cognate signal activating VpsS is as yet unidentified.
Conclusions
ARTICLE SECTIONS
We showed here that clustering of V. cholerae in response to cationic polymers leads to autoinduction due to a rapid increase of local autoinducer concentration in the vicinity of aggregated bacteria. Moreover, we demonstrate that stimulation of further autoinducer synthesis is also observed and involves at least two of the four known quorum sensing systems, CAI-1 and AI-2. We speculate that the third quorum sensing pathway, which proceeds through the intracellular hybrid sensor kinase VpsS, (14,27) is also activated and leads to the production of biofilm in response to polymer-driven aggregation. Our previous work together with the data presented here rules out membrane disruption and nutrient limitation within clusters, respectively, as cues leading to the phenotypes observed here. Our future work will aim to further dissect the pathway(s) triggered in response to polymer exposure to clarify whether VpsS is indeed involved and activated in response to polymers. Polymeric materials that inhibit bacterial dissemination, both mechanically and transcriptionally, may be useful for applications to enhance wastewater treatment for the decontamination of water from V. cholerae.
Materials and Methods
ARTICLE SECTIONS
Bacterial Strains and Culture Conditions
V. cholerae El Tor strains used in this study (Table 1) were derived from A1552 (29) and E7946. (30) The E. coli K12 strains JCB387 (31) DH5α (32) and SM10 λpir (33) were used for general cloning and conjugation procedures. Strains were propagated at 37 °C in lysogeny broth (LB) supplemented with 10 μg/μL of tetracycline or 30 μg/μL of kanamycin for selection when required. Plasmids were introduced into V. cholerae strains by triparental mating with E. coli DH5α carrying the desired plasmid (donor) and E. coli SM10 (helper strain) carrying the conjugative machinery on pRK2013. Cultures were mixed at a volumetric ratio of 1:2:2 of recipient:helper:donor in 250 μL and spotted onto brain-heart infusion (BHI) agar for incubation overnight at 37 °C. Spots of bacteria were dislodged after an overnight incubation and resuspended in 3 mL of sterile PBS. Then, 100 μL of serial dilutions were plated onto TCBS plates containing 10 μg/μL of tetracycline. The resulting colonies were checked by PCR in the case of pRW50T constructs, and pBB1 transconjugants were screened for luminescence (Table 2).
Table 1. Bacterial Strains Used in This Study
| strain | description or genotype | source or ref |
|---|---|---|
| Vibrio cholerae | ||
| A1552 | wild-type; O1 biovar El Tor serotype Inaba | (29) |
| E7956 | wild-type; O1 biovar El Tor serotype Ogawa | (30) |
| BH1651 | luxOD47E | (15) |
| BH1578 | ΔluxSΔcqsA | (36) |
| DH231 | ΔluxSΔcqsS | (37) |
| WN1103 | ΔluxQΔcqsA | (37) |
| E7956 Δcrp | Δcrp KanR | gift from D. Grainger |
| NP5005 | A1552 pRW50T containing upstream region of aphA promoter; TetR | (12) |
| Escherichia coli | ||
| DH5α | donor and maintenance of pBB1 | (32) |
| JCB387 | donor and maintenance of pRW50T | (31) |
| SM10 | helper strain; λpir pRK2013; KanR | (33) |
Table 2. Plasmids Used in This Study
| plasmid | description | source or ref |
|---|---|---|
| pRW50T | pRW50 derivative with a oriT sequence from pRK2; TetR | gift from D. Grainger |
| pRW50T-rbmA | pRW50T containing 273 bp of the upstream region of rbmA, cloned between EcoRI and HindIII restriction sites; TetR | this study |
| pRW50T-vpsR | pRW50T containing 195 bp of the upstream region of vpsR, cloned between EcoRI and HindIII restriction sites; TetR | this study |
| pRW50T-aphA | pRW50T containing the upstream region of aphA, cloned between EcoRI and HindIII sites; TetR | (12) |
| pBB1 | luxCDABE cosmid; TetR | (35) |
β-Galactosidase Assays
pRW50T derivative construction was described before. (12) Regions encoding aphA, rbmA, or vpsR promoters were amplified by PCR and cloned into pRW50T using EcoRI and HindIII sites. The insertion was checked by PCR using external primers. Measurement of β-galactosidase activity as a readout for transcriptional activity was done as previously described (34) with some modifications to accommodate testing of aggregated bacteria. Small cultures of reporter strains were grown in the absence or presence of polymers P1 and P2 and incubated overnight at 37 °C with shaking. Clustered bacteria were split in two and either used for transcriptional assays or washed with high salt PBS (200 mM NaCl) to disrupt aggregation and enable OD600 measurements.
Luminescence Assays
Luminescence assays were done using V. cholerae pBB1 transconjugants. The pBB1 cosmid (35) was introduced into V. cholerae strains by triparental mating in the same conditions as for pRW50T. Overnight cultures of V. cholerae pBB1 were adjusted to OD600 of 0.5, 0.1, and 0.01 in artificial marine water with 10 μg/μL of tetracycline. Polymers P1 and P2 were added at concentrations of 0.005, 0.05, and 0.5 mg mL–1 in DMEM or AMW in 200 μL final volume using a dark-wall clear-bottom 96-well plate. The plate was incubated up to 15 h at 37 °C with shaking a 200 rpm, whereas luminescence and OD600 were recorded every 30 min using a FLUOstar Omega plate reader. The following assays were done with bacterial cultures with OD600 adjusted to 0.2. Cells were recovered after the assay and washed with high-salt PBS containing 200 mM NaCl to disrupt charge-based aggregation and plated onto LB with tetracycline to determine the viability. Plates were imaged using a BioRad Gel Doc XR System, and the images were processed with ImageJ.
Luminescence Time-Lapse Imaging
Overnight culture of V. cholerae A1552 pBB1 was diluted to an OD600 of 0.2 in artificial marine water or clear DMEM with 10 μg/μL of tetracycline and polymers at concentrations of 0.005, 0.05, and 0.5 mg mL–1. Samples were prepared in 200 μL using a glass-bottom 96-well plate and incubated at 37 °C with 5% CO2 in a microscope imaging chamber. Images were taken every 30 min with 10 s of exposure at 40× magnification using an Evolve 512 EMCCD camera mounted on a Nikon-Eclipse TE2000-U microscope. Image acquisition was done using Nikon NIS-Elements software, and final images were processed with ImageJ. Pixel intensity was determined from several clusters within frame using ImageJ.
Super-Resolution Microscopy of Bacterial Clusters
V. cholerae A1552 was incubated with 0.05 mg mL–1 P1 in PBS for 1 h. For membrane integrity to be visualized, the sample was stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies) for 10 min at RT. The sample was mounted with ProLong Gold Antifade Mountant and covered with a coverslip. Images were taken on a Nikon N-SIM super-resolution microscope fitted with SR Apo TIRF 100× lens at 100 ms exposure. Deconvolution was carried out using the Nikon NIS elements software.
Luminescence Assays Using V. cholerae BH1578 pBB1 as a Reporter
V. cholerae BH1578 pBB1 was used to determine the effect of polymers on the production of autoinducers. V. cholerae strains at an OD600 of 0.2 were clustered with polymers at concentrations of 0.005, 0.05, and 0.5 mg mL–1 in artificial marine water. Supernatants were recovered by centrifugation and used to resuspend V. cholerae BH1578 pBB1 previously adjusted to an OD600 of 0.2 in 200 μL. Luminescence was recorded at 37 °C using a FLUOStar Omega plate reader. Similarly, V. cholerae strains and V. cholerae BH1578 pBB1 were cocultured in 200 μL final volume, and polymers were added at concentrations of 0.005, 0.05, and 0.5 mg mL–1 in artificial marine water. Both strains were adjusted to a final OD600 of 0.1 each (0.2 total density). Incubation was done at 37 °C with shaking at 200 rpm, and luminescence and OD600 were measured every 30 min using a FLUOStar Omega plate reader.
Author Information
ARTICLE SECTIONS
- Francisco Fernandez-Trillo - Institute of Microbiology and Infection and School of Chemistry, , and , University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom;
http://orcid.org/0000-0002-6680-5683;
- Anne-Marie Krachler - University of Texas Health Science Center at Houston, McGovern Medical School, Department of Microbiology and Molecular Genetics, 6431 Fannin Street, Houston, Texas 77030, United States;http://orcid.org/0000-0002-0936-0016;
Acknowledgments
ARTICLE SECTIONS
We thank B. Bassler for the generous gift of quorum sensing mutants BH1578, DH23, and WN1103 and the cosmid pBB1. We thank H. Kaplan and the Krachler and Fernandez-Trillo laboratories for critical reading of the manuscript and for suggestions on how to improve this study. This work was supported by University of Birmingham Fellowships (to A.M.K. and F.F.-T.), Wellcome Trust grant 177ISSFPP (to A.M.K. and F.F.-T), BBSRC grants BB/M021513/1 (to A.M.K.) and BB/L007916/1 (to A.M.K.), a CONICYT fellowship (to N.P.-S.), BBSRC MIBTP scholarship BB/M01116X/1 (to O.C.), and a UT Systems Science and Technology Acquisition and Retention Award (to A.M.K.).
References
ARTICLE SECTIONS
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- 3Tew, G. N., Liu, D., Chen, B., Doerksen, R. J., Kaplan, J., Carroll, P. J., Klein, M. L., and DeGrado, W. F. (2002) De novo design of biomimetic antimicrobial polymers. Proc. Natl. Acad. Sci. U. S. A. 99, 5110– 5114, DOI: 10.1073/pnas.082046199Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtFKmtbs%253D&md5=662afd62c508b7baa9b6c6dea2f8f856De novo design of biomimetic antimicrobial polymersTew, Gregory N.; Liu, Dahui; Chen, Bin; Doerksen, Robert J.; Kaplan, Justin; Carroll, Patrick J.; Klein, Michael L.; DeGrado, William F.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (8), 5110-5114CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The design of polymers and oligomers that mimic the complex structures and remarkable biol. properties of proteins is an important endeavor with both fundamental and practical implications. Recently, a no. of nonnatural peptides with designed sequences have been elaborated to provide biol. active structures; in particular, facially amphiphilic peptides built from β-amino acids have been shown to mimic both the structures as well as the biol. function of natural antimicrobial peptides such as magainins and cecropins. However, these natural peptides as well as their β-peptide analogs are expensive to prep. and difficult to produce on a large scale, limiting their potential use to certain pharmaceutical applications. We therefore have designed a series of facially amphiphilic arylamide polymers that capture the phys. and biol. properties of this class of antimicrobial peptides, but are easy to prep. from inexpensive monomers. The design process was aided by mol. calcns. with d. functional theory-computed torsional potentials. This new class of amphiphilic polymers may be applied in situations where inexpensive antimicrobial agents are required.
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- 6Devi, U. V., Puri, P., Sharma, N. N., and Ananthasubramanian, M. (2014) Electrokinetics of Cells in Dielectrophoretic Separation: A Biological Perspective. BioNanoScience 4, 276– 287, DOI: 10.1007/s12668-014-0140-yGoogle ScholarThere is no corresponding record for this reference.
