Interactions of the c-di-GMP riboswitch with its second messenger ligand

The c-di-GMP [bis-(3′–5′)-cyclic dimeric guanosine monophosphate] riboswitch is a macromolecular target in the c-di-GMP second messenger signalling pathway. It regulates many genes related to c-di-GMP metabolism as well as genes involved in bacterial motility, virulence and biofilm formation. The riboswitch makes asymmetric contacts to the bases and phosphate backbone of this symmetric dinucleotide. The phylogenetics suggested and mutagenesis has confirmed that this is a flexible motif where variants can make alternative interactions with each of the guanine bases of c-di-GMP. A mutant riboswitch has been designed that can bind a related molecule, c-di-AMP, confirming the most important contacts made to the ligand. The binding kinetics reveal that this is a kinetically controlled riboswitch and mutations to the riboswitch lead to increases in the off-rate. This riboswitch is therefore flexible in sequence as well as kinetic properties.

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Cyclic di-GMP Regulates TfoY inVibrio choleraeTo Control Motility by both Transcriptional and Posttranscriptional Mechanisms

Journal of Bacteriology ◽

10.1128/jb.00578-17 ◽

2018 ◽

Vol 200 (7) ◽

Cited By ~ 18

Author(s):

Benjamin R. Pursley ◽

Michael M. Maiden ◽

Meng-Lun Hsieh ◽

Nicolas L. Fernandez ◽

Geoffrey B. Severin ◽

...

Keyword(s):

Transcription Factor ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Noncoding Rna ◽

Molecular Mechanisms ◽

Second Messenger ◽

Bacterial Motility ◽

Content Type ◽

Intracellular Concentrations ◽

Messenger Molecule

ABSTRACT3′,5′-Cyclic diguanylic acid (c-di-GMP) is a bacterial second messenger molecule that is a key global regulator inVibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. One such regulatory factor that may respond to c-di-GMP is the Vc2 c-di-GMP-binding riboswitch that is hypothesized to control the expression of the downstream putative transcription factor TfoY. Although much is known about the physical and structural properties of the Vc2 riboswitch aptamer, the nature of its expression and function inV. choleraehas not been investigated. Here, we show that Vc2 functions as an off switch to inhibit TfoY production at intermediate and high concentrations of c-di-GMP. At low c-di-GMP concentrations, TfoY production is induced to stimulate dispersive motility. We also observed increased transcription oftfoYat high intracellular concentrations of c-di-GMP, but this induction is independent of the Vc2 riboswitch and occurs via transcriptional control of promoters upstream oftfoYby the previously identified c-di-GMP dependent transcription factor VpsR. Our results show that TfoY is induced by c-di-GMP at both low and high intracellular concentrations of c-di-GMP via posttranscriptional and transcriptional mechanisms, respectively. This regulation contributes to the formation of three distinct c-di-GMP signaling states inV. cholerae.IMPORTANCEThe bacterial pathogenVibrio choleraemust transition between life in aquatic environmental reservoirs and life in the gastrointestinal tract. Biofilm formation and bacterial motility, and their control by the second messenger molecule c-di-GMP, play integral roles in this adaptation. Here, we define the third major mechanism by which c-di-GMP controls bacterial motility. This pathway utilizes a noncoding RNA element known as a riboswitch that, when bound to c-di-GMP, inhibits the expression of the transcription factor TfoY. TfoY production switchesV. choleraemotility from a dense to a dispersive state. Our results suggest that the c-di-GMP signaling network ofV. choleraecan exist in at least three distinct states to regulate biofilm formation and motility.

