scholarly journals VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae

2020 ◽  
Vol 202 (18) ◽  
Author(s):  
Meng-Lun Hsieh ◽  
Christopher M. Waters ◽  
Deborah M. Hinton
Keyword(s):  
Rna Polymerase ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Core Promoter ◽  
Class Ii ◽  
Class I ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Content Type

ABSTRACT Vibrio cholerae biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the Vibrio polysaccharides (vps) operons I and II as well as the cluster of ribomatrix (rbm) genes. Transcriptional control of these genes is a complex process that requires several activators/repressors and the ubiquitous signaling molecule, cyclic di-GMP (c-di-GMP). Previously, we demonstrated that VpsR directly activates RNA polymerase containing σ70 (σ70-RNAP) at the vpsL promoter (PvpsL), which precedes the vps-II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex. Using in vitro transcription, electrophoretic mobility shift assays, and DNase I footprinting, we show here that VpsR also directly activates σ70-RNAP transcription from other promoters within the biofilm formation cluster, including PvpsU, at the beginning of the vps-I operon, PrbmA, at the start of the rbm cluster, and PrbmF, which lies upstream of the divergent rbmF and rbmE genes. In this capacity, we find that VpsR is able to behave both as a class II activator, which functions immediately adjacent/overlapping the core promoter sequence (PvpsL and PvpsU), and as a class I activator, which functions farther upstream (PrbmA and PrbmF). Because these promoters vary in VpsR-DNA binding affinity in the absence and presence of c-di-GMP, we speculate that VpsR’s mechanism of activation is dependent on both the concentration of VpsR and the level of c-di-GMP to increase transcription, resulting in finely tuned regulation. IMPORTANCE Vibrio cholerae, the bacterial pathogen that is responsible for the disease cholera, uses biofilms to aid in survival, dissemination, and persistence. VpsR, which directly senses the second messenger c-di-GMP, is a major regulator of this process. Together with c-di-GMP, VpsR directly activates transcription by RNA polymerase containing σ70 from the vpsL biofilm biogenesis promoter. Using biochemical methods, we demonstrate for the first time that VpsR/c-di-GMP directly activates σ70-RNA polymerase at the first genes of the vps and ribomatrix operons. In this regard, it functions as either a class I or class II activator. Our results broaden the mechanism of c-di-GMP-dependent transcription activation and the specific role of VpsR in biofilm formation.

scholarly journals In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

2011 ◽  
Vol 56 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Marisa H. Miceli ◽  
Stella M. Bernardo ◽  
T. S. Neil Ku ◽  
Carla Walraven ◽  
Samuel A. Lee
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Biofilm Formation ◽  
High Dose ◽  
Dependent Manner ◽  
Planktonic Cells ◽  
Content Type ◽  
Heparin Sodium ◽  
The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

scholarly journals Cyclic di-GMP Positively Regulates DNA Repair in Vibrio cholerae

2018 ◽  
Vol 200 (15) ◽  
Author(s):  
Nicolas L. Fernandez ◽  
Disha Srivastava ◽  
Amanda L. Ngouajio ◽  
Christopher M. Waters
Keyword(s):  
Dna Repair ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Binding Assays ◽  
Gene Encoding ◽  
Repair Gene ◽  
Content Type ◽  
Dna Repair Gene ◽  
High Concentrations

ABSTRACT In Vibrio cholerae, high intracellular cyclic di-GMP (c-di-GMP) concentration are associated with a biofilm lifestyle, while low intracellular c-di-GMP concentrations are associated with a motile lifestyle. c-di-GMP also regulates other behaviors, such as acetoin production and type II secretion; however, the extent of phenotypes regulated by c-di-GMP is not fully understood. We recently determined that the sequence upstream of the DNA repair gene encoding 3-methyladenine glycosylase (tag) was positively induced by c-di-GMP, suggesting that this signaling system might impact DNA repair pathways. We identified a DNA region upstream of tag that is required for transcriptional induction by c-di-GMP. We further showed that c-di-GMP induction of tag expression was dependent on the c-di-GMP-dependent biofilm regulators VpsT and VpsR. In vitro binding assays and heterologous host expression studies show that VpsT acts directly at the tag promoter in response to c-di-GMP to induce tag expression. Last, we determined that strains with high c-di-GMP concentrations are more tolerant of the DNA-damaging agent methyl methanesulfonate. Our results indicate that the regulatory network of c-di-GMP in V. cholerae extends beyond biofilm formation and motility to regulate DNA repair through the VpsR/VpsT c-di-GMP-dependent cascade. IMPORTANCE Vibrio cholerae is a prominent human pathogen that is currently causing a pandemic outbreak in Haiti, Yemen, and Ethiopia. The second messenger molecule cyclic di-GMP (c-di-GMP) mediates the transitions in V. cholerae between a sessile biofilm-forming state and a motile lifestyle, both of which are important during V. cholerae environmental persistence and human infections. Here, we report that in V. cholerae c-di-GMP also controls DNA repair. We elucidate the regulatory pathway by which c-di-GMP increases DNA repair, allowing this bacterium to tolerate high concentrations of mutagens at high intracellular levels of c-di-GMP. Our work suggests that DNA repair and biofilm formation may be linked in V. cholerae.