- 7Xue, X., Pasparakis, G., Halliday, N., Winzer, K., Howdle, S. M., Cramphorn, C. J., Cameron, N. R., Gardner, P. M., Davis, B. G., Fernandez-Trillo, F., and Alexander, C. (2011) Synthetic polymers for simultaneous bacterial sequestration and quorum sense interference. Angew. Chem., Int. Ed. 50, 9852– 9856, DOI: 10.1002/anie.201103130Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFylsrnK&md5=3472a95b9621f634db83487efbab5f67Synthetic polymers for simultaneous bacterial sequestration and quorum sense interferenceXue, Xuan; Pasparakis, George; Halliday, Nigel; Winzer, Klaus; Howdle, Steven M.; Cramphorn, Christopher J.; Cameron, Neil R.; Gardner, Paul M.; Davis, Benjamin G.; Fernandez-Trillo, Francisco; Alexander, CameronAngewandte Chemie, International Edition (2011), 50 (42), 9852-9856, S9852/1-S9852/19CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors prepd. polymers with dual function in binding and deactivating population responses in Vibrio harveyi. The ability to sequester bacteria, while at the same time interfering with their quorum sensing response, is potentially a powerful route to novel diagnostics and antimicrobials.
- 8Lui, L. T., Xue, X., Sui, C., Brown, A., Pritchard, D. I., Halliday, N., Winzer, K., Howdle, S. M., Fernandez-Trillo, F., Krasnogor, N., and Alexander, C. (2013) Bacteria clustering by polymers induces the expression of quorum-sensing-controlled phenotypes. Nat. Chem. 5, 1058– 1065, DOI: 10.1038/nchem.1793Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslCmsb7N&md5=b06d866a3d9fb34a5647299f2d42a73bBacteria clustering by polymers induces the expression of quorum-sensing-controlled phenotypesLui, Leong T.; Xue, Xuan; Sui, Cheng; Brown, Alan; Pritchard, David I.; Halliday, Nigel; Winzer, Klaus; Howdle, Steven M.; Fernandez-Trillo, Francisco; Krasnogor, Natalio; Alexander, CameronNature Chemistry (2013), 5 (12), 1058-1065CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Bacteria deploy a range of chemistries to regulate their behavior and respond to their environment. Quorum sensing is one method by which bacteria use chem. reactions to modulate pre-infection behavior such as surface attachment. Polymers that can interfere with bacterial adhesion or the chem. reactions used for quorum sensing are therefore a potential means to control bacterial population responses. Here, we report how polymeric 'bacteria sequestrants', designed to bind to bacteria through electrostatic interactions and therefore inhibit bacterial adhesion to surfaces, induce the expression of quorum-sensing-controlled phenotypes as a consequence of cell clustering. A combination of polymer and anal. chem., biol. assays and computational modeling has been used to characterize the feedback between bacteria clustering and quorum sensing signaling. We have also derived design principles and chem. strategies for controlling bacterial behavior at the population level.
- 9Louzao, I., Sui, C., Winzer, K., Fernandez-Trillo, F., and Alexander, C. (2015) Cationic polymer mediated bacterial clustering: Cell-adhesive properties of homo- and copolymers. Eur. J. Pharm. Biopharm. 95, 47– 62, DOI: 10.1016/j.ejpb.2015.05.026Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVegtLrI&md5=9ce0ea651a2e1ad22b57cf3a6008725bCationic polymer mediated bacterial clustering: Cell-adhesive properties of homo- and copolymersLouzao, Iria; Sui, Cheng; Winzer, Klaus; Fernandez-Trillo, Francisco; Alexander, CameronEuropean Journal of Pharmaceutics and Biopharmaceutics (2015), 95 (Part_A), 47-62CODEN: EJPBEL; ISSN:0939-6411. (Elsevier B.V.)New anti-infective materials are needed urgently as alternatives to conventional biocides. It has recently been established that polymer materials designed to bind to the surface of bacteria can induce the formation of cell clusters which enhance the expression of quorum sensing controlled phenotypes. These materials are relevant for anti-infective strategies as they have the potential to inhibit adhesion while at the same time modulating Quorum Sensing (QS) controlled virulence. Here we carefully evaluate the role that charge and catechol moieties in these polymers play on the binding. We investigate the ability of the cationic polymers poly(N-[3-(dimethylamino)propyl] methacrylamide) (pDMAPMAm, P1), poly(N-dopamine methacrylamide-co-N-[3-(dimethylamino)propyl] methacrylamide) (pDMAm-co-pDMAPMAm, P2) and p(3,4-dihydroxy-L-phenylalanine methacrylamide), p(L-DMAm, P3) to cluster a range of bacteria, such as Staphylococcus aureus (Gram-pos.), Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa (Gram-neg.) under conditions of varying pH (6, 7 and 8) and polymer concn. (0.1 and 0.5 mg/mL). We identify that clustering ability is strongly dependent on the balance between charge and hydrophobicity. Moreover, our results suggest that catechol moieties have a pos. effect on adhesive properties, but only in the presence of cationic residues such as for P2. Overall, our results highlight the subtle interplay between dynamic natural surfaces and synthetic materials, as well as the need to consider synergistic structure-property relationship when designing antimicrobial polymers.
- 10Zhang, P., Lu, H., Chen, H., Zhang, J., Liu, L., Lv, F., and Wang, S. (2016) Cationic Conjugated Polymers-Induced Quorum Sensing of Bacteria Cells. Anal. Chem. 88, 2985– 2988, DOI: 10.1021/acs.analchem.5b03920Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjtFKhur8%253D&md5=6092fce0bcc90074c09bb5381ae44236Cationic conjugated polymers-induced quorum sensing of bacterial cellsZhang, Pengbo; Lu, Huan; Chen, Hui; Zhang, Jiangyan; Liu, Libing; Lv, Fengting; Wang, ShuAnalytical Chemistry (Washington, DC, United States) (2016), 88 (6), 2985-2988CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Bacteria quorum sensing (QS) has attracted significant interest for understanding cell-cell communication and regulating biol. functions. In this work, the authors demonstrate that water-sol. cationic conjugated polymers (PFP-G2) can interact with bacteria to form aggregates through electrostatic interactions. With bacteria coated in the aggregate, PFP-G2 can induce the bacterial QS system and prolong the time duration of QS signal mols. [autoinducer-2 (AI-2)] prodn. The prolonged AI-2 can bind with specific protein and continuously regulate downstream gene expression. Consequently, the bacteria show a higher survival rate against antibiotics, resulting in decreased antimicrobial susceptibility. Also, AI-2 induced by PFP-G2 can stimulate 55.54 ± 12.03% more biofilm in E. coli. This method can be used to understand cell-cell communication and regulate biol. functions, such as the prodn. of signaling mols., antibiotics, other microbial metabolites, and even virulence.
- 11Leire, E., Amaral, S. P., Louzao, I., Winzer, K., Alexander, C., Fernandez-Megia, E., and Fernandez-Trillo, F. (2016) Dendrimer mediated clustering of bacteria: improved aggregation and evaluation of bacterial response and viability. Biomater. Sci. 4, 998– 1006, DOI: 10.1039/C6BM00079GGoogle Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmvFegurw%253D&md5=e20849a4149396d95d3a87205d818fd4Dendrimer mediated clustering of bacteria: improved aggregation and evaluation of bacterial response and viabilityLeire, Emma; Amaral, Sandra P.; Louzao, Iria; Winzer, Klaus; Alexander, Cameron; Fernandez-Megia, Eduardo; Fernandez-Trillo, FranciscoBiomaterials Science (2016), 4 (6), 998-1006CODEN: BSICCH; ISSN:2047-4849. (Royal Society of Chemistry)Here, we evaluate how cationic gallic acid-triethylene glycol (GATG) dendrimers interact with bacteria and their potential to develop new antimicrobials. We demonstrate that GATG dendrimers functionalised with primary amines in their periphery can induce the formation of clusters in Vibrio harveyi, an opportunistic marine pathogen, in a generation dependent manner. Moreover, these cationic GATG dendrimers demonstrate an improved ability to induce cluster formation when compared to poly(N-[3-(dimethylamino)propyl]methacrylamide) [p(DMAPMAm)], a cationic linear polymer previously shown to cluster bacteria. Viability of the bacteria within the formed clusters and evaluation of quorum sensing controlled phenotypes (i.e. light prodn. in V. harveyi) suggest that GATG dendrimers may be activating microbial responses by maintaining a high concn. of quorum sensing signals inside the clusters while increasing permeability of the microbial outer membranes. Thus, the reported GATG dendrimers constitute a valuable platform for the development of novel antimicrobial materials that can target microbial viability and/or virulence.
- 12Perez-Soto, N., Moule, L., Crisan, D. N., Insua, I., Taylor-Smith, L. M., Voelz, K., Fernandez-Trillo, F., and Krachler, A. M. (2017) Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation in Vibrio cholerae and downregulate the expression of virulence genes. Chem. Sci. 8, 5291– 5298, DOI: 10.1039/C7SC00615BGoogle Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXotVelur8%253D&md5=0b127e8af1bc266eb73de2a55c799ff8Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation in Vibrio cholerae and downregulate the expression of virulence genesPerez-Soto, Nicolas; Moule, Lauren; Crisan, Daniel N.; Insua, Ignacio; Taylor-Smith, Leanne M.; Voelz, Kerstin; Fernandez-Trillo, Francisco; Krachler, Anne MarieChemical Science (2017), 8 (8), 5291-5298CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Here we report the first application of non-bactericidal synthetic polymers to modulate the physiol. of a bacterial pathogen. Poly(N-[3-(dimethylamino)propyl] methacrylamide) (P1) and poly(N-(3-aminopropyl)methacrylamide) (P2), cationic polymers that bind to the surface of V. cholerae, the infectious agent causing cholera disease, can sequester the pathogen into clusters. Upon clustering, V. cholerae transitions to a sessile lifestyle, characterised by increased biofilm prodn. and the repression of key virulence factors such as the cholera toxin (CTX). Moreover, clustering the pathogen results in the minimisation of adherence and toxicity to intestinal epithelial cells. Our results suggest that the redn. in toxicity is assocd. with the redn. to the no. of free bacteria, but also the downregulation of toxin prodn. Finally we demonstrate that these polymers can reduce colonisation of zebrafish larvae upon ingestion of water contaminated with V. cholerae. Overall, our results suggest that the physiol. of this pathogen can be modulated without the need to genetically manipulate the microorganism and that this modulation is an off-target effect that results from the intrinsic ability of the pathogen to sense and adapt to its environment. We believe these findings pave the way towards a better understanding of the interactions between pathogenic bacteria and polymeric materials and will underpin the development of novel antimicrobial polymers.
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- 15Svenningsen, S. L., Waters, C. M., and Bassler, B. L. (2008) A negative feedback loop involving small RNAs accelerates Vibrio cholerae’s transition out of quorum-sensing mode. Genes Dev. 22, 226– 238, DOI: 10.1101/gad.1629908Google ScholarThere is no corresponding record for this reference.
- 16Teschler, J. K., Zamorano-Sanchez, D., Utada, A. S., Warner, C. J., Wong, G. C., Linington, R. G., and Yildiz, F. H. (2015) Living in the matrix: assembly and control of Vibrio cholerae biofilms. Nat. Rev. Microbiol. 13, 255– 268, DOI: 10.1038/nrmicro3433Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1WltbrF&md5=544d369ed02f8d23512dc85888425803Living in the matrix: assembly and control of Vibrio cholerae biofilmsTeschler, Jennifer K.; Zamorano-Sanchez, David; Utada, Andrew S.; Warner, Christopher J. A.; Wong, Gerard C. L.; Linington, Roger G.; Yildiz, Fitnat H.Nature Reviews Microbiology (2015), 13 (5), 255-268CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are assocd. with biotic and abiotic surfaces and are composed of aggregates of cells that are encased by a self-produced or acquired extracellular matrix. Vibrio cholerae has been studied as a model organism for understanding biofilm formation in environmental pathogens, as it spends much of its life cycle outside of the human host in the aquatic environment. Given the important role of biofilm formation in the V. cholerae life cycle, the mol. mechanisms underlying this process and the signals that trigger biofilm assembly or dispersal have been areas of intense study over the past 20 years. In this Review, V. cholerae surface attachment, various matrix components and the regulatory networks controlling biofilm formation are discussed.