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Cyclic di-GMP Increases Catalase Production and Hydrogen Peroxide Tolerance inVibrio cholerae

10.1101/631275 ◽

2019 ◽

Author(s):

Nicolas L. Fernandez ◽

Christopher M. Waters

Keyword(s):

Catalase Activity ◽

Biofilm Formation ◽

Salt Water ◽

Bacterial Pathogen ◽

Second Messenger ◽

Signaling Network ◽

Guanosine Monophosphate ◽

Dependent Manner ◽

Flagellar Motility ◽

Environmental Persistence

AbstractVibrio choleraeis a Gram-negative bacterial pathogen that causes the disease cholera, which affects nearly 1 million people each year. In between outbreaks,V. choleraeresides 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 ofV. choleraeis the ability to form multicellular communities, called biofilms, that often adhere to biotic and abiotic sources. Biofilm formation inV. choleraeis positively regulated by the dinucleotide second messenger cyclic dimeric guanosine monophosphate (c-di-GMP). While most research on the c-di-GMP regulon has focused on biofilm formation or motility, we hypothesized 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 approximately 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 genekatB. Moreover, c-di-GMP mediated regulation of catalase activity andkatBexpression required the c-di-GMP dependent transcription factors VpsT and VpsR. Lastly, we found that high c-di-GMP increased survival after H2O2challenge in akatB, vpsR, andvpsTdependent manner. Our results indicate antioxidant production is regulated by c-di-GMP inV. choleraeuncovering a new node in the growing VpsT and VpsR c-di-GMP signaling network.ImportanceAs a result of infection withV. cholerae, patients become dehydrated leading to death if not properly treated. The marine environment is the natural reservoir forV. choleraewhere it can survive alterations in temperature, salinity, and oxygen. The second messenger molecule c-di-GMP is an important signal regulating host and marine environmental persistence because it controls whetherV. choleraewill form a biofilm or disperse through flagellar motility. In this work, we demonstrate another function of c-di-GMP inV. choleraebiology: promoting tolerance to the reactive oxygen species H2O2through 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.

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Putrescine and its metabolic precursor arginine promote biofilm and c-di-GMP synthesis in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00297-21 ◽

2021 ◽

Author(s):

Zhexian Liu ◽

Sarzana S. Hossain ◽

Zayda Morales Moreira ◽

Cara H. Haney

Keyword(s):

Pseudomonas Aeruginosa ◽

Immune Responses ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Bacterial Pathogen ◽

Guanosine Monophosphate ◽

Genetic Approach ◽

Chronic Infections ◽

Host Immune Responses ◽

Primary Mechanism

Pseudomonas aeruginosa , an opportunistic bacterial pathogen can synthesize and catabolize a number of small cationic molecules known as polyamines. In several clades of bacteria polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, L-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or through a metabolic derivative. Here we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa . Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that L-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation, but via a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and L-arginine induced a significant increase in the intracellular level of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor arginine promote biofilm and c-di-GMP synthesis in P. aeruginosa . Importance: Biofilm formation allows bacteria to physically attach to a surface, confers tolerance to antimicrobial agents, and promotes resistance to host immune responses. As a result, regulation of biofilm is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switching that favors chronic infection.

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More than Enzymes That Make or Break Cyclic Di-GMP—Local Signaling in the Interactome of GGDEF/EAL Domain Proteins of Escherichia coli

mBio ◽

10.1128/mbio.01639-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 60

Author(s):

Olga Sarenko ◽

Gisela Klauck ◽

Franziska M. Wilke ◽

Vanessa Pfiffer ◽

Anja M. Richter ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Second Messenger ◽

