Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

Two-component systems play important roles in the physiology of many bacterial pathogens.Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression ofvps(Vibriopolysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of thealmEFGgenes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of thealmEFGoperon. Similarly to acarRmutant, strains lackingalmE,almF, andalmGexhibited enhanced polymyxin B sensitivity. We also observed that strains lackingalmEor thealmEFGoperon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance inV. cholerae.

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The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.00139-17 ◽

2017 ◽

Vol 199 (18) ◽

Cited By ~ 15

Author(s):

Jennifer K. Teschler ◽

Andrew T. Cheng ◽

Fitnat H. Yildiz

Keyword(s):

Signal Transduction ◽

Vibrio Cholerae ◽

Histidine Kinase ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Response Regulator ◽

Systematic Analysis ◽

Content Type ◽

Two Component ◽

Two Component Signal Transduction

ABSTRACT Two-component signal transduction systems (TCSs), typically composed of a sensor histidine kinase (HK) and a response regulator (RR), are the primary mechanism by which pathogenic bacteria sense and respond to extracellular signals. The pathogenic bacterium Vibrio cholerae is no exception and harbors 52 RR genes. Using in-frame deletion mutants of each RR gene, we performed a systematic analysis of their role in V. cholerae biofilm formation. We determined that 7 RRs impacted the expression of an essential biofilm gene and found that the recently characterized RR, VxrB, regulates the expression of key structural and regulatory biofilm genes in V. cholerae. vxrB is part of a 5-gene operon, which contains the cognate HK vxrA and three genes of unknown function. Strains carrying ΔvxrA and ΔvxrB mutations are deficient in biofilm formation, while the ΔvxrC mutation enhances biofilm formation. The overexpression of VxrB led to a decrease in motility. We also observed a small but reproducible effect of the absence of VxrB on the levels of cyclic di-GMP (c-di-GMP). Our work reveals a new function for the Vxr TCS as a regulator of biofilm formation and suggests that this regulation may act through key biofilm regulators and the modulation of cellular c-di-GMP levels. IMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, providing protection from environmental stresses and contributing to the transmission of V. cholerae to the human host. V. cholerae can utilize two-component systems (TCS), composed of a histidine kinase (HK) and a response regulator (RR), to regulate biofilm formation in response to external cues. We performed a systematic analysis of V. cholerae RRs and identified a new regulator of biofilm formation, VxrB. We demonstrated that the VxrAB TCS is essential for robust biofilm formation and that this system may regulate biofilm formation via its regulation of key biofilm regulators and cyclic di-GMP levels. This research furthers our understanding of the role that TCSs play in the regulation of V. cholerae biofilm formation.

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Vibrio cholerae LeuO Links the ToxR Regulon to Expression of Lipid A Remodeling Genes

Infection and Immunity ◽

10.1128/iai.00445-16 ◽

2016 ◽

Vol 84 (11) ◽

pp. 3161-3171 ◽

Cited By ~ 12

Author(s):

X. Renee Bina ◽

Mondraya F. Howard ◽

Vanessa M. Ante ◽

James E. Bina

Keyword(s):