- 17Engebrecht, J. and Silverman, M. (1984) Identification of genes and gene products necessary for bacterial bioluminescence. Proc. Natl. Acad. Sci. U. S. A. 81, 4154– 4158, DOI: 10.1073/pnas.81.13.4154Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXltVGjtrc%253D&md5=33573f2cf44ee14fcd05e35c6692c01aIdentification of genes and gene products necessary for bacterial bioluminescenceEngebrecht, JoAnne; Silverman, MichaelProceedings of the National Academy of Sciences of the United States of America (1984), 81 (13), 4154-8CODEN: PNASA6; ISSN:0027-8424.Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymic activities necessary for light prodn. A genetic anal. to identify the luminescence genes (lux) that reside on this recombinant plasmid was conducted. Mutations at gene lux were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by complementation in trans with a series of polar transposon insertions on other plasmids. The lux genes were defined by complementation of lux gene defects on pairs of plasmids in trans in E. coli. Hybrid plasmids were also used to direct the synthesis of polypeptides in the E. coli minicell system. Seven lux genes and the corresponding gene products were identified from the complementation anal. and the minicell programming expts. These genes, in the order of their position on a linear map, and the apparent mol. wts. of the gene products are luxR (27,000), luxI (25,000), luxC (53,000), luxD (33,000), luxA (40,000), luxB (38,000), and luxE (42,000). From the luminescence phenotypes of E. coli contg. mutant plasmids, functions were assigned to these genes: luxA, luxB, luxC, luxD, and luxE encode enzymes for light prodn. and luxR and luxI encode regulatory functions.
- 18Silva, A. J. and Benitez, J. A. (2004) Transcriptional regulation of Vibrio cholerae hemagglutinin/protease by the cyclic AMP receptor protein and RpoS. J. Bact. 186, 6374– 6382, DOI: 10.1128/JB.186.19.6374-6382.2004Google ScholarThere is no corresponding record for this reference.
- 19Liang, W., Pascual-Montano, A., Silva, A. J., and Benitez, J. A. (2007) The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio cholerae. Microbiology 153, 2964– 2975, DOI: 10.1099/mic.0.2007/006668-0Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFKrtL7M&md5=595ba66391284d90749052f3bfffae72The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio choleraeLiang, Weili; Pascual-Montano, Alberto; Silva, Anisia J.; Benitez, Jorge A.Microbiology (Reading, United Kingdom) (2007), 153 (9), 2964-2975CODEN: MROBEO; ISSN:1350-0872. (Society for General Microbiology)Vibrio cholerae is the causative agent of cholera, which continues to be a major public health concern in Asia, Africa and Latin America. The bacterium can persist outside the human host and alternates between planktonic and biofilm community lifestyles. Transition between the different lifestyles is mediated by multiple signal transduction pathways including quorum sensing. Expression of the Zn-metalloprotease hemagglutinin (HA)/protease is subject to a dual regulation which involves the quorum sensing regulator HapR and the cAMP receptor protein. In a previous study, the authors obsd. that a mutant defective in the cAMP-receptor protein (CRP) expressed lower levels of HapR. To further investigate the role of CRP in modulating HapR and other signal transduction pathways, they performed global gene expression profiling of a Δcrp mutant of El Tor biotype V. cholerae. Here, they show that CRP is required for the biosynthesis of cholera autoinducer 1 (CAI-1) and affects the expression of multiple HapR-regulated genes. As expected, the Δcrp mutant produced more cholera toxin and enhanced biofilm. Expression of flagellar genes, reported to be affected in ΔhapR mutants, was diminished in the Δcrp mutant. However, an epistasis anal. indicated that cAMP-CRP affects motility by a mechanism independent of HapR. Inactivation of crp inhibited the expression of multiple genes reported to be strongly induced in vivo and to affect the ability of V. cholerae to colonize the small intestine and cause disease. These genes included ompU, ompT and ompW encoding outer membrane proteins, the alternative sigma factor σE required for intestinal colonization, and genes involved in anaerobic energy metab. The results indicate that CRP plays a crucial role in the V. cholerae life cycle by affecting quorum sensing and multiple genes required for survival of V. cholerae in the human host and the environment.
- 20Dunlap, P. V. and Greenberg, E. P. (1988) Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-luxR protein regulatory circuit. J. Bacteriol. 170, 4040– 4046, DOI: 10.1128/jb.170.9.4040-4046.1988Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXlvVCntb4%253D&md5=1babdf2cff6b3a1744642321fc3a7e73Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-LuxR protein regulatory circuitDunlap, Paul V.; Greenberg, E. P.Journal of Bacteriology (1988), 170 (9), 4040-6CODEN: JOBAAY; ISSN:0021-9193.Expression of the V. fischeri luminescence genes (lux genes) requires 2 transcriptional activators: the V. fischeri luxR gene product with autoinducer and the cAMP receptor protein (CRP) with cAMP. It has been established that autoinducer and the luxR gene product are required for transcriptional activation of the luxICDABE operon, which contains a gene required for autoinducer synthesis and genes required for light emission. Transcriptional control of the lux genes in Escherichia coli was examd. using catabolite repression mutants carrying lux DNA-contg. plasmids. Transcriptional fusions between the lacZ gene on Mu dI and luxR were used to assess luxR promoter activity, and the luxAB genes (which encode the 2 luciferase subunits) were used as a natural reporter of luxICDABE promoter activity. A plasmid contg. luxR under control of the cAMP-CRP-independent tac promoter was constructed to direct the synthesis of the luxR gene product in cells contg. compatible luxR::Mu dI insertion mutant plasmids. In E. coli, cAMP-CRP activated transcription of luxR and concurrently decreased luxICDABE transcription. In the presence of relatively high levels of the luxR gene product, cAMP and CRP were not required for induction of the luxICDABE operon. As shown previously and in this research, the luxR gene product in the presence of autoinducer activated transcription of the luxICDABE operon and also decreased luxR transcription. Apparently, control of the V. ficheri luminescence genes involves a regulatory circuit in which cAMP and CRP activate luxR transcription and in turn the luxR gene product activates transcription of the operon responsible for light emission (luxICDABE). Furthermore, in lux gene regulation, cAMP-CRP and autoinducer-LuxR protein appear to function as transcriptional antagonists.
- 21Hurley, A. and Bassler, B. L. (2017) Asymmetric regulation of quorum-sensing receptors drives autoinducer-specific gene expression programs in Vibrio cholerae. PLoS Genet. 13, e1006826 DOI: 10.1371/journal.pgen.1006826Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1Knsb7O&md5=562bf009052bdcd7cf14954239f93b8dAsymmetric regulation of quorum-sensing receptors drives autoinducer-specific gene expression programs in Vibrio choleraeHurley, Amanda; Bassler, Bonnie L.PLoS Genetics (2017), 13 (5), e1006826/1-e1006826/28CODEN: PGLEB5; ISSN:1553-7404. (Public Library of Science)Quorum sensing (QS) is a mechanism of chem. communication that bacteria use to monitor cell-population d. and coordinate group behaviors. QS relies on the prodn., detection, and group-wide response to extracellular signal mols. called autoinducers. Vibrio cholerae employs parallel QS circuits that converge into a shared signaling pathway. At high cell d., the CqsS and LuxPQ QS receptors detect the intra-genus and inter-species autoinducers CAI-1 and AI-2, resp., to repress virulence factor prodn. and biofilm formation. We show that pos. feedback, mediated by the QS pathway, increases CqsS but not LuxQ levels during the transition into QS-mode, which amplifies the CAI-1 input into the pathway relative to the AI-2 input. Asym. feedback on CqsS enables responses exclusively to the CAI-1 autoinducer. Because CqsS exhibits the dominant QS signaling role in V. cholerae, agonism of CqsS with synthetic compds. could be used to control pathogenicity and host dispersal. We identify nine compds. that share no structural similarity to CAI-1, yet potently agonize CqsS via inhibition of CqsS autokinase activity.
- 22Zhu, J., Miller, M. B., Vance, R. E., Dziejman, M., Bassler, B. L., and Mekalanos, J. J. (2002) Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proc. Natl. Acad. Sci. U. S. A. 99, 3129– 3134, DOI: 10.1073/pnas.052694299Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xit1Cqs7o%253D&md5=4c5783f0dd172edec01536143c9f2778Quorum-sensing regulators control virulence gene expression in Vibrio choleraeZhu, Jun; Miller, Melissa B.; Vance, Russell E.; Dziejman, Michelle; Bassler, Bonnie L.; Mekalanos, John J.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (5), 3129-3134CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The prodn. of virulence factors including cholera toxin and the toxin-coregulated pilus in the human pathogen Vibrio cholerae is strongly influenced by environmental conditions. The well-characterized ToxR signal transduction cascade is responsible for sensing and integrating the environmental information and controlling the virulence regulon. We show here that, in addn. to the known components of the ToxR signaling circuit, quorum-sensing regulators are involved in regulation of V. cholerae virulence. We focused on the regulators LuxO and HapR because homologs of these two proteins control quorum sensing in the closely related luminous marine bacterium Vibrio harveyi. Using an infant mouse model, we found that a luxO mutant is severely defective in colonization of the small intestine. Gene arrays were used to profile transcription in the V. cholerae wild type and the luxO mutant. These studies revealed that the ToxR regulon is repressed in the luxO mutant, and that this effect is mediated by another neg. regulator, HapR. We show that LuxO represses hapR expression early in log-phase growth, and constitutive expression of hapR blocks ToxR-regulon expression. Addnl., LuxO and HapR regulate a variety of other cellular processes including motility, protease prodn., and biofilm formation. Together these data suggest a role for quorum sensing in modulating expression of blocks of virulence genes in a reciprocal fashion in vivo.
- 23Yildiz, F. H., Dolganov, N. A., and Schoolnik, G. K. (2001) VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of vps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibrio cholerae O1 El Tor. J. Bact. 183, 1716– 1726, DOI: 10.1128/JB.183.5.1716-1726.2001Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhtlOqsr0%253D&md5=e363df534db04fa70f5e011b2513fa86VpsR, a member of the response regulators of the two-component regulatory systems, is required for expression of vps biosynthesis genes and EPSETr-associated phenotypes in Vibrio cholerae O1 E1 TorYildiz, Fitnat H.; Dolganov, Nadia A.; Schoolnik, Gary K.Journal of Bacteriology (2001), 183 (5), 1716-1726CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPSETr) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of vpsR, which codes for a homolog of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic back-ground yielded a nonreverting smooth-colony morphotype that produced no detectable EPSETr and did not form an architecturally mature biofilm. Anal. of two genes, vpsA and vpsL, within the vps cluster of EPSETr biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires vpsR. These results show that VpsR functions as a pos. regulator of vpsA and vpsL and thus acts to pos. regulates EPSETr prodn. and biofilm formation.
- 24Fong, J. C., Karplus, K., Schoolnik, G. K., and Yildiz, F. H. (2006) Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae. J. Bact. 188, 1049– 1059, DOI: 10.1128/JB.188.3.1049-1059.2006Google ScholarThere is no corresponding record for this reference.