Bacterial Biofilm ◽

Interaction Patterns ◽

Systematic Analysis ◽

E Coli ◽

Diguanylate Cyclases ◽

Hub Proteins ◽

Effector Target

ABSTRACT The bacterial second messenger bis-(3′-5′)-cyclic diguanosine monophosphate (c-di-GMP) ubiquitously promotes bacterial biofilm formation. Intracellular pools of c-di-GMP seem to be dynamically negotiated by diguanylate cyclases (DGCs, with GGDEF domains) and specific phosphodiesterases (PDEs, with EAL or HD-GYP domains). Most bacterial species possess multiple DGCs and PDEs, often with surprisingly distinct and specific output functions. One explanation for such specificity is “local” c-di-GMP signaling, which is believed to involve direct interactions between specific DGC/PDE pairs and c-di-GMP-binding effector/target systems. Here we present a systematic analysis of direct protein interactions among all 29 GGDEF/EAL domain proteins of Escherichia coli . Since the effects of interactions depend on coexpression and stoichiometries, cellular levels of all GGDEF/EAL domain proteins were also quantified and found to vary dynamically along the growth cycle. Instead of detecting specific pairs of interacting DGCs and PDEs, we discovered a tightly interconnected protein network of a specific subset or “supermodule” of DGCs and PDEs with a coregulated core of five hyperconnected hub proteins. These include the DGC/PDE proteins representing the c-di-GMP switch that turns on biofilm matrix production in E. coli . Mutants lacking these core hub proteins show drastic biofilm-related phenotypes but no changes in cellular c-di-GMP levels. Overall, our results provide the basis for a novel model of local c-di-GMP signaling in which a single strongly expressed master PDE, PdeH, dynamically eradicates global effects of several DGCs by strongly draining the global c-di-GMP pool and thereby restricting these DGCs to serving as local c-di-GMP sources that activate specific colocalized effector/target systems. IMPORTANCE c-di-GMP signaling in bacteria is believed to occur via changes in cellular c-di-GMP levels controlled by antagonistic and potentially interacting pairs of diguanylate cyclases (DGCs) and c-di-GMP phosphodiesterases (PDEs). Our systematic analysis of protein-protein interaction patterns of all 29 GGDEF/EAL domain proteins of E. coli , together with our measurements of cellular c-di-GMP levels, challenges both aspects of this current concept. Knocking out distinct DGCs and PDEs has drastic effects on E. coli biofilm formation without changing the cellular c-di-GMP level. In addition, rather than generally coming in interacting DGC/PDE pairs, a subset of DGCs and PDEs operates as central interaction hubs in a larger "supermodule," with other DGCs and PDEs behaving as “lonely players” without contacts to other c-di-GMP-related enzymes. On the basis of these data, we propose a novel concept of “local” c-di-GMP signaling in bacteria with multiple enzymes that make or break the second messenger c-di-GMP.

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Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.00795-15 ◽

2015 ◽

Vol 198 (3) ◽

pp. 521-535 ◽

Cited By ~ 28

Author(s):

Simon Schäper ◽

Elizaveta Krol ◽

Dorota Skotnicka ◽

Volkhard Kaever ◽

Rolf Hilker ◽

...

Keyword(s):

Biofilm Formation ◽

Sinorhizobium Meliloti ◽

Acid Stress ◽

Second Messenger ◽

Lifestyle Changes ◽

Single Domain ◽

Receptor Protein ◽

Leguminous Plant ◽

Content Type

ABSTRACTSinorhizobium melilotiundergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG inS. melilotiRm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation ofN-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of theS. melilotigenespleD,SMb20523,SMb20447,SMc01464, andSMc03178encoding putative DGCs and ofSMb21517encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, anS. melilotistrain that did not produce detectable levels of cdG (cdG0) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. TheSMc01790-SMc01796locus, homologous to theAgrobacterium tumefaciensuppABCDEFcluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility.IMPORTANCEWe present the first systematic genome-wide investigation of the role of 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of anS. melilotistrain unable to produce cdG (cdG0) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility ofS. melilotiand uncovered a single-domain PilZ protein as regulator of motility.

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A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP

Journal of Experimental Medicine ◽

10.1084/jem.20082874 ◽

2009 ◽

Vol 206 (9) ◽

pp. 1899-1911 ◽

Cited By ~ 202

Author(s):

Sarah M. McWhirter ◽

Roman Barbalat ◽

Kathryn M. Monroe ◽

Mary F. Fontana ◽

Mamoru Hyodo ◽

...

Keyword(s):

Mammalian Cells ◽

Map Kinases ◽

Second Messenger ◽

Innate Immune ◽

Guanosine Monophosphate ◽

Host Cells ◽

Type I Interferons ◽

Interferon Response ◽

Type I

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.