scholarly journals A MarR-Type Regulator Directly Activates Transcription from the Brucella abortus virB Promoter by Sharing a Redundant Role with HutC

2012 ◽  
Vol 194 (23) ◽  
pp. 6431-6440 ◽  
Author(s):  
Rodrigo Sieira ◽  
Gastón M. Arocena ◽  
Angeles Zorreguieta ◽  
Diego J. Comerci ◽  
Rodolfo A. Ugalde
Keyword(s):  
Transcription Factors ◽  
Dna Sequences ◽  
Cell Envelope ◽  
Regulatory Element ◽  
Dependent Manner ◽  
Secretion Systems ◽  
Mobility Shift ◽  
Content Type ◽  
Environmental Signals

ABSTRACTType IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genusBrucellaessentially depends on the integrity of the T4SS-encodingvirBoperon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of thevirBgenes that were isolated from total protein extracts ofBrucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of thevirBpromoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNAin vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activatedvirBexpression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of thevirBpromoter and is probably involved in coordinating gene expression in response to specific environmental signals.

scholarly journals Determinants of the C-Terminal Domain of the Escherichia coli RNA Polymerase α Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters

2002 ◽  
Vol 184 (8) ◽  
pp. 2273-2280 ◽  
Author(s):  
Nigel J. Savery ◽  
Georgina S. Lloyd ◽  
Stephen J. W. Busby ◽  
Mark S. Thomas ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class Ii ◽  
Class I ◽  
Receptor Protein ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Terminal Domain

ABSTRACT Alanine scanning of the Escherichia coli RNA polymerase α subunit C-terminal domain (αCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of αCTD affected class I CRP-dependent transcription from the CC(−61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for αCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in αCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.

scholarly journals Cyclic di-GMP Increases Catalase Production and Hydrogen Peroxide Tolerance in Vibrio cholerae

2019 ◽  
Vol 85 (18) ◽  
Author(s):  
Nicolas L. Fernandez ◽  
Christopher M. Waters
Keyword(s):  
Vibrio Cholerae ◽  
Catalase Activity ◽  
Biofilm Formation ◽  
Salt Water ◽  
Bacterial Pathogen ◽  
Second Messenger ◽  
Signaling Network ◽  
Dependent Manner ◽  
Environmental Persistence ◽  
Content Type

ABSTRACT Vibrio cholerae is a Gram-negative bacterial pathogen that causes the disease cholera, which affects nearly 1 million people each year. In between outbreaks, V. cholerae resides in fresh and salt water environments, where it is able to persist through changes in temperature, oxygen, and salinity. One key characteristic that promotes environmental persistence of V. cholerae is the ability to form multicellular communities, called biofilms, that often adhere to biotic and abiotic sources. Biofilm formation in V. cholerae is positively regulated by the dinucleotide second messenger cyclic dimeric GMP (c-di-GMP). While most research on the c-di-GMP regulon has focused on biofilm formation or motility, we hypothesized that the c-di-GMP signaling network encompassed a larger set of effector functions than reported. We found that high intracellular c-di-GMP increased catalase activity ∼4-fold relative to strains with unaltered c-di-GMP. Genetic studies demonstrated that c-di-GMP mediated catalase activity was due to increased expression of the catalase-encoding gene katB. Moreover, c-di-GMP mediated regulation of catalase activity and katB expression required the c-di-GMP dependent transcription factors VpsT and VpsR. Lastly, we found that high c-di-GMP increased survival after H2O2 challenge in a katB-, vpsR-, and vpsT-dependent manner. Our results indicate that antioxidant production is regulated by c-di-GMP uncovering a new node in the growing VpsT and VpsR c-di-GMP signaling network of V. cholerae. IMPORTANCE As a result of infection with V. cholerae, patients become dehydrated, leading to death if not properly treated. The aquatic environment is the natural reservoir for V. cholerae, where it can survive alterations in temperature, salinity, and oxygen. The second messenger molecule c-di-GMP is an important signal regulating host and aquatic environmental persistence because it controls whether V. cholerae will form a biofilm or disperse through flagellar motility. In this work, we demonstrate another function of c-di-GMP in V. cholerae biology: promoting tolerance to the reactive oxygen species H2O2 through the differential regulation of catalase expression. Our results suggest a mechanism where c-di-GMP simultaneously controls biofilm formation and antioxidant production, which could promote persistence in human and marine environments.