- 25Fong, J. C. and Yildiz, F. H. (2007) The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae. J. Bact. 189, 2319– 2330, DOI: 10.1128/JB.01569-06Google ScholarThere is no corresponding record for this reference.
- 26Friedrich, W. F. and Greenberg, E. P. (1983) Glucose repression of luminescence and luciferase in Vibrio fischeri. Arch. Microbiol. 134, 87– 91, DOI: 10.1007/BF00407937Google ScholarThere is no corresponding record for this reference.
- 27Shikuma, N. J., Fong, J. C., Odell, L. S., Perchuk, B. S., Laub, M. T., and Yildiz, F. H. (2009) Overexpression of VpsS, a hybrid sensor kinase, enhances biofilm formation in Vibrio cholerae. J. Bact. 191, 5147– 5158, DOI: 10.1128/JB.00401-09Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVSis7%252FK&md5=09ab9467ac38b1ec1d4aaef474a97fd1Overexpression of VpsS, a hybrid sensor kinase, enhances biofilm formation in Vibrio choleraeShikuma, Nicholas J.; Fong, Jiunn C. N.; Odell, Lindsay S.; Perchuk, Barrett S.; Laub, Michael T.; Yildiz, Fitnat H.Journal of Bacteriology (2009), 191 (16), 5147-5158CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Vibrio cholerae causes the disease cholera and inhabits aquatic environments. One key factor in the environmental survival of V. cholerae is its ability to form matrix-enclosed, surface-assocd. microbial communities known as biofilms. Mature biofilms rely on Vibrio polysaccharide to connect cells to each other and to a surface. The authors previously described a core regulatory network, which consists of two pos. transcriptional regulators, VpsR and VpsT, and a neg. transcriptional regulator HapR, that controls biofilm formation by regulating the expression of vps genes. In this study, they report the identification of a sensor histidine kinase, VpsS, which can control biofilm formation and activates the expression of vps genes. VpsS required the response regulator VpsR to activate vps expression. VpsS is a hybrid sensor histidine kinase that is predicted to contain both histidine kinase and response regulator domains, but it lacks a histidine phosphotransferase (HPT) domain. VpsS acts through the HPT protein LuxU, which is involved in a quorum sensing signal transduction network in V. cholerae. In vitro anal. of phosphotransfer relationships revealed that LuxU can specifically reverse phosphotransfer to CqsS, LuxQ, and VpsS. Furthermore, mutational and phenotypic analyses revealed that VpsS requires the response regulator LuxO to activate vps expression, and LuxO pos. regulates the transcription of vpsR and vpsT. The induction of vps expression via VpsS was also shown to occur independent of HapR. Thus, VpsS utilizes components of the quorum-sensing pathway to modulate biofilm formation in V. cholerae.
- 28Zamorano-Sanchez, D., Fong, J. C., Kilic, S., Erill, I., and Yildiz, F. H. (2015) Identification and characterization of VpsR and VpsT binding sites in Vibrio cholerae. J. Bacteriol. 197, 1221– 1235, DOI: 10.1128/JB.02439-14Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvV2gu74%253D&md5=992251ead5c1980021fcf6a94d65153fIdentification and characterization of VpsR and VpsT binding sites in Vibrio choleraeZamorano-Sanchez, David; Fong, Jiunn C. N.; Kilic, Sefa; Erill, Ivan; Yildiz, Fitnat H.Journal of Bacteriology (2015), 197 (7), 1221-1235CODEN: JOBAAY; ISSN:1098-5530. (American Society for Microbiology)The ability to form biofilms is crit. for environmental survival and transmission of Vibrio cholerae, a facultative human pathogen responsible for the disease cholera. Biofilm formation is controlled by several transcriptional regulators and alternative sigma factors. In this study, we report that the two main pos. regulators of biofilm formation, VpsR and VpsT, bind to nonoverlapping target sequences in the regulatory region of vpsL in vitro. VpsR binds to a proximal site (the R1 box) as well as a distal site (the R2 box) with respect to the transcriptional start site identified upstream of vpsL. The VpsT binding site (the T box) is located between the R1 and R2 boxes. While mutations in the T and R boxes resulted in a decrease in vpsL expression, deletion of the T and R2 boxes resulted in an increase in vpsL expression. Anal. of the role of H-NS in vpsL expression revealed that deletion of hns resulted in enhanced vpsL expression. The level of vpsL expression was higher in an hns vpsT double mutant than in the parental strain but lower than that in an hns mutant. In silico anal. of the regulatory regions of the VpsR and VpsT targets resulted in the identification of conserved recognition motifs for VpsR and VpsT and revealed that operons involved in biofilm formation and vpsT are coregulated by VpsR and VpsT. Furthermore, a comparative genomics anal. revealed substantial variability in the promoter region of the vpsT and vpsL genes among extant V. cholerae isolates, suggesting that regulation of biofilm formation is under active selection.
- 29Yildiz, F. H. and Schoolnik, G. K. (1998) Role of rpoS in stress survival and virulence of Vibrio cholerae. J. Bact. 180, 773– 784Google ScholarThere is no corresponding record for this reference.
- 30Miller, V. L., Dirita, V. J., and Mekalanos, J. J. (1989) Identification of Toxs, a Regulatory Gene Whose Product Enhances Toxr-Mediated Activation of the Cholera-Toxin Promoter. J. Bacteriol. 171, 1288– 1293, DOI: 10.1128/jb.171.3.1288-1293.1989Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXhs1Ojt7k%253D&md5=a3a60f22540cd735ec589582859c8e98Identification of toxS, a regulatory gene whose product enhances toxR-mediated activation of the cholera toxin promoterMiller, Virginia L.; DiRita, Victor J.; Mekalanos, John J.Journal of Bacteriology (1989), 171 (3), 1288-93CODEN: JOBAAY; ISSN:0021-9193.The cloning of the toxS gene from Vibrio cholerae E1 Tor strain E7946 is described. This gene lies downstream from the toxR gene, which encodes the transcriptional activator for the cholera toxin (ctx) operon in V. cholerae. ToxS acts in conjunction with ToxR to activate expression of the ctx operon in Escherichia coli. The classical strain 569B, which is attenuated for virulence but which synthesizes high levels of cholera toxin in vitro, carries a deletion of 1.2 kilobase pairs of DNA, downstream from the toxR gene, which removes toxS. Evidence is presented that toxS is the downstream gene in an operon with toxR.
- 31Page, L., Griffiths, L., and Cole, J. A. (1990) Different physiological roles of two independent pathways for nitrite reduction to ammonia by enteric bacteria. Arch. Microbiol. 154, 349– 354, DOI: 10.1007/BF00276530Google ScholarThere is no corresponding record for this reference.
- 32Taylor, R. G., Walker, D. C., and McInnes, R. R. (1993) E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing. Nucleic Acids Res. 21, 1677– 1678, DOI: 10.1093/nar/21.7.1677Google ScholarThere is no corresponding record for this reference.
- 33Simon, R., Priefer, U., and Puhler, A. (1983) A Broad Host Range Mobilization System for Invivo Genetic-Engineering - Transposon Mutagenesis in Gram-Negative Bacteria. Bio/Technology 1, 784– 791, DOI: 10.1038/nbt1183-784Google ScholarThere is no corresponding record for this reference.
- 34Bell, A. I., Gaston, K. L., Cole, J. A., and Busby, S. J. (1989) Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP. Nucleic Acids Res. 17, 3865– 3874, DOI: 10.1093/nar/17.10.3865Google ScholarThere is no corresponding record for this reference.
- 35Bassler, B. L., Wright, M., Showalter, R. E., and Silverman, M. R. (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol. Microbiol. 9, 773– 786, DOI: 10.1111/j.1365-2958.1993.tb01737.xGoogle Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXkt1Wn&md5=57332ec8fdd0c196772ae1b23b5e5d0aIntercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescenceBassler, Bonnie L.; Wright, Miriam; Showalter, Richard E.; Silverman, Michael R.Molecular Microbiology (1993), 9 (4), 773-86CODEN: MOMIEE; ISSN:0950-382X.D.-dependent expression of luminescence in V. harveyi is regulated by the concn. of an extracellular signal mol. (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants encoded functions necessary for the synthesis of, and response to, a signal mol. Sequence anal. of the region encoding these functions revealed 3 open reading frames, two (luxL and luxM) that are required for prodn. of an autoinducer substance and a 3rd (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of 2-component, signal transduction proteins. The phenotypes of mutants with luxL, luxM, and luxN defects indicated that an addnl. signal-response system controlling d.-dependent expression of luminescence remains to be identified.
- 36Hammer, B. K. and Bassler, B. L. (2007) Regulatory small RNAs circumvent the conventional quorum sensing pathway in pandemic Vibrio cholerae. Proc. Natl. Acad. Sci. U. S. A. 104, 11145– 11149, DOI: 10.1073/pnas.0703860104Google ScholarThere is no corresponding record for this reference.
- 37Ng, W. L., Wei, Y., Perez, L. J., Cong, J., Long, T., Koch, M., Semmelhack, M. F., Wingreen, N. S., and Bassler, B. L. (2010) Probing bacterial transmembrane histidine kinase receptor-ligand interactions with natural and synthetic molecules. Proc. Natl. Acad. Sci. U. S. A. 107, 5575– 5580, DOI: 10.1073/pnas.1001392107Google ScholarThere is no corresponding record for this reference.
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Abstract
Figure 1
Figure 1. Polymers enhance quorum sensing in Vibrio cholerae. Chemical structures of P1 (A) and P2 (B). N-SIM super-resolution image of LIVE/DEAD-stained V. cholerae A1552 clustered by P1 (C). V. cholerae El Tor strains A1552 (D, E, H) or E7646 (F, G, I) containing the luminescence reporter pBB1 were adjusted to an OD600 of 0.2 following 16 h of growth and incubated in AMW alone (black) or AMW-containing polymer P1 (D, F) or P2 (E, G) at concentrations of 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL–1. Luminescence was recorded every 30 min for 10 h and plotted as means ± SEM from at least three biological replicates. For the effect of polymers on bacterial viability to be tested, samples were removed after 10 h, serially diluted, and plated on LB (H, I).
Figure 2
Figure 2. Bacterial density shapes the kinetics of quorum induction in response to polymer. Cultures of V. cholerae A1552 containing pBB1 were grown for 16 h and diluted into AMW alone (black) or AMW containing 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL–1 of P1 (A–C) or P2 (D–F). Bacterial densities were adjusted to result in OD600 values of 0.5 (A, D), 0.05 (B, E), and 0.005 (C, F), respectively. Luminescence and OD600 were recorded every 30 min for 10 h, and values are means ± SEM from at least three biological replicates. No significant growth was detected over this time frame at either initial density of 0.5 (G) or 0.005 (H).
Figure 3
Figure 3. Polymers enhance quorum sensing in V. cholerae. V. cholerae A1552 containing pBB1 was grown for 16 h and then adjusted to an OD600 of 0.2 in the presence of P1 (A–C), P2 (D–F), or AMW alone (G). Polymers were adjusted to final concentrations of 0.005 (A, D), 0.05 (B, E), or 0.5 (C, F) mg mL–1 in AMW. Luminescence of samples was imaged every 30 min for 15 h, and representative images for each time point are shown from 0 h (top left) to 15 h (bottom right) of each panel. Luminescence intensities over time were analyzed by quantifying pixel intensities, and means ± SEM from at least three biological replicates are shown for P1 (left) and P2 (right panel) (H).
