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Bacterial biofilms pose a significant challenge in clinical environments due to their inherent lack of susceptibility to antibiotic treatment. It is widely recognized that most pathogenic bacterial strains in the clinical setting persist in the biofilm state, and are the root cause of many recrudescent infections. Discovery and development of compounds capable of either inhibiting biofilm formation or initiating biofilm dispersal may provide new therapeutic avenues for reducing the number of hospital acquired, biofilm-mediated infections. We now report the application of our recently reported image-based, high-throughput screen to the discovery of microbially-derived natural products with biofilm inhibitory activity against Vibrio cholerae. Examination of a prefractionated library of microbially-derived marine natural products has lead to the identification of a new biofilm inhibitor that is structurally unrelated to previously reported inhibitors and is one of the most potent inhibitors reported to date against V. cholerae. Combination of this compound with sub-MIC concentrations of a number of clinically relevant antibiotics was shown to improve the biofilm inhibitory efficacy of this new compound compared to monotherapy treatments, and provides evidence for the potential therapeutic benefit of biofilm inhibitors in treating persistent biofilm-mediated infections.

The second messenger nucleotide cyclic dimeric guanosine monophosphate (c-di-GMP) governs many cellular processes in the facultative human pathogen Vibrio cholerae. This organism copes with changing environmental conditions in aquatic environments and during transitions to and from human hosts. Modulation of c-di-GMP allows V. cholerae to shift between motile and sessile stages of life, thus allowing adaptation to stressors and environmental conditions during its transmission cycle. The V. cholerae genome encodes a large set of proteins predicted to degrade and produce c-di-GMP. A subset of these enzymes has been demonstrated to control cellular processes – particularly motility, biofilm formation, and virulence – through transcriptional, post-transcriptional, and translational mechanisms. Recent studies have identified and characterized enzymes that modulate or sense c-di-GMP levels and have lead towards mechanistic understanding of c-di-GMP regulatory circuits in V. cholerae.

The ability to sense and adapt to temperature fluctuation is critical to the aquatic survival, transmission, and infectivity of Vibrio cholerae , the causative agent of the disease cholera. Little information is available on the physiological changes that occur when V. cholerae experiences temperature shifts. The genome-wide transcriptional profile of V. cholerae upon 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 gene cspV , 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 lacking cspV had significant defects for attachment and type VI-mediated killing on the surface of the aquatic crustacean Daphnia magna . Collectively, these studies reveal that cspV is a major regulator of the temperature downshift response and plays an important role in controlling cellular processes crucial to the infectious cycle of V. cholerae . IMPORTANCE Little 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 of V. cholerae and cholera outbreaks. In this study, we showed that V. cholerae reprograms 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.

Infectious diseases kill nearly 9 million people annually. Bacterial pathogens are responsible for a large proportion of these diseases, and the bacterial agents of pneumonia, diarrhea, and tuberculosis are leading causes of death and disability worldwide. Increasingly, the crucial role of nonhost environments in the life cycle of bacterial pathogens is being recognized. Heightened scrutiny has been given to the biological processes impacting pathogen dissemination and survival in the natural environment, because these processes are essential for the transmission of pathogenic bacteria to new hosts. This chapter focuses on the model environmental pathogen Vibrio cholerae to describe recent advances in our understanding of how pathogens survive between hosts and to highlight the processes necessary to support the cycle of environmental survival, transmission, and dissemination. We describe the physiological and molecular responses of V. cholerae to changing environmental conditions, focusing on its survival in aquatic reservoirs between hosts and its entry into and exit from human hosts.

Vibrio cholerae O1 El Tor strains have been responsible for pandemic cholera since 1961. These strains have evolved over time, spreading globally in three separate waves. Wave 3 is caused by altered El Tor (AET) variant strains, which include the strain with the signature ctxB7 allele that was introduced in 2010 into Haiti, where it caused a devastating epidemic. In this study, we used phenotypic analysis to compare an early isolate from the Haiti epidemic to wave 1 El Tor isolates commonly used for research. It is demonstrated that the Haiti isolate has increased production of cholera toxin (CT) and hemolysin, increased motility, and a reduced ability to form biofilms. This strain also outcompetes common wave 1 El Tor isolates for colonization of infant mice, indicating that it has increased virulence. Monitoring of CT production and motility in additional wave 3 isolates revealed that this phenotypic variation likely evolved over time rather than in a single genetic event. Analysis of available whole-genome sequences and phylogenetic analyses suggested that increased virulence arose from positive selection for mutations found in known and putative regulatory genes, including hns and vieA , diguanylate cyclase genes, and genes belonging to the lysR and gntR regulatory families. Overall, the studies presented here revealed that V. cholerae virulence potential can evolve and that the currently prevalent wave 3 AET strains are both phenotypically distinct from and more virulent than many El Tor isolates.