scholarly journals Diguanylate Cyclases AdrA and STM1987 Regulate Salmonella enterica Exopolysaccharide Production during Plant Colonization in an Environment-Dependent Manner

2015 ◽  
Vol 82 (4) ◽  
pp. 1237-1248 ◽  
Author(s):  
Kimberly N. Cowles ◽  
David K. Willis ◽  
Tyler N. Engel ◽  
Jeffrey B. Jones ◽  
Jeri D. Barak
Keyword(s):  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Plant Colonization ◽  
Dependent Manner ◽  
Cellulose Production ◽  
Content Type ◽  
Colanic Acid ◽  
Diguanylate Cyclases ◽  
Colonization Factors

ABSTRACTIncreasing evidence indicates that despite exposure to harsh environmental stresses,Salmonella entericasuccessfully persists on plants, utilizing fresh produce as a vector to animal hosts. Among the importantS. entericaplant colonization factors are those involved in biofilm formation.S. entericabiofilm formation is controlled by the signaling molecule cyclic di-GMP and represents a sessile lifestyle on surfaces that protects the bacterium from environmental factors. Thus, the transition from a motile, planktonic lifestyle to a sessile lifestyle may represent a vital step in bacterial success. This study examined the mechanisms ofS. entericaplant colonization, including the role of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), the enzymes involved in cyclic di-GMP metabolism. We found that two biofilm components, cellulose and curli, are differentially required at distinct stages in root colonization and that the DGC STM1987 regulates cellulose production in this environment independent of AdrA, the DGC that controls the majority ofin vitrocellulose production. In addition, we identified a new function for AdrA in the transcriptional regulation of colanic acid and demonstrated thatadrAand colanic acid biosynthesis are associated withS. entericadesiccation tolerance on the leaf surface. Finally, two PDEs with known roles in motility, STM1344 and STM1697, had competitive defects in the phyllosphere, suggesting that regulation of motility is crucial forS. entericasurvival in this niche. Our results indicate that specific conditions influence the contribution of individual DGCs and PDEs to bacterial success, perhaps reflective of differential responses to environmental stimuli.

scholarly journals Staphylococcus epidermidis SrrAB Regulates Bacterial Growth and Biofilm Formation Differently under Oxic and Microaerobic Conditions

2014 ◽  
Vol 197 (3) ◽  
pp. 459-476 ◽  
Author(s):  
Youcong Wu ◽  
Yang Wu ◽  
Tao Zhu ◽  
Haiyan Han ◽  
Huayong Liu ◽  
...  
Keyword(s):  
Electron Transport ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Electron Transport Chain ◽  
Regulatory Function ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Content Type ◽  
Transport Chain ◽  
Microaerobic Conditions