Figure 4
Figure 4. Polymer-mediated luminescence is not due to nutrient starvation within clusters. V. cholerae E7646 Δcrp containing the luminescence reporter pBB1 was grown for 16 h and then diluted to an OD600 of 0.2 in AMW alone (black) or AMW containing P1 (A) or P2 (B) at concentrations of 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL–1. Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown. (C) V. cholerae E7646 wild-type containing pBB1 was grown for 16 h and adjusted to an OD600 of 0.2 in AMW containing 1% glucose alone (orange) or in the presence of 0.5 mg mL–1 of P1 (light blue) or P2 (dark blue). Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown.
Figure 5
Figure 5. Polymer-mediated enhancement of quorum sensing is mainly driven by CAI-1. V. cholerae A1552 wild-type (dark) and quorum sensing mutants containing pBB1 were grown for 16 h and then diluted into AMW to give equal cell densities and a total OD600 of 0.2. Strains were grown together in AMW alone (black) or AMW containing P1 at 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL–1. Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown. Mutants grown in coculture with the wild-type were (A) DH231 (ΔluxSΔcqsS) producing no AI-2, (B) WN1103 (ΔluxQΔcqsA) producing no CAI-1, (C) BH1578 (ΔluxSΔcqsA) producing no AI-2 or CAI-1, and (D) BH1651 (luxOD47E).
Figure 6
Figure 6. Enhanced quorum sensing is driven by enhanced production of autoinducers in response to polymers. Cultures of V. cholerae were adjusted to an OD of 0.2 and grown for 16 h in LB medium, and the supernatants were harvested, filtered, and incubated with V. cholerae BH1578 containing pBB1. Strains used to harvest supernatants were (A) wild-type A1552, (B) DH231 (ΔluxSΔcqs), (C) WN1103 (ΔluxQΔcqsA), and (D) BH1578 ((ΔluxSΔcqsA). Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown. V. cholerae wild-type was adjusted to an OD600 of 0.2 in AMW alone or AMW containing 0.005–0.5 mg mL–1 P1 (E) or P2 (F), and the supernatants were harvested and filtered 16 h later. For their autoinducer content to be determined, the supernatants were incubated with the reporter strain V. cholerae BH1578 containing pBB1. Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown.
Figure 7
Figure 7. Polymer-mediated quorum induction overrides the canonical biofilm dissipation program in V. cholerae. V. cholerae wild-type A1552 containing pRW50T lacZ reporters for the promoters of rbmA (A) or vpsR (B) were grown for 16 h and then diluted into AMW alone or containing 0.005 or 0.05 mg mL–1 P1 or P2 as indicated to give an OD600 of 0.2. Following 16 h of incubation, clustered bacteria were removed and either processed for β-galactosidase assays or treated with high-salt PBS to disperse the cultures for OD600 measurements. Transcriptional activities were calculated and normalized to untreated cultures. Shown are means ± SEM and individual measurements for three biological replicates. Statistical significance was determined by ANOVA and a Dunnett’s multiple comparison test and is depicted as ****p-values ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, and ns for not significant (p ≥ 0.05). (C) V. cholerae wild-type A1552 containing pRW50T lacZ reporter for the aphA promoter was grown in AMW alone (orange) or containing P1 (light blue) at 0.05 mg mL–1 or P2 (dark blue) at 0.5 mg mL–1 for 18 h. Clustered bacteria were removed at indicated times and either processed for β-galactosidase assays or treated with high-salt PBS to disperse the cultures for OD600 measurements. Transcriptional activities were calculated and normalized to the activities of untreated cultures at 2 h. Shown are means ± SEM and individual measurements for three biological replicates.
References
ARTICLE SECTIONSThis article references 37 other publications.
- 1Hartlieb, M., Williams, E. G. L., Kuroki, A., Perrier, S., and Locock, K. E. S. (2017) Antimicrobial Polymers: Mimicking Amino Acid Functionality, Sequence Control and Three-dimensional Structure of Host-defense Peptides. Curr. Med. Chem. 24, 2115– 2140, DOI: 10.2174/09298673246661701161223221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVKltLvL&md5=98d1a6e17f1d974eca9413803fa124e4Antimicrobial Polymers: Mimicking Amino Acid Functionality, Sequence Control and Three-dimensional Structure of Host-defense PeptidesHartlieb, Matthias; Williams, Elizabeth G. L.; Kuroki, Agnes; Perrier, Sebastien; Locock, Katherine E. S.Current Medicinal Chemistry (2017), 24 (19), 2115-2140CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)A review. Peptides and proteins control and direct all aspects of cellular function and communication. Having been honed by nature for millions of years, they also typically display an unsurpassed specificity for their biol. targets. This underlies the continued focus on peptides as promising drug candidates. However, the development of peptides into viable drugs is hampered by their lack of chem. and pharmacokinetic stability and the cost of large scale prodn. One method to overcome such hindrances is to develop polymer systems that are able to retain the important structural features of these biol. active peptides, while being cheaper and easier to produce and manipulate chem. This review illustrates these principles using examples of polymers designed to mimic antimicrobial host-defense peptides. The host-defense peptides have been identified as some of the most important leads for the next generation of antibiotics as they typically exhibit broad spectrum antimicrobial ability, low toxicity toward human cells and little susceptibility to currently known mechanisms of bacterial resistance. Their movement from the bench to clinic is yet to be realized, however, due to the limitations of these peptides as drugs. The literature provides a no. of examples of polymers that have been able to mimic these peptides through all levels of structure, starting from specific amino acid sidechains, through to more global features such as overall charge, mol. wt. and threedimensional structure (e.g. α-helical). The resulting optimized polymers are able retain the activity profile of the peptides, but within a synthetic macromol. construct that may be better suited to the development of a new generation of antimicrobial therapeutics. Such work has not only produced important new leads to combat the growing threat of antibiotic resistance, but may also open up new ways for polymers to mimic other important classes of biol. active peptides.
- 2Palermo, E. F. and Kuroda, K. (2009) Chemical structure of cationic groups in amphiphilic polymethacrylates modulates the antimicrobial and hemolytic activities. Biomacromolecules 10, 1416– 1428, DOI: 10.1021/bm900044x2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXktF2gtrs%253D&md5=6b75f157a30f80f42a767af49a6797aaChemical Structure of Cationic Groups in Amphiphilic Polymethacrylates Modulates the Antimicrobial and Hemolytic ActivitiesPalermo, Edmund F.; Kuroda, KenichiBiomacromolecules (2009), 10 (6), 1416-1428CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)A library of amphiphilic random copolymers contg. cationic and hydrophobic side chains were prepd. by copolymn. of amine-functionalized methacrylate monomers with various ratios of an alkyl methacrylate. Primary or tertiary amine groups, or quaternary ammonium groups, were utilized as the source of cationic charge in each copolymer series. The antimicrobial and hemolytic activities of these copolymers are reported, enabling a systematic assessment of the effect different amine groups exert on the biol. activity of the polymers. It was shown that the copolymer compn. of amphiphilic copolymers contg. primary or tertiary amine groups can be tuned to achieve potent antimicrobial activity while minimizing red blood cell lysis. On the other hand, the copolymers contg. quaternary ammonium groups required a greater amt. of hydrophobic comonomer to express activity and showed generally lower selectivity for E. coli vs. human red blood cells. Potentiometric titrn. data revealed the fraction of the primary or tertiary amine groups in the polymers, which are deprotonated (basic) at physiol. pH. Measurements of the bactericidal and hemolytic activities in buffers of pH varying from 6 to 8 showed the impact of polymer ionization on biol. activity. A decrease in the fraction of amine groups that are cationic, from α = 1.0 to 0.7, caused an enhancement of antimicrobial and hemolytic activity. As this value was decreased further to α = 0.5, loss of activity was obsd. The activities of polymers contg. quaternary ammonium groups were shown to be pH-independent.
- 3Tew, G. N., Liu, D., Chen, B., Doerksen, R. J., Kaplan, J., Carroll, P. J., Klein, M. L., and DeGrado, W. F. (2002) De novo design of biomimetic antimicrobial polymers. Proc. Natl. Acad. Sci. U. S. A. 99, 5110– 5114, DOI: 10.1073/pnas.0820461993https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtFKmtbs%253D&md5=662afd62c508b7baa9b6c6dea2f8f856De novo design of biomimetic antimicrobial polymersTew, Gregory N.; Liu, Dahui; Chen, Bin; Doerksen, Robert J.; Kaplan, Justin; Carroll, Patrick J.; Klein, Michael L.; DeGrado, William F.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (8), 5110-5114CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The design of polymers and oligomers that mimic the complex structures and remarkable biol. properties of proteins is an important endeavor with both fundamental and practical implications. Recently, a no. of nonnatural peptides with designed sequences have been elaborated to provide biol. active structures; in particular, facially amphiphilic peptides built from β-amino acids have been shown to mimic both the structures as well as the biol. function of natural antimicrobial peptides such as magainins and cecropins. However, these natural peptides as well as their β-peptide analogs are expensive to prep. and difficult to produce on a large scale, limiting their potential use to certain pharmaceutical applications. We therefore have designed a series of facially amphiphilic arylamide polymers that capture the phys. and biol. properties of this class of antimicrobial peptides, but are easy to prep. from inexpensive monomers. The design process was aided by mol. calcns. with d. functional theory-computed torsional potentials. This new class of amphiphilic polymers may be applied in situations where inexpensive antimicrobial agents are required.
- 4Cevc, G. (1990) Membrane electrostatics. Biochim. Biophys. Acta, Rev. Biomembr. 1031, 311– 382, DOI: 10.1016/0304-4157(90)90015-54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXmt1KntLY%253D&md5=ae4c57f27d94d64bd33872ec5a85dc5cMembrane electrostaticsCevc, GregorBiochimica et Biophysica Acta, Reviews on Biomembranes (1990), 1031 (3), 311-82CODEN: BRBMC5; ISSN:0304-4157.A review, with 613 refs., on electrostatic phenomena in biol. membranes. The topics discussed include electrostatic models, interfacial structure, interfacial polarity and the dielec. const., hydration effects, ion distribution and binding, thermodn. of charged membranes, and biol. significance.
- 5Sonohara, R., Muramatsu, N., Ohshima, H., and Kondo, T. (1995) Difference in surface properties between Escherichia coli and Staphylococcus aureus as revealed by electrophoretic mobility measurements. Biophys. Chem. 55, 273– 277, DOI: 10.1016/0301-4622(95)00004-H5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXmsFGnu78%253D&md5=cabb5ba75a8bb10a00328ef733394d1bDifference in surface properties between Escherichia coli and Staphylococcus aureus as revealed by electrophoretic mobility measurementsSonohara, Ritsu; Muramatsu, Nobuhiro; Ohshima, Hiroyuki; Kondo, TamotsuBiophysical Chemistry (1995), 55 (3), 273-7CODEN: BICIAZ; ISSN:0301-4622. (Elsevier)Electrophoretic mobilities of Escherichia coli and Staphylococcus aureus were measured in media of different pH values and ionic strengths at 310 K and the results were analyzed via a new mobility formula (Ohshima, H. et al. 1989) which was derived on the assumptions of uniform charge distribution in the cell surface layer of finite thickness and ion-penetrability in the layer. E. coli was shown to have a more neg. charged and less soft surface than that of S. aureus. It is suggested that electrophoretic mobility measurement can be used to detect the difference in surface structure between gram-pos. and gram-neg. bacteria.