Transcription Factor ◽

Vibrio Cholerae ◽

Bile Salts ◽

Lipid A ◽

Diarrheal Disease ◽

Regulatory System ◽

Polymyxin B ◽

Environmental Cues ◽

Cationic Antimicrobial Peptide ◽

Content Type

Vibrio choleraeis an intestinal pathogen that causes the diarrheal disease cholera. Colonization of the intestine depends upon the expression of genes that allowV. choleraeto overcome host barriers, including low pH, bile acids, and the innate immune system. ToxR is a major contributor to this process. ToxR is a membrane-spanning transcription factor that coordinates gene expression in response to environmental cues. In previous work we showed that ToxR upregulatedleuOexpression in response to bile salts. LeuO is a LysR family transcription factor that contributes to acid tolerance, bile resistance, and biofilm formation inV. cholerae. Here, we investigated the function of ToxR and LeuO in cationic antimicrobial peptide (CAMP) resistance. We report that ToxR and LeuO contribute to CAMP resistance by regulatingcarRStranscription. CarRS is a two-component regulatory system that positively regulatesalmEFGexpression. AlmEFG confers CAMP resistance by glycinylation of lipid A. We found that the expression ofcarRSandalmEFGand the polymyxin B MIC increased in mutants lackingtoxRSorleuO. Conversely,leuOoverexpression decreased the polymyxin B MIC. Furthermore, we found that LeuO directly bound to thecarRSpromoter and that ToxR-dependent activation ofleuOtranscription regulatedcarRStranscription in response to bile salts. Our results suggest that LeuO functions downstream of ToxR to modulatecarRSexpression in response to environmental cues. This study extends the functional role of ToxR and LeuO in environmental adaptation to include cell surface remodeling and CAMP resistance.

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Identification of a Second Two-Component Signal Transduction System That Controls Fosfomycin Tolerance and Glycerol-3-Phosphate Uptake

Journal of Bacteriology ◽

10.1128/jb.02491-14 ◽

2014 ◽

Vol 197 (5) ◽

pp. 861-871 ◽

Cited By ~ 4

Author(s):

Kumiko Kurabayashi ◽

Yuko Hirakawa ◽

Koichi Tanimoto ◽

Haruyoshi Tomita ◽

Hidetada Hirakawa

Keyword(s):

Phosphate Uptake ◽

Response Regulator ◽

Anaerobic Respiration ◽

Regulatory System ◽

Carbon Substrate ◽

Two Component System ◽

Component System ◽

Content Type ◽

Two Component ◽

Biological Cost

Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagicEscherichia coli(EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression oftorR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression ofglpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens.

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Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

Journal of Bacteriology ◽

10.1128/jb.00023-10 ◽

2010 ◽

Vol 192 (8) ◽

pp. 2044-2052 ◽

Cited By ~ 31

Author(s):

Jyl S. Matson ◽

Hyun Ju Yoo ◽

Kristina Hakansson ◽

Victor J. DiRita

Keyword(s):

Antimicrobial Peptides ◽

Vibrio Cholerae ◽

Lipid A ◽

Polymyxin B ◽

Mass Spectrometry Analysis ◽

Peptide Antibiotic ◽

Wild Type ◽

Spectrometry Analysis ◽

Antimicrobial Peptide Resistance ◽

El Tor

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.

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Hik36–Hik43 and Rre6 act as a two-component regulatory system to control cell aggregation in Synechocystis sp. PCC6803

Scientific Reports ◽

10.1038/s41598-020-76264-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kota Kera ◽

Yuichiro Yoshizawa ◽

Takehiro Shigehara ◽

Tatsuya Nagayama ◽

Masaru Tsujii ◽

...

Keyword(s):

Biofilm Formation ◽

Morphological Changes ◽

Response Regulator ◽

Control Cell ◽

Regulatory System ◽

Cell Aggregation ◽

Wild Type ◽

Type Iv ◽

Two Component ◽

In The Wild

Abstract In response to environmental stress the model cyanobacterium, Synechocystis sp. PCC6803 can switch from a planktonic state to autoaggregation and biofilm formation. The precise mechanism of this transition remains unknown. Here we investigated the role of a candidate two-component regulatory system (TCS) in controlling morphological changes, as a way to understand the intermediate molecular steps that are part of the signaling pathway. A bacterial two-hybrid assay showed that the response regulator Rre6 formed a TCS together with a split histidine kinase consisting of Hik36 and Hik43. Individual disruption mutants displayed autoaggregation in a static culture. In contrast, unlike in the wild type, high salinity did not induce biofilm formation in Δhik36, Δhik43 and Δrre6. The expression levels of exopolysaccharide (EPS) production genes were higher in Δhik36 and Δhik43, compared with the wild type, but lower in Δrre6, suggesting that the TCS regulated EPS production in Synechocystis. Rre6 interacted physically with the motor protein PilT2, that is a component of the type IV pilus system. This interaction was enhanced in a phosphomimic version of Rre6. Taken together, Hik36–Hik43–Rre6 function as an upstream component of the pili-related signal transduction cascade and control the prevention of cell adhesion and biofilm formation.