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Pumilacidin-Like Lipopeptides Derived from Marine Bacterium Bacillus sp. Strain 176 Suppress the Motility of Vibrio alginolyticus

Applied and Environmental Microbiology ◽

10.1128/aem.00450-17 ◽

2017 ◽

Vol 83 (12) ◽

Cited By ~ 21

Author(s):

Pengyuan Xiu ◽

Rui Liu ◽

Dechao Zhang ◽

Chaomin Sun

Keyword(s):

Cell Death ◽

Antibiotic Resistance ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Marine Bacterium ◽

Vibrio Alginolyticus ◽

Bacterial Motility ◽

Great Promise ◽

Content Type ◽

Cyclic Lipopeptides

ABSTRACT Bacterial motility is a crucial factor during the invasion and colonization processes of pathogens, which makes it an attractive therapeutic drug target. Here, we isolated a marine bacterium (Vibrio alginolyticus strain 178) from a seamount in the tropical West Pacific that exhibits vigorous motility on agar plates and severe pathogenicity to zebrafish. We found that V. alginolyticus 178 motility was significantly suppressed by another marine bacterium, Bacillus sp. strain 176, isolated from the same niche. We isolated, purified, and characterized two different cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The two related CLPs have a pumilacidin-like structure and were both effective inhibitors of V. alginolyticus 178 motility. The CLPs differ by only one methylene group in their fatty acid chains. In addition to motility suppression, the CLPs also induced cell aggregation in the medium and reduced adherence of V. alginolyticus 178 to glass substrates. Notably, upon CLP treatment, the expression levels of two V. alginolyticus flagellar assembly genes (flgA and flgP) dropped dramatically. Moreover, the CLPs inhibited biofilm formation in several other strains of pathogenic bacteria without inducing cell death. This study indicates that CLPs from Bacillus sp. 176 show promise as antimicrobial lead compounds targeting bacterial motility and biofilm formation with a low potential for eliciting antibiotic resistance. IMPORTANCE Pathogenic bacteria often require motility to establish infections and subsequently spread within host organisms. Thus, motility is an attractive therapeutic target for the development of novel antibiotics. We found that cyclic lipopeptides (CLPs) produced by marine bacterium Bacillus sp. strain 176 dramatically suppress the motility of the pathogenic bacterium Vibrio alginolyticus strain 178, reduce biofilm formation, and promote cellular aggregation without inducing cell death. These findings suggest that CLPs hold great promise as potential drug candidates targeting bacterial motility and biofilm formation with a low overall potential for triggering antibiotic resistance.

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Enhanced propagation of motile bacteria on surfaces due to forward scattering

Nature Communications ◽

10.1038/s41467-019-12010-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Stanislaw Makarchuk ◽

Vasco C. Braz ◽

Nuno A. M. Araújo ◽

Lena Ciric ◽

Giorgio Volpe

Keyword(s):

Biofilm Formation ◽

Flat Surface ◽

Experimental Studies ◽

Forward Scattering ◽

Bacterial Motility ◽

Complex Environments ◽

Natural Habitats ◽

Near Surface ◽

Propulsion Mechanism ◽

Motile Bacteria

Abstract How motile bacteria move near a surface is a problem of fundamental biophysical interest and is key to the emergence of several phenomena of biological, ecological and medical relevance, including biofilm formation. Solid boundaries can strongly influence a cell’s propulsion mechanism, thus leading many flagellated bacteria to describe long circular trajectories stably entrapped by the surface. Experimental studies on near-surface bacterial motility have, however, neglected the fact that real environments have typical microstructures varying on the scale of the cells’ motion. Here, we show that micro-obstacles influence the propagation of peritrichously flagellated bacteria on a flat surface in a non-monotonic way. Instead of hindering it, an optimal, relatively low obstacle density can significantly enhance cells’ propagation on surfaces due to individual forward-scattering events. This finding provides insight on the emerging dynamics of chiral active matter in complex environments and inspires possible routes to control microbial ecology in natural habitats.