- 6Devi, U. V., Puri, P., Sharma, N. N., and Ananthasubramanian, M. (2014) Electrokinetics of Cells in Dielectrophoretic Separation: A Biological Perspective. BioNanoScience 4, 276– 287, DOI: 10.1007/s12668-014-0140-yThere is no corresponding record for this reference.
- 7Xue, X., Pasparakis, G., Halliday, N., Winzer, K., Howdle, S. M., Cramphorn, C. J., Cameron, N. R., Gardner, P. M., Davis, B. G., Fernandez-Trillo, F., and Alexander, C. (2011) Synthetic polymers for simultaneous bacterial sequestration and quorum sense interference. Angew. Chem., Int. Ed. 50, 9852– 9856, DOI: 10.1002/anie.2011031307https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFylsrnK&md5=3472a95b9621f634db83487efbab5f67Synthetic polymers for simultaneous bacterial sequestration and quorum sense interferenceXue, Xuan; Pasparakis, George; Halliday, Nigel; Winzer, Klaus; Howdle, Steven M.; Cramphorn, Christopher J.; Cameron, Neil R.; Gardner, Paul M.; Davis, Benjamin G.; Fernandez-Trillo, Francisco; Alexander, CameronAngewandte Chemie, International Edition (2011), 50 (42), 9852-9856, S9852/1-S9852/19CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors prepd. polymers with dual function in binding and deactivating population responses in Vibrio harveyi. The ability to sequester bacteria, while at the same time interfering with their quorum sensing response, is potentially a powerful route to novel diagnostics and antimicrobials.
- 8Lui, L. T., Xue, X., Sui, C., Brown, A., Pritchard, D. I., Halliday, N., Winzer, K., Howdle, S. M., Fernandez-Trillo, F., Krasnogor, N., and Alexander, C. (2013) Bacteria clustering by polymers induces the expression of quorum-sensing-controlled phenotypes. Nat. Chem. 5, 1058– 1065, DOI: 10.1038/nchem.17938https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslCmsb7N&md5=b06d866a3d9fb34a5647299f2d42a73bBacteria clustering by polymers induces the expression of quorum-sensing-controlled phenotypesLui, Leong T.; Xue, Xuan; Sui, Cheng; Brown, Alan; Pritchard, David I.; Halliday, Nigel; Winzer, Klaus; Howdle, Steven M.; Fernandez-Trillo, Francisco; Krasnogor, Natalio; Alexander, CameronNature Chemistry (2013), 5 (12), 1058-1065CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Bacteria deploy a range of chemistries to regulate their behavior and respond to their environment. Quorum sensing is one method by which bacteria use chem. reactions to modulate pre-infection behavior such as surface attachment. Polymers that can interfere with bacterial adhesion or the chem. reactions used for quorum sensing are therefore a potential means to control bacterial population responses. Here, we report how polymeric 'bacteria sequestrants', designed to bind to bacteria through electrostatic interactions and therefore inhibit bacterial adhesion to surfaces, induce the expression of quorum-sensing-controlled phenotypes as a consequence of cell clustering. A combination of polymer and anal. chem., biol. assays and computational modeling has been used to characterize the feedback between bacteria clustering and quorum sensing signaling. We have also derived design principles and chem. strategies for controlling bacterial behavior at the population level.
- 9Louzao, I., Sui, C., Winzer, K., Fernandez-Trillo, F., and Alexander, C. (2015) Cationic polymer mediated bacterial clustering: Cell-adhesive properties of homo- and copolymers. Eur. J. Pharm. Biopharm. 95, 47– 62, DOI: 10.1016/j.ejpb.2015.05.0269https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVegtLrI&md5=9ce0ea651a2e1ad22b57cf3a6008725bCationic polymer mediated bacterial clustering: Cell-adhesive properties of homo- and copolymersLouzao, Iria; Sui, Cheng; Winzer, Klaus; Fernandez-Trillo, Francisco; Alexander, CameronEuropean Journal of Pharmaceutics and Biopharmaceutics (2015), 95 (Part_A), 47-62CODEN: EJPBEL; ISSN:0939-6411. (Elsevier B.V.)New anti-infective materials are needed urgently as alternatives to conventional biocides. It has recently been established that polymer materials designed to bind to the surface of bacteria can induce the formation of cell clusters which enhance the expression of quorum sensing controlled phenotypes. These materials are relevant for anti-infective strategies as they have the potential to inhibit adhesion while at the same time modulating Quorum Sensing (QS) controlled virulence. Here we carefully evaluate the role that charge and catechol moieties in these polymers play on the binding. We investigate the ability of the cationic polymers poly(N-[3-(dimethylamino)propyl] methacrylamide) (pDMAPMAm, P1), poly(N-dopamine methacrylamide-co-N-[3-(dimethylamino)propyl] methacrylamide) (pDMAm-co-pDMAPMAm, P2) and p(3,4-dihydroxy-L-phenylalanine methacrylamide), p(L-DMAm, P3) to cluster a range of bacteria, such as Staphylococcus aureus (Gram-pos.), Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa (Gram-neg.) under conditions of varying pH (6, 7 and 8) and polymer concn. (0.1 and 0.5 mg/mL). We identify that clustering ability is strongly dependent on the balance between charge and hydrophobicity. Moreover, our results suggest that catechol moieties have a pos. effect on adhesive properties, but only in the presence of cationic residues such as for P2. Overall, our results highlight the subtle interplay between dynamic natural surfaces and synthetic materials, as well as the need to consider synergistic structure-property relationship when designing antimicrobial polymers.
- 10Zhang, P., Lu, H., Chen, H., Zhang, J., Liu, L., Lv, F., and Wang, S. (2016) Cationic Conjugated Polymers-Induced Quorum Sensing of Bacteria Cells. Anal. Chem. 88, 2985– 2988, DOI: 10.1021/acs.analchem.5b0392010https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjtFKhur8%253D&md5=6092fce0bcc90074c09bb5381ae44236Cationic conjugated polymers-induced quorum sensing of bacterial cellsZhang, Pengbo; Lu, Huan; Chen, Hui; Zhang, Jiangyan; Liu, Libing; Lv, Fengting; Wang, ShuAnalytical Chemistry (Washington, DC, United States) (2016), 88 (6), 2985-2988CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Bacteria quorum sensing (QS) has attracted significant interest for understanding cell-cell communication and regulating biol. functions. In this work, the authors demonstrate that water-sol. cationic conjugated polymers (PFP-G2) can interact with bacteria to form aggregates through electrostatic interactions. With bacteria coated in the aggregate, PFP-G2 can induce the bacterial QS system and prolong the time duration of QS signal mols. [autoinducer-2 (AI-2)] prodn. The prolonged AI-2 can bind with specific protein and continuously regulate downstream gene expression. Consequently, the bacteria show a higher survival rate against antibiotics, resulting in decreased antimicrobial susceptibility. Also, AI-2 induced by PFP-G2 can stimulate 55.54 ± 12.03% more biofilm in E. coli. This method can be used to understand cell-cell communication and regulate biol. functions, such as the prodn. of signaling mols., antibiotics, other microbial metabolites, and even virulence.
- 11Leire, E., Amaral, S. P., Louzao, I., Winzer, K., Alexander, C., Fernandez-Megia, E., and Fernandez-Trillo, F. (2016) Dendrimer mediated clustering of bacteria: improved aggregation and evaluation of bacterial response and viability. Biomater. Sci. 4, 998– 1006, DOI: 10.1039/C6BM00079G11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmvFegurw%253D&md5=e20849a4149396d95d3a87205d818fd4Dendrimer mediated clustering of bacteria: improved aggregation and evaluation of bacterial response and viabilityLeire, Emma; Amaral, Sandra P.; Louzao, Iria; Winzer, Klaus; Alexander, Cameron; Fernandez-Megia, Eduardo; Fernandez-Trillo, FranciscoBiomaterials Science (2016), 4 (6), 998-1006CODEN: BSICCH; ISSN:2047-4849. (Royal Society of Chemistry)Here, we evaluate how cationic gallic acid-triethylene glycol (GATG) dendrimers interact with bacteria and their potential to develop new antimicrobials. We demonstrate that GATG dendrimers functionalised with primary amines in their periphery can induce the formation of clusters in Vibrio harveyi, an opportunistic marine pathogen, in a generation dependent manner. Moreover, these cationic GATG dendrimers demonstrate an improved ability to induce cluster formation when compared to poly(N-[3-(dimethylamino)propyl]methacrylamide) [p(DMAPMAm)], a cationic linear polymer previously shown to cluster bacteria. Viability of the bacteria within the formed clusters and evaluation of quorum sensing controlled phenotypes (i.e. light prodn. in V. harveyi) suggest that GATG dendrimers may be activating microbial responses by maintaining a high concn. of quorum sensing signals inside the clusters while increasing permeability of the microbial outer membranes. Thus, the reported GATG dendrimers constitute a valuable platform for the development of novel antimicrobial materials that can target microbial viability and/or virulence.
- 12Perez-Soto, N., Moule, L., Crisan, D. N., Insua, I., Taylor-Smith, L. M., Voelz, K., Fernandez-Trillo, F., and Krachler, A. M. (2017) Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation in Vibrio cholerae and downregulate the expression of virulence genes. Chem. Sci. 8, 5291– 5298, DOI: 10.1039/C7SC00615B12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXotVelur8%253D&md5=0b127e8af1bc266eb73de2a55c799ff8Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation in Vibrio cholerae and downregulate the expression of virulence genesPerez-Soto, Nicolas; Moule, Lauren; Crisan, Daniel N.; Insua, Ignacio; Taylor-Smith, Leanne M.; Voelz, Kerstin; Fernandez-Trillo, Francisco; Krachler, Anne MarieChemical Science (2017), 8 (8), 5291-5298CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Here we report the first application of non-bactericidal synthetic polymers to modulate the physiol. of a bacterial pathogen. Poly(N-[3-(dimethylamino)propyl] methacrylamide) (P1) and poly(N-(3-aminopropyl)methacrylamide) (P2), cationic polymers that bind to the surface of V. cholerae, the infectious agent causing cholera disease, can sequester the pathogen into clusters. Upon clustering, V. cholerae transitions to a sessile lifestyle, characterised by increased biofilm prodn. and the repression of key virulence factors such as the cholera toxin (CTX). Moreover, clustering the pathogen results in the minimisation of adherence and toxicity to intestinal epithelial cells. Our results suggest that the redn. in toxicity is assocd. with the redn. to the no. of free bacteria, but also the downregulation of toxin prodn. Finally we demonstrate that these polymers can reduce colonisation of zebrafish larvae upon ingestion of water contaminated with V. cholerae. Overall, our results suggest that the physiol. of this pathogen can be modulated without the need to genetically manipulate the microorganism and that this modulation is an off-target effect that results from the intrinsic ability of the pathogen to sense and adapt to its environment. We believe these findings pave the way towards a better understanding of the interactions between pathogenic bacteria and polymeric materials and will underpin the development of novel antimicrobial polymers.