SrrAB expression inStaphylococcus epidermidisstrain 1457 (SE1457) was upregulated during a shift from oxic to microaerobic conditions. AnsrrAdeletion (ΔsrrA) mutant was constructed for studying the regulatory function of SrrAB. The deletion resulted in retarded growth and abolished biofilm formation bothin vitroandin vivoand under both oxic and microaerobic conditions. Associated with the reduced biofilm formation, the ΔsrrAmutant produced much less polysaccharide intercellular adhesion (PIA) and showed decreased initial adherence capacity. Microarray analysis showed that thesrrAmutation affected transcription of 230 genes under microaerobic conditions, and 51 genes under oxic conditions. Quantitative real-time PCR confirmed this observation and showed downregulation of genes involved in maintaining the electron transport chain by supporting cytochrome and quinol-oxidase assembly (e.g.,qoxBandctaA) and in anaerobic metabolism (e.g.,pflBAandnrdD). In the ΔsrrAmutant, the expression of the biofilm formation-related geneicaRwas upregulated under oxic conditions and downregulated under microaerobic conditions, whereasicaAwas downregulated under both conditions. An electrophoretic mobility shift assay further revealed that phosphorylated SrrA bound to the promoter regions oficaR,icaA,qoxB, andpflBA, as well as its own promoter region. These findings demonstrate that inS. epidermidisSrrAB is an autoregulator and regulates biofilm formation in anica-dependent manner. Under oxic conditions, SrrAB modulates electron transport chain activity by positively regulatingqoxBACDtranscription. Under microaerobic conditions, it regulates fermentation processes and DNA synthesis by modulating the expression of both thepfloperon andnrdDG.

scholarly journals Identification and Characterization of VpsR and VpsT Binding Sites in Vibrio cholerae

2015 ◽  
Vol 197 (7) ◽  
pp. 1221-1235 ◽  
Author(s):  
David Zamorano-Sánchez ◽  
Jiunn C. N. Fong ◽  
Sefa Kilic ◽  
Ivan Erill ◽  
Fitnat H. Yildiz
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Structural Features ◽  
Content Type ◽  
Environmental Survival ◽  
Proximal Site ◽  
Target Sequences

ABSTRACTThe ability to form biofilms is critical for environmental survival and transmission ofVibrio 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 positive regulators of biofilm formation, VpsR and VpsT, bind to nonoverlapping target sequences in the regulatory region ofvpsL 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 ofvpsL. 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 invpsLexpression, deletion of the T and R2 boxes resulted in an increase invpsLexpression. Analysis of the role of H-NS invpsLexpression revealed that deletion ofhnsresulted in enhancedvpsLexpression. The level ofvpsLexpression was higher in anhns vpsTdouble mutant than in the parental strain but lower than that in anhnsmutant.In silicoanalysis 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 andvpsTare coregulated by VpsR and VpsT. Furthermore, a comparative genomics analysis revealed substantial variability in the promoter region of thevpsTandvpsLgenes among extantV. choleraeisolates, suggesting that regulation of biofilm formation is under active selection.IMPORTANCEVibrio choleraecauses cholera and is a natural inhabitant of aquatic environments. One critical factor that is important for environmental survival and transmission ofV. choleraeis the microbe's ability to form biofilms, which are surface-associated communities encased in a matrix composed of the exopolysaccharide VPS (Vibriopolysaccharide), proteins, and nucleic acids. Two proteins, VpsR and VpsT, positively regulate VPS production and biofilm formation. We characterized the structural features of the promoter of thevpsLgene, determined the target sequences recognized by VpsT and VpsR, and analyzed their distribution and conservation patterns in multipleV. choleraeisolates. This work fills a fundamental gap in our understanding of the regulatory mechanisms employed by the master regulators VpsR and VpsT in controlling biofilm matrix production.

scholarly journals Positive Autoregulation ofmrkHIby the Cyclic Di-GMP-Dependent MrkH Protein in the Biofilm Regulatory Circuit of Klebsiella pneumoniae

2015 ◽  
Vol 197 (9) ◽  
pp. 1659-1667 ◽  
Author(s):  
Jason W. H. Tan ◽  
Jonathan J. Wilksch ◽  
Dianna M. Hocking ◽  
Nancy Wang ◽  
Yogitha N. Srikhanta ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Rna Polymerase ◽  
Dna Sequence ◽  
Biofilm Formation ◽  
Transcriptional Activator ◽  
Dependent Manner ◽  
Α Subunit ◽  
Content Type ◽  
Regulatory Circuit ◽  
Type 3