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A Conserved Two-Component Signal Transduction System Controls the Response to Phosphate Starvation in Bifidobacterium breve UCC2003

Applied and Environmental Microbiology ◽

10.1128/aem.00804-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5258-5269 ◽

Cited By ~ 14

Author(s):

Pablo Alvarez-Martin ◽

Matilde Fernández ◽

Mary O'Connell-Motherway ◽

Kerry Joan O'Connell ◽

Nicolas Sauvageot ◽

...

Keyword(s):

Response Regulator ◽

Regulatory Protein ◽

Gc Content ◽

Regulatory System ◽

Bioinformatic Analysis ◽

Insertion Mutant ◽

Recognition Sequence ◽

Content Type ◽

Bifidobacterium Breve ◽

Two Component

ABSTRACTThis work reports on the identification and molecular characterization of the two-component regulatory system (2CRS) PhoRP, which controls the response to inorganic phosphate (Pi) starvation inBifidobacterium breveUCC2003. The response regulator PhoP was shown to bind to the promoter region ofpstSCAB, specifying a predicted Pitransporter system, as well as that ofphoU, which encodes a putative Pi-responsive regulatory protein. This interaction is assumed to cause transcriptional modulation under conditions of Pilimitation. Our data suggest that thephoRPgenes are subject to positive autoregulation and, together withpstSCABand presumablyphoU, represent the complete regulon controlled by thephoRP-encoded 2CRS inB. breveUCC2003. Determination of the minimal PhoP binding region combined with bioinformatic analysis revealed the probable recognition sequence of PhoP, designated here as the PHO box, which together withphoRPis conserved among many high-GC-content Gram-positive bacteria. The importance of thephoRP2CRS in the response ofB. breveto Pistarvation conditions was confirmed by analysis of aB. brevephoPinsertion mutant which exhibited decreased growth under phosphate-limiting conditions compared to its parent strain UCC2003.

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A Two-Component Regulatory System Controls Autoregulated Serpin Expression in Bifidobacterium breve UCC2003

Applied and Environmental Microbiology ◽

10.1128/aem.01776-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 7032-7041 ◽

Cited By ~ 19

Author(s):

Pablo Alvarez-Martin ◽

Mary O'Connell Motherway ◽

Francesca Turroni ◽

Elena Foroni ◽

Marco Ventura ◽

...

Keyword(s):

Response Regulator ◽

Regulatory System ◽

Site Directed Mutagenesis ◽

Insertion Mutant ◽

Recognition Sequence ◽

Mobility Shift ◽

Content Type ◽

Bifidobacterium Breve ◽

Two Component

ABSTRACTThis work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded byserRK, which is believed to control the expression of theser2003locus inBifidobacterium breveUCC2003. Theser2003locus consists of two genes, Bbr_1319 (sagA) and Bbr_1320 (serU), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region ofser2003, and the probable recognition sequence of SerR was determined by a combinatorial approach ofin vitrosite-directed mutagenesis coupled to transcriptional fusion and electrophoretic mobility shift assays (EMSAs). The importance of theserRK2CRS in the response ofB. breveto protease-mediated induction was confirmed by generating aB. breve serRinsertion mutant, which was shown to exhibit alteredser2003transcriptional induction patterns compared to the parent strain, UCC2003. Interestingly, the analysis of aB. breve serUmutant revealed that the SerRK signaling pathway appears to include a SerU-dependent autoregulatory loop.

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Characterization of a Four-Component Regulatory System Controlling Bacteriocin Production in Streptococcus gallolyticus

mBio ◽

10.1128/mbio.03187-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Alexis Proutière ◽

Laurence du Merle ◽

Bruno Périchon ◽

Hugo Varet ◽

Myriam Gominet ◽

...