- 13Miller, M. B., Skorupski, K., Lenz, D. H., Taylor, R. K., and Bassler, B. L. (2002) Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae. Cell 110, 303– 314, DOI: 10.1016/S0092-8674(02)00829-213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xmtl2rsrg%253D&md5=847780f5da47636697033411d1e743f1Parallel quorum sensing systems converge to regulate virulence in Vibrio choleraeMiller, Melissa B.; Skorupski, Karen; Lenz, Derrick H.; Taylor, Ronald K.; Bassler, Bonnie L.Cell (Cambridge, MA, United States) (2002), 110 (3), 303-314CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The marine bacterium Vibrio harveyi possesses 2 quorum sensing systems (System 1 and System 2) that regulate bioluminescence. Although the V. cholerae genome sequence reveals that a V. harveyi-like System 2 exists, it does not predict the existence of a V. harveyi-like System 1 or any obvious quorum sensing-controlled target genes. In this report we identify and characterize the genes encoding an addnl. V. cholerae autoinducer synthase and its cognate sensor. Anal. of double mutants indicates that a 3rd as yet unidentified sensory circuit exists in V. cholerae. This quorum sensing app. is unusually complex, as it is composed of ≥3 parallel signaling channels. We show that in V. cholerae these communication systems converge to control virulence.
- 14Jung, S. A., Chapman, C. A., and Ng, W. L. (2015) Quadruple quorum-sensing inputs control Vibrio cholerae virulence and maintain system robustness. PLoS Pathog. 11, e1004837 DOI: 10.1371/journal.ppat.1004837There is no corresponding record for this reference.
- 15Svenningsen, S. L., Waters, C. M., and Bassler, B. L. (2008) A negative feedback loop involving small RNAs accelerates Vibrio cholerae’s transition out of quorum-sensing mode. Genes Dev. 22, 226– 238, DOI: 10.1101/gad.1629908There is no corresponding record for this reference.
- 16Teschler, J. K., Zamorano-Sanchez, D., Utada, A. S., Warner, C. J., Wong, G. C., Linington, R. G., and Yildiz, F. H. (2015) Living in the matrix: assembly and control of Vibrio cholerae biofilms. Nat. Rev. Microbiol. 13, 255– 268, DOI: 10.1038/nrmicro343316https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1WltbrF&md5=544d369ed02f8d23512dc85888425803Living in the matrix: assembly and control of Vibrio cholerae biofilmsTeschler, Jennifer K.; Zamorano-Sanchez, David; Utada, Andrew S.; Warner, Christopher J. A.; Wong, Gerard C. L.; Linington, Roger G.; Yildiz, Fitnat H.Nature Reviews Microbiology (2015), 13 (5), 255-268CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are assocd. with biotic and abiotic surfaces and are composed of aggregates of cells that are encased by a self-produced or acquired extracellular matrix. Vibrio cholerae has been studied as a model organism for understanding biofilm formation in environmental pathogens, as it spends much of its life cycle outside of the human host in the aquatic environment. Given the important role of biofilm formation in the V. cholerae life cycle, the mol. mechanisms underlying this process and the signals that trigger biofilm assembly or dispersal have been areas of intense study over the past 20 years. In this Review, V. cholerae surface attachment, various matrix components and the regulatory networks controlling biofilm formation are discussed.
- 17Engebrecht, J. and Silverman, M. (1984) Identification of genes and gene products necessary for bacterial bioluminescence. Proc. Natl. Acad. Sci. U. S. A. 81, 4154– 4158, DOI: 10.1073/pnas.81.13.415417https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXltVGjtrc%253D&md5=33573f2cf44ee14fcd05e35c6692c01aIdentification of genes and gene products necessary for bacterial bioluminescenceEngebrecht, JoAnne; Silverman, MichaelProceedings of the National Academy of Sciences of the United States of America (1984), 81 (13), 4154-8CODEN: PNASA6; ISSN:0027-8424.Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymic activities necessary for light prodn. A genetic anal. to identify the luminescence genes (lux) that reside on this recombinant plasmid was conducted. Mutations at gene lux were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by complementation in trans with a series of polar transposon insertions on other plasmids. The lux genes were defined by complementation of lux gene defects on pairs of plasmids in trans in E. coli. Hybrid plasmids were also used to direct the synthesis of polypeptides in the E. coli minicell system. Seven lux genes and the corresponding gene products were identified from the complementation anal. and the minicell programming expts. These genes, in the order of their position on a linear map, and the apparent mol. wts. of the gene products are luxR (27,000), luxI (25,000), luxC (53,000), luxD (33,000), luxA (40,000), luxB (38,000), and luxE (42,000). From the luminescence phenotypes of E. coli contg. mutant plasmids, functions were assigned to these genes: luxA, luxB, luxC, luxD, and luxE encode enzymes for light prodn. and luxR and luxI encode regulatory functions.
- 18Silva, A. J. and Benitez, J. A. (2004) Transcriptional regulation of Vibrio cholerae hemagglutinin/protease by the cyclic AMP receptor protein and RpoS. J. Bact. 186, 6374– 6382, DOI: 10.1128/JB.186.19.6374-6382.2004There is no corresponding record for this reference.
- 19Liang, W., Pascual-Montano, A., Silva, A. J., and Benitez, J. A. (2007) The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio cholerae. Microbiology 153, 2964– 2975, DOI: 10.1099/mic.0.2007/006668-019https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFKrtL7M&md5=595ba66391284d90749052f3bfffae72The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio choleraeLiang, Weili; Pascual-Montano, Alberto; Silva, Anisia J.; Benitez, Jorge A.Microbiology (Reading, United Kingdom) (2007), 153 (9), 2964-2975CODEN: MROBEO; ISSN:1350-0872. (Society for General Microbiology)Vibrio cholerae is the causative agent of cholera, which continues to be a major public health concern in Asia, Africa and Latin America. The bacterium can persist outside the human host and alternates between planktonic and biofilm community lifestyles. Transition between the different lifestyles is mediated by multiple signal transduction pathways including quorum sensing. Expression of the Zn-metalloprotease hemagglutinin (HA)/protease is subject to a dual regulation which involves the quorum sensing regulator HapR and the cAMP receptor protein. In a previous study, the authors obsd. that a mutant defective in the cAMP-receptor protein (CRP) expressed lower levels of HapR. To further investigate the role of CRP in modulating HapR and other signal transduction pathways, they performed global gene expression profiling of a Δcrp mutant of El Tor biotype V. cholerae. Here, they show that CRP is required for the biosynthesis of cholera autoinducer 1 (CAI-1) and affects the expression of multiple HapR-regulated genes. As expected, the Δcrp mutant produced more cholera toxin and enhanced biofilm. Expression of flagellar genes, reported to be affected in ΔhapR mutants, was diminished in the Δcrp mutant. However, an epistasis anal. indicated that cAMP-CRP affects motility by a mechanism independent of HapR. Inactivation of crp inhibited the expression of multiple genes reported to be strongly induced in vivo and to affect the ability of V. cholerae to colonize the small intestine and cause disease. These genes included ompU, ompT and ompW encoding outer membrane proteins, the alternative sigma factor σE required for intestinal colonization, and genes involved in anaerobic energy metab. The results indicate that CRP plays a crucial role in the V. cholerae life cycle by affecting quorum sensing and multiple genes required for survival of V. cholerae in the human host and the environment.
- 20Dunlap, P. V. and Greenberg, E. P. (1988) Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-luxR protein regulatory circuit. J. Bacteriol. 170, 4040– 4046, DOI: 10.1128/jb.170.9.4040-4046.198820https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXlvVCntb4%253D&md5=1babdf2cff6b3a1744642321fc3a7e73Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-LuxR protein regulatory circuitDunlap, Paul V.; Greenberg, E. P.Journal of Bacteriology (1988), 170 (9), 4040-6CODEN: JOBAAY; ISSN:0021-9193.Expression of the V. fischeri luminescence genes (lux genes) requires 2 transcriptional activators: the V. fischeri luxR gene product with autoinducer and the cAMP receptor protein (CRP) with cAMP. It has been established that autoinducer and the luxR gene product are required for transcriptional activation of the luxICDABE operon, which contains a gene required for autoinducer synthesis and genes required for light emission. Transcriptional control of the lux genes in Escherichia coli was examd. using catabolite repression mutants carrying lux DNA-contg. plasmids. Transcriptional fusions between the lacZ gene on Mu dI and luxR were used to assess luxR promoter activity, and the luxAB genes (which encode the 2 luciferase subunits) were used as a natural reporter of luxICDABE promoter activity. A plasmid contg. luxR under control of the cAMP-CRP-independent tac promoter was constructed to direct the synthesis of the luxR gene product in cells contg. compatible luxR::Mu dI insertion mutant plasmids. In E. coli, cAMP-CRP activated transcription of luxR and concurrently decreased luxICDABE transcription. In the presence of relatively high levels of the luxR gene product, cAMP and CRP were not required for induction of the luxICDABE operon. As shown previously and in this research, the luxR gene product in the presence of autoinducer activated transcription of the luxICDABE operon and also decreased luxR transcription. Apparently, control of the V. ficheri luminescence genes involves a regulatory circuit in which cAMP and CRP activate luxR transcription and in turn the luxR gene product activates transcription of the operon responsible for light emission (luxICDABE). Furthermore, in lux gene regulation, cAMP-CRP and autoinducer-LuxR protein appear to function as transcriptional antagonists.
- 21Hurley, A. and Bassler, B. L. (2017) Asymmetric regulation of quorum-sensing receptors drives autoinducer-specific gene expression programs in Vibrio cholerae. PLoS Genet. 13, e1006826 DOI: 10.1371/journal.pgen.100682621https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1Knsb7O&md5=562bf009052bdcd7cf14954239f93b8dAsymmetric regulation of quorum-sensing receptors drives autoinducer-specific gene expression programs in Vibrio choleraeHurley, Amanda; Bassler, Bonnie L.PLoS Genetics (2017), 13 (5), e1006826/1-e1006826/28CODEN: PGLEB5; ISSN:1553-7404. (Public Library of Science)Quorum sensing (QS) is a mechanism of chem. communication that bacteria use to monitor cell-population d. and coordinate group behaviors. QS relies on the prodn., detection, and group-wide response to extracellular signal mols. called autoinducers. Vibrio cholerae employs parallel QS circuits that converge into a shared signaling pathway. At high cell d., the CqsS and LuxPQ QS receptors detect the intra-genus and inter-species autoinducers CAI-1 and AI-2, resp., to repress virulence factor prodn. and biofilm formation. We show that pos. feedback, mediated by the QS pathway, increases CqsS but not LuxQ levels during the transition into QS-mode, which amplifies the CAI-1 input into the pathway relative to the AI-2 input. Asym. feedback on CqsS enables responses exclusively to the CAI-1 autoinducer. Because CqsS exhibits the dominant QS signaling role in V. cholerae, agonism of CqsS with synthetic compds. could be used to control pathogenicity and host dispersal. We identify nine compds. that share no structural similarity to CAI-1, yet potently agonize CqsS via inhibition of CqsS autokinase activity.