ABSTRACTKlebsiella pneumoniaeis an important cause of nosocomial infections, primarily through the formation of surface-associated biofilms to promote microbial colonization on host tissues. Expression of type 3 fimbriae byK. pneumoniaefacilitates surface adherence, a process strongly activated by the cyclic di-GMP (c-di-GMP)-dependent transcriptional activator MrkH. In this study, we demonstrated the critical importance of MrkH in facilitatingK. pneumoniaeattachment on a variety of medically relevant materials and demonstrated the mechanism by which bacteria activate expression of type 3 fimbriae to colonize these materials. Sequence analysis revealed a putative MrkH recognition DNA sequence (“MrkH box”; TATCAA) located in the regulatory region of themrkHIoperon. Mutational analysis, electrophoretic mobility shift assay, and quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA sequence to autoregulatemrkHIexpression in a c-di-GMP-dependent manner. A half-turn deletion, but not a full-turn deletion, between the MrkH box and the −35 promoter element rendered MrkH ineffective in activatingmrkHIexpression, implying that a direct interaction between MrkH and RNA polymerase exists.In vivoanalyses showed that residues L260, R265, N268, C269, E273, and I275 in the C-terminal domain of the RNA polymerase α subunit are involved in the positive control ofmrkHIexpression by MrkH and revealed the regions of MrkH required for DNA binding and transcriptional activation. Taken together, the data suggest a model whereby c-di-GMP-dependent MrkH recruits RNA polymerase to themrkHIpromoter to autoactivatemrkHexpression. Increased MrkH production subsequently drivesmrkABCDFexpression when activated by c-di-GMP, leading to biosynthesis of type 3 fimbriae and biofilm formation.IMPORTANCEBacterial biofilms can cause persistent infections that are refractory to antimicrobial treatments. This study investigated how a commonly encountered hospital-acquired pathogen,Klebsiella pneumoniae, controls the expression of MrkH, the principal regulator of type 3 fimbriae and biofilm formation. We discovered a regulatory circuit whereby MrkH acts as a c-di-GMP-dependent transcriptional activator of both the gene cluster of type 3 fimbriae and themrkHIoperon. In this positive-feedback loop, whereby MrkH activates its own production,K. pneumoniaehas evolved a mechanism to ensure rapid MrkH production, expression of type 3 fimbriae, and subsequent biofilm formation under favorable conditions. Deciphering the molecular mechanisms of biofilm formation by bacterial pathogens is important for the development of innovative treatment strategies for biofilm infections.

scholarly journals Response of Vibrio cholerae to Low-Temperature Shifts: CspV Regulation of Type VI Secretion, Biofilm Formation, and Association with Zooplankton

2016 ◽  
Vol 82 (14) ◽  
pp. 4441-4452 ◽  
Author(s):  
Loni Townsley ◽  
Marilou P. Sison Mangus ◽  
Sanjin Mehic ◽  
Fitnat H. Yildiz
Keyword(s):  
Low Temperature ◽  
Vibrio Cholerae ◽  
Causative Agent ◽  
Biofilm Formation ◽  
Cold Shock ◽  
Diarrheal Disease ◽  
Dependent Manner ◽  
Natural Habitats ◽  
Content Type ◽  
Type Vi Secretion

ABSTRACTThe ability to sense and adapt to temperature fluctuation is critical to the aquatic survival, transmission, and infectivity ofVibrio cholerae, the causative agent of the disease cholera. Little information is available on the physiological changes that occur whenV. choleraeexperiences temperature shifts. The genome-wide transcriptional profile ofV. choleraeupon a shift in human body temperature (37°C) to lower temperatures, 15°C and 25°C, which mimic those found in the aquatic environment, was determined. Differentially expressed genes included those involved in the cold shock response, biofilm formation, type VI secretion, and virulence. Analysis of a mutant lacking the cold shock genecspV, which was upregulated >50-fold upon a low-temperature shift, revealed that it regulates genes involved in biofilm formation and type VI secretion. CspV controls biofilm formation through modulation of the second messenger cyclic diguanylate and regulates type VI-mediated interspecies killing in a temperature-dependent manner. Furthermore, a strain lackingcspVhad significant defects for attachment and type VI-mediated killing on the surface of the aquatic crustaceanDaphnia magna. Collectively, these studies reveal thatcspVis a major regulator of the temperature downshift response and plays an important role in controlling cellular processes crucial to the infectious cycle ofV. cholerae.IMPORTANCELittle is known about how human pathogens respond and adapt to ever-changing parameters of natural habitats outside the human host and how environmental adaptation alters dissemination.Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, experiences fluctuations in temperature in its natural aquatic habitats and during the infection process. Furthermore, temperature is a critical environmental signal governing the occurrence ofV. choleraeand cholera outbreaks. In this study, we showed thatV. choleraereprograms its transcriptome in response to fluctuations in temperature, which results in changes to biofilm formation and type VI secretion system activation. These processes in turn impact environmental survival and the virulence potential of this pathogen.

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