Keyword(s):

Response Regulator ◽

Regulatory System ◽

Opportunistic Pathogen ◽

Bacteriocin Production ◽

Two Component System ◽

Promoter Regions ◽

Component System ◽

Content Type ◽

Streptococcus Gallolyticus ◽

Two Component

ABSTRACT Bacteriocins are natural antimicrobial peptides produced by bacteria to kill closely related competitors. The opportunistic pathogen Streptococcus gallolyticus subsp. gallolyticus was recently shown to outcompete commensal enterococci of the murine microbiota under tumoral conditions thanks to the production of a two-peptide bacteriocin named gallocin. Here, we identified four genes involved in the regulatory control of gallocin in S. gallolyticus subsp. gallolyticus UCN34 that encode a histidine kinase/response regulator two-component system (BlpH/BlpR), a secreted peptide (GSP [gallocin-stimulating peptide]), and a putative regulator of unknown function (BlpS). While BlpR is a typical 243-amino-acid (aa) response regulator possessing a phospho-receiver domain and a LytTR DNA-binding domain, BlpS is a 108-aa protein containing only a LytTR domain. Our results showed that the secreted peptide GSP activates the dedicated two-component system BlpH/BlpR to induce gallocin transcription. A genome-wide transcriptome analysis indicates that this regulatory system (GSP-BlpH/BlpR) is specific for bacteriocin production. Importantly, as opposed to BlpR, BlpS was shown to repress gallocin gene transcription. A conserved operator DNA sequence of 30 bp was found in all promoter regions regulated by BlpR and BlpS. Electrophoretic mobility shift assays (EMSA) and footprint assays showed direct and specific binding of BlpS and BlpR to various regulated promoter regions in a dose-dependent manner on this conserved sequence. Gallocin expression appears to be tightly controlled in S. gallolyticus subsp. gallolyticus by quorum sensing and antagonistic activity of 2 LytTR-containing proteins. Competition experiments in gut microbiota medium and 5% CO2 to mimic intestinal conditions demonstrate that gallocin is functional under these in vivo-like conditions. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus, formerly known as Streptococcus bovis biotype I, is an opportunistic pathogen causing septicemia and endocarditis in the elderly often associated with asymptomatic colonic neoplasia. Recent studies indicate that S. gallolyticus subsp. gallolyticus is both a driver and a passenger of colorectal cancer. We previously showed that S. gallolyticus subsp. gallolyticus produces a bacteriocin, termed gallocin, enabling colonization of the colon under tumoral conditions by outcompeting commensal members of the murine microbiota such as Enterococcus faecalis. Here, we identified and extensively characterized a four-component system that regulates gallocin production. Gallocin gene transcription is activated by a secreted peptide pheromone (GSP) and a two-component signal transduction system composed of a transmembrane histidine kinase receptor (BlpH) and a cytosolic response regulator (BlpR). Finally, a DNA-binding protein (BlpS) was found to repress gallocin genes transcription, likely by antagonizing BlpR. Understanding gallocin regulation is crucial to prevent S. gallolyticus subsp. gallolyticus colon colonization under tumoral conditions.

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The Vancomycin Resistance-Associated Regulatory System VraSR Modulates Biofilm Formation of Staphylococcus epidermidis in an ica -Dependent Manner

mSphere ◽

10.1128/msphere.00641-21 ◽

2021 ◽

Author(s):

Youcong Wu ◽

Yuanyuan Meng ◽

Lian Qian ◽

Baixing Ding ◽

Haiyan Han ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

Regulatory System ◽

Vancomycin Resistance ◽

Dependent Manner ◽

Content Type ◽

Two Component ◽

Complex Procedure ◽

Hospital Acquired

S. epidermidis is a leading cause of hospital-acquired catheter-related infections, and its pathogenicity depends mostly on its ability to form biofilms on implants. The biofilm formation is a complex procedure that involves multiple regulating factors. Here, we show that a vancomycin resistance-associated two-component regulatory system, VraSR, plays an important role in modulating S. epidermidis biofilm formation and tolerance to stress.