- 22Zhu, J., Miller, M. B., Vance, R. E., Dziejman, M., Bassler, B. L., and Mekalanos, J. J. (2002) Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proc. Natl. Acad. Sci. U. S. A. 99, 3129– 3134, DOI: 10.1073/pnas.05269429922https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xit1Cqs7o%253D&md5=4c5783f0dd172edec01536143c9f2778Quorum-sensing regulators control virulence gene expression in Vibrio choleraeZhu, Jun; Miller, Melissa B.; Vance, Russell E.; Dziejman, Michelle; Bassler, Bonnie L.; Mekalanos, John J.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (5), 3129-3134CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The prodn. of virulence factors including cholera toxin and the toxin-coregulated pilus in the human pathogen Vibrio cholerae is strongly influenced by environmental conditions. The well-characterized ToxR signal transduction cascade is responsible for sensing and integrating the environmental information and controlling the virulence regulon. We show here that, in addn. to the known components of the ToxR signaling circuit, quorum-sensing regulators are involved in regulation of V. cholerae virulence. We focused on the regulators LuxO and HapR because homologs of these two proteins control quorum sensing in the closely related luminous marine bacterium Vibrio harveyi. Using an infant mouse model, we found that a luxO mutant is severely defective in colonization of the small intestine. Gene arrays were used to profile transcription in the V. cholerae wild type and the luxO mutant. These studies revealed that the ToxR regulon is repressed in the luxO mutant, and that this effect is mediated by another neg. regulator, HapR. We show that LuxO represses hapR expression early in log-phase growth, and constitutive expression of hapR blocks ToxR-regulon expression. Addnl., LuxO and HapR regulate a variety of other cellular processes including motility, protease prodn., and biofilm formation. Together these data suggest a role for quorum sensing in modulating expression of blocks of virulence genes in a reciprocal fashion in vivo.
- 23Yildiz, F. H., Dolganov, N. A., and Schoolnik, G. K. (2001) VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of vps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibrio cholerae O1 El Tor. J. Bact. 183, 1716– 1726, DOI: 10.1128/JB.183.5.1716-1726.200123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhtlOqsr0%253D&md5=e363df534db04fa70f5e011b2513fa86VpsR, a member of the response regulators of the two-component regulatory systems, is required for expression of vps biosynthesis genes and EPSETr-associated phenotypes in Vibrio cholerae O1 E1 TorYildiz, Fitnat H.; Dolganov, Nadia A.; Schoolnik, Gary K.Journal of Bacteriology (2001), 183 (5), 1716-1726CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPSETr) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of vpsR, which codes for a homolog of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic back-ground yielded a nonreverting smooth-colony morphotype that produced no detectable EPSETr and did not form an architecturally mature biofilm. Anal. of two genes, vpsA and vpsL, within the vps cluster of EPSETr biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires vpsR. These results show that VpsR functions as a pos. regulator of vpsA and vpsL and thus acts to pos. regulates EPSETr prodn. and biofilm formation.
- 24Fong, J. C., Karplus, K., Schoolnik, G. K., and Yildiz, F. H. (2006) Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae. J. Bact. 188, 1049– 1059, DOI: 10.1128/JB.188.3.1049-1059.2006There is no corresponding record for this reference.
- 25Fong, J. C. and Yildiz, F. H. (2007) The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae. J. Bact. 189, 2319– 2330, DOI: 10.1128/JB.01569-06There is no corresponding record for this reference.
- 26Friedrich, W. F. and Greenberg, E. P. (1983) Glucose repression of luminescence and luciferase in Vibrio fischeri. Arch. Microbiol. 134, 87– 91, DOI: 10.1007/BF00407937There is no corresponding record for this reference.
- 27Shikuma, N. J., Fong, J. C., Odell, L. S., Perchuk, B. S., Laub, M. T., and Yildiz, F. H. (2009) Overexpression of VpsS, a hybrid sensor kinase, enhances biofilm formation in Vibrio cholerae. J. Bact. 191, 5147– 5158, DOI: 10.1128/JB.00401-0927https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVSis7%252FK&md5=09ab9467ac38b1ec1d4aaef474a97fd1Overexpression of VpsS, a hybrid sensor kinase, enhances biofilm formation in Vibrio choleraeShikuma, Nicholas J.; Fong, Jiunn C. N.; Odell, Lindsay S.; Perchuk, Barrett S.; Laub, Michael T.; Yildiz, Fitnat H.Journal of Bacteriology (2009), 191 (16), 5147-5158CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Vibrio cholerae causes the disease cholera and inhabits aquatic environments. One key factor in the environmental survival of V. cholerae is its ability to form matrix-enclosed, surface-assocd. microbial communities known as biofilms. Mature biofilms rely on Vibrio polysaccharide to connect cells to each other and to a surface. The authors previously described a core regulatory network, which consists of two pos. transcriptional regulators, VpsR and VpsT, and a neg. transcriptional regulator HapR, that controls biofilm formation by regulating the expression of vps genes. In this study, they report the identification of a sensor histidine kinase, VpsS, which can control biofilm formation and activates the expression of vps genes. VpsS required the response regulator VpsR to activate vps expression. VpsS is a hybrid sensor histidine kinase that is predicted to contain both histidine kinase and response regulator domains, but it lacks a histidine phosphotransferase (HPT) domain. VpsS acts through the HPT protein LuxU, which is involved in a quorum sensing signal transduction network in V. cholerae. In vitro anal. of phosphotransfer relationships revealed that LuxU can specifically reverse phosphotransfer to CqsS, LuxQ, and VpsS. Furthermore, mutational and phenotypic analyses revealed that VpsS requires the response regulator LuxO to activate vps expression, and LuxO pos. regulates the transcription of vpsR and vpsT. The induction of vps expression via VpsS was also shown to occur independent of HapR. Thus, VpsS utilizes components of the quorum-sensing pathway to modulate biofilm formation in V. cholerae.
- 28Zamorano-Sanchez, D., Fong, J. C., Kilic, S., Erill, I., and Yildiz, F. H. (2015) Identification and characterization of VpsR and VpsT binding sites in Vibrio cholerae. J. Bacteriol. 197, 1221– 1235, DOI: 10.1128/JB.02439-1428https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvV2gu74%253D&md5=992251ead5c1980021fcf6a94d65153fIdentification and characterization of VpsR and VpsT binding sites in Vibrio choleraeZamorano-Sanchez, David; Fong, Jiunn C. N.; Kilic, Sefa; Erill, Ivan; Yildiz, Fitnat H.Journal of Bacteriology (2015), 197 (7), 1221-1235CODEN: JOBAAY; ISSN:1098-5530. (American Society for Microbiology)The ability to form biofilms is crit. for environmental survival and transmission of Vibrio cholerae, a facultative human pathogen responsible for the disease cholera. Biofilm formation is controlled by several transcriptional regulators and alternative sigma factors. In this study, we report that the two main pos. regulators of biofilm formation, VpsR and VpsT, bind to nonoverlapping target sequences in the regulatory region of vpsL in vitro. VpsR binds to a proximal site (the R1 box) as well as a distal site (the R2 box) with respect to the transcriptional start site identified upstream of vpsL. The VpsT binding site (the T box) is located between the R1 and R2 boxes. While mutations in the T and R boxes resulted in a decrease in vpsL expression, deletion of the T and R2 boxes resulted in an increase in vpsL expression. Anal. of the role of H-NS in vpsL expression revealed that deletion of hns resulted in enhanced vpsL expression. The level of vpsL expression was higher in an hns vpsT double mutant than in the parental strain but lower than that in an hns mutant. In silico anal. of the regulatory regions of the VpsR and VpsT targets resulted in the identification of conserved recognition motifs for VpsR and VpsT and revealed that operons involved in biofilm formation and vpsT are coregulated by VpsR and VpsT. Furthermore, a comparative genomics anal. revealed substantial variability in the promoter region of the vpsT and vpsL genes among extant V. cholerae isolates, suggesting that regulation of biofilm formation is under active selection.
- 29Yildiz, F. H. and Schoolnik, G. K. (1998) Role of rpoS in stress survival and virulence of Vibrio cholerae. J. Bact. 180, 773– 784There is no corresponding record for this reference.
- 30Miller, V. L., Dirita, V. J., and Mekalanos, J. J. (1989) Identification of Toxs, a Regulatory Gene Whose Product Enhances Toxr-Mediated Activation of the Cholera-Toxin Promoter. J. Bacteriol. 171, 1288– 1293, DOI: 10.1128/jb.171.3.1288-1293.198930https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXhs1Ojt7k%253D&md5=a3a60f22540cd735ec589582859c8e98Identification of toxS, a regulatory gene whose product enhances toxR-mediated activation of the cholera toxin promoterMiller, Virginia L.; DiRita, Victor J.; Mekalanos, John J.Journal of Bacteriology (1989), 171 (3), 1288-93CODEN: JOBAAY; ISSN:0021-9193.The cloning of the toxS gene from Vibrio cholerae E1 Tor strain E7946 is described. This gene lies downstream from the toxR gene, which encodes the transcriptional activator for the cholera toxin (ctx) operon in V. cholerae. ToxS acts in conjunction with ToxR to activate expression of the ctx operon in Escherichia coli. The classical strain 569B, which is attenuated for virulence but which synthesizes high levels of cholera toxin in vitro, carries a deletion of 1.2 kilobase pairs of DNA, downstream from the toxR gene, which removes toxS. Evidence is presented that toxS is the downstream gene in an operon with toxR.
- 31Page, L., Griffiths, L., and Cole, J. A. (1990) Different physiological roles of two independent pathways for nitrite reduction to ammonia by enteric bacteria. Arch. Microbiol. 154, 349– 354, DOI: 10.1007/BF00276530There is no corresponding record for this reference.
- 32Taylor, R. G., Walker, D. C., and McInnes, R. R. (1993) E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing. Nucleic Acids Res. 21, 1677– 1678, DOI: 10.1093/nar/21.7.1677There is no corresponding record for this reference.
- 33Simon, R., Priefer, U., and Puhler, A. (1983) A Broad Host Range Mobilization System for Invivo Genetic-Engineering - Transposon Mutagenesis in Gram-Negative Bacteria. Bio/Technology 1, 784– 791, DOI: 10.1038/nbt1183-784There is no corresponding record for this reference.
- 34Bell, A. I., Gaston, K. L., Cole, J. A., and Busby, S. J. (1989) Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP. Nucleic Acids Res. 17, 3865– 3874, DOI: 10.1093/nar/17.10.3865There is no corresponding record for this reference.
- 35Bassler, B. L., Wright, M., Showalter, R. E., and Silverman, M. R. (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol. Microbiol. 9, 773– 786, DOI: 10.1111/j.1365-2958.1993.tb01737.x35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXkt1Wn&md5=57332ec8fdd0c196772ae1b23b5e5d0aIntercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescenceBassler, Bonnie L.; Wright, Miriam; Showalter, Richard E.; Silverman, Michael R.Molecular Microbiology (1993), 9 (4), 773-86CODEN: MOMIEE; ISSN:0950-382X.D.-dependent expression of luminescence in V. harveyi is regulated by the concn. of an extracellular signal mol. (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants encoded functions necessary for the synthesis of, and response to, a signal mol. Sequence anal. of the region encoding these functions revealed 3 open reading frames, two (luxL and luxM) that are required for prodn. of an autoinducer substance and a 3rd (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of 2-component, signal transduction proteins. The phenotypes of mutants with luxL, luxM, and luxN defects indicated that an addnl. signal-response system controlling d.-dependent expression of luminescence remains to be identified.
- 36Hammer, B. K. and Bassler, B. L. (2007) Regulatory small RNAs circumvent the conventional quorum sensing pathway in pandemic Vibrio cholerae. Proc. Natl. Acad. Sci. U. S. A. 104, 11145– 11149, DOI: 10.1073/pnas.0703860104There is no corresponding record for this reference.
- 37Ng, W. L., Wei, Y., Perez, L. J., Cong, J., Long, T., Koch, M., Semmelhack, M. F., Wingreen, N. S., and Bassler, B. L. (2010) Probing bacterial transmembrane histidine kinase receptor-ligand interactions with natural and synthetic molecules. Proc. Natl. Acad. Sci. U. S. A. 107, 5575– 5580, DOI: 10.1073/pnas.1001392107There is no corresponding record for this reference.