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CRISPR-Cas9 knockout of qseB induced asynchrony between motility and biofilm formation in Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/cjm-2019-0100 ◽

2019 ◽

Vol 65 (9) ◽

pp. 691-702 ◽

Cited By ~ 3

Author(s):

Yi Gou ◽

Weiqi Liu ◽

Jing Jing Wang ◽

Ling Tan ◽

Bin Hong ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Motility ◽

Biofilm Formation ◽

Response Regulator ◽

Signal Transmission ◽

Bacterial Motility ◽

Type I ◽

E Coli ◽

Real Time Quantitative Pcr ◽

In The Wild

Generally, cell motility and biofilm formation are tightly regulated. The QseBC two-component system (TCS) serves as a bridge for bacterial signal transmission, in which the protein QseB acts as a response regulator bacterial motility, biofilm formation, and virulence. The mechanisms that govern the interaction between QseBC and their functions have been studied in general, but the regulatory role of QseB on bacterial motility and biofilm formation is unknown. In this study, the CRISPR-Cas9 system was used to construct the Escherichia coli MG1655ΔqseB strain (strain ΔqseB), and the effects of the qseB gene on changes in motility and biofilm formation in the wild type (WT) were determined. The motility assay results showed that the ΔqseB strain had higher (p < 0.05) motility than the WT strain. However, there was no difference in the formation of biofilm between the ΔqseB and WT strains. Real-time quantitative PCR illustrated that deletion of qseB in the WT strain downregulated expression of the type I pili gene fimA. Therefore, we might conclude that the ΔqseB induced the downregulation of fimA, which led to asynchrony between motility and biofilm formation in E. coli, providing new insight into the functional importance of QseB in regulating cell motility and biofilm formation.

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Homologous c-di-GMP-Binding Scr Transcription Factors Orchestrate Biofilm Development in Vibrio parahaemolyticus

Journal of Bacteriology ◽

10.1128/jb.00723-19 ◽

2020 ◽

Vol 202 (6) ◽

Author(s):

John H. Kimbrough ◽

J. Thomas Cribbs ◽

Linda L. McCarter

Keyword(s):

Transcription Factors ◽

Biofilm Formation ◽

Vibrio Parahaemolyticus ◽

Matrix Protein ◽

Second Messenger ◽

Minor Role ◽

Binding Motif ◽

Swarming Motility ◽

Content Type ◽

Biofilm Development

ABSTRACT The marine bacterium and human pathogen Vibrio parahaemolyticus rapidly colonizes surfaces by using swarming motility and forming robust biofilms. Entering one of the two colonization programs, swarming motility or sessility, involves differential regulation of many genes, resulting in a dramatic shift in physiology and behavior. V. parahaemolyticus has evolved complex regulation to control these two processes that have opposing outcomes. One mechanism relies on the balance of the second messenger c-di-GMP, where high c-di-GMP favors biofilm formation. V. parahaemolyticus possesses four homologous regulators, the Scr transcription factors, that belong in a Vibrio-specific family of W[F/L/M][T/S]R motif transcriptional regulators, some members of which have been demonstrated to bind c-di-GMP. In this work, we explore the role of these Scr regulators in biofilm development. We show that each protein binds c-di-GMP, that this binding requires a critical R in the binding motif, and that the biofilm-relevant activities of CpsQ, CpsS, and ScrO but not ScrP are dependent upon second messenger binding. ScrO and CpsQ are the primary drivers of biofilm formation, as biofilms are eliminated when both of these regulators are absent. ScrO is most important for capsule expression. CpsQ is most important for RTX-matrix protein expression, although it contributes to capsule expression when c-di-GMP levels are high. Both regulators contribute to O-antigen ligase expression. ScrP works oppositely in a minor role to repress the ligase gene. CpsS plays a regulatory checkpointing role by negatively modulating expression of these biofilm-pertinent genes under fluctuating c-di-GMP conditions. Our work further elucidates the multifactorial network that contributes to biofilm development in V. parahaemolyticus. IMPORTANCE Vibrio parahaemolyticus can inhabit open ocean, chitinous shells, and the human gut. Such varied habitats and the transitions between them require adaptable regulatory networks controlling energetically expensive behaviors, including swarming motility and biofilm formation, which are promoted by low and high concentrations of the signaling molecule c-di-GMP, respectively. Here, we describe four homologous c-di-GMP-binding Scr transcription factors in V. parahaemolyticus. Members of this family of regulators are present in many vibrios, yet their numbers and the natures of their activities differ across species. Our work highlights the distinctive roles that these transcription factors play in dynamically controlling biofilm formation and architecture in V. parahaemolyticus and serves as a powerful example of regulatory network evolution and diversification.

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