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ResDE Two-Component Regulatory System Mediates Oxygen Limitation-Induced Biofilm Formation byBacillus amyloliquefaciensSQR9

Applied and Environmental Microbiology ◽

10.1128/aem.02744-17 ◽

2018 ◽

Vol 84 (8) ◽

pp. e02744-17 ◽

Cited By ~ 4

Author(s):

Xuan Zhou ◽

Nan Zhang ◽

Liming Xia ◽

Qing Li ◽

Jiahui Shao ◽

...

Keyword(s):

Plant Growth ◽

Biofilm Formation ◽

Root Colonization ◽

Oxygen Deficiency ◽

Regulatory System ◽

Regulatory Function ◽

Content Type ◽

Environmental Signals ◽

Link Type ◽

Two Component

ABSTRACTEfficient biofilm formation and root colonization capabilities facilitate the ability of beneficial plant rhizobacteria to promote plant growth and antagonize soilborne pathogens. Biofilm formation by plant-beneficialBacillusstrains is triggered by environmental cues, including oxygen deficiency, but the pathways that sense these environmental signals and regulate biofilm formation have not been thoroughly elucidated. In this study, we showed that the ResDE two-component regulatory system in the plant growth-promoting rhizobacteriumBacillus amyloliquefaciensstrain SQR9 senses the oxygen deficiency signal and regulates biofilm formation. ResE is activated by sensing the oxygen limitation-induced reduction of the NAD+/NADH pool through its PAS domain, stimulating its kinase activity, and resulting in the transfer of a phosphoryl group to ResD. The phosphorylated ResD directly binds to the promoter regions of theqoxABCDandctaCDEFoperons to improve the biosynthesis of terminal oxidases, which can interact with KinB to activate biofilm formation. These results not only revealed the novel regulatory function of the ResDE two-component system but also contributed to the understanding of the complicated regulatory network governingBacillusbiofilm formation. This research may help to enhance the root colonization and the plant-beneficial efficiency of SQR9 and otherBacillusrhizobacteria used in agriculture.IMPORTANCEBacillusspp. are widely used as bioinoculants for plant growth promotion and disease suppression. The exertion of their plant-beneficial functions is largely dependent on their root colonization, which is closely related to their biofilm formation capabilities. On the other hand,Bacillusis the model bacterium for biofilm study, and the process and molecular network of biofilm formation are well characterized (B. Mielich-Süss and D. Lopez, Environ Microbiol 17:555–565, 2015,https://doi.org/10.1111/1462-2920.12527; L. S. Cairns, L. Hobley, and N. R. Stanley-Wall, Mol Microbiol 93:587–598, 2014,https://doi.org/10.1111/mmi.12697; H. Vlamakis, C. Aguilar, R. Losick, and R. Kolter, Genes Dev 22:945–953, 2008,https://doi.org/10.1101/gad.1645008; S. S. Branda, A. Vik, L. Friedman, and R. Kolter, Trends Microbiol 13:20–26, 2005,https://doi.org/10.1016/j.tim.2004.11.006; C. Aguilar, H. Vlamakis, R. Losick, and R. Kolter, Curr Opin Microbiol 10:638–643, 2007,https://doi.org/10.1016/j.mib.2007.09.006; S. S. Branda, J. E. González-Pastor, S. Ben-Yehuda, R. Losick, and R. Kolter, Proc Natl Acad Sci U S A 98:11621–11626, 2001,https://doi.org/10.1073/pnas.191384198). However, the identification and sensing of environmental signals triggeringBacillusbiofilm formation need further research. Here, we report that the oxygen deficiency signal inducingBacillusbiofilm formation is sensed by the ResDE two-component regulatory system. Our results not only revealed the novel regulatory function of the ResDE two-component regulatory system but also identified the sensing system of a biofilm-triggering signal. This knowledge can help to enhance the biofilm formation and root colonization of plant-beneficialBacillusstrains and also provide new insights of bacterial biofilm formation regulation.

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