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.

Vibrio cholerae inhabits aquatic environments and colonizes the human digestive tract to cause the disease cholera. In these environments, V. cholerae copes with fluctuations in salinity and osmolarity by producing and transporting small, organic, highly soluble molecules called compatible solutes, which counteract extracellular osmotic pressure. Currently, it is unclear how V. cholerae regulates the expression of genes important for the biosynthesis or transport of compatible solutes in response to changing salinity or osmolarity conditions. Through a genome-wide transcriptional analysis of the salinity response of V. cholerae, we identified a transcriptional regulator we name CosR for compatible solute regulator. The expression of cosR is regulated by ionic strength and not osmolarity. A transcriptome analysis of a ΔcosR mutant revealed that CosR represses genes involved in ectoine biosynthesis and compatible solute transport in a salinity-dependent manner. When grown in salinities similar to estuarine environments, CosR activates biofilm formation and represses motility independently of its function as an ectoine regulator. This is the first study to characterize a compatible solute regulator in V. cholerae and couples the regulation of osmotic tolerance with biofilm formation and motility.

Summary Vibrio cholerae causes the severe diarrhoeal disease cholera. A cascade of regulators controls expression of virulence determinants in V. cholerae at both transcriptional and post-transcriptional levels. ToxT is the direct transcription activator of the major virulence genes in V. cholerae. Here we describe TarA, a highly conserved, small regulatory RNA, whose transcription is activated by ToxT from toxboxes present upstream of the ToxT-activated gene tcpI. TarA regulates ptsG, encoding a major glucose transporter in V. cholerae. Cells overexpressing TarA exhibit decreased steady-state levels of ptsG mRNA and grow poorly in glucose-minimal media. A mutant lacking the ubiquitous regulatory protein Hfq expresses diminished TarA levels, indicating that TarA likely interacts with Hfq to regulate gene expression. RNAhybrid analysis of TarA and the putative ptsG mRNA leader suggests potential productive base-pairing between these two RNA molecules. A V. cholerae mutant lacking TarA is compromised for infant mouse colonization in competition with wild type, suggesting a role in the in vivo fitness of V. cholerae. Although somewhat functionally analogous to SgrS of Escherichia coli, TarA does not encode a regulatory peptide, and its expression is activated by the virulence gene pathway in V. cholerae and not by glycolytic intermediates.

Vibrio cholerae switches between free-living motile and surface-attached sessile lifestyles. Cyclic diguanylate (c-di-GMP) is a signaling molecule controlling such lifestyle changes. C-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a GGDEF domain and is degraded by phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain. We constructed in-frame deletions of all V. cholerae genes encoding proteins with GGDEF and/or EAL domains and screened mutants for altered motility phenotypes. Of 52 mutants tested, four mutants exhibited an increase in motility, while three mutants exhibited a decrease in motility. We further characterized one mutant lacking VC0137 ( cdgJ ), which encodes an EAL domain protein. Cellular c-di-GMP quantifications and in vitro enzymatic activity assays revealed that CdgJ functions as a PDE. The cdgJ mutant had reduced motility and exhibited a small decrease in flaA expression; however, it was able to produce a flagellum. This mutant had enhanced biofilm formation and vps gene expression compared to that of the wild type, indicating that CdgJ inversely regulates motility and biofilm formation. Genetic interaction analysis revealed that at least four DGCs, together with CdgJ, control motility in V. cholerae .

Biofilm formation enhances the survival and persistence of the facultative human pathogenVibrio choleraein natural ecosystems and its transmission during seasonal cholera outbreaks. A major component of theV. choleraebiofilm matrix is theVibriopolysaccharide (VPS), which is essential for development of three-dimensional biofilm structures. Thevpsgenes are clustered in two regions, thevps-I cluster (vpsU,vpsA–K, VC0916–27) and thevps-II cluster (vpsL–Q, VC0934–39), separated by an intergenic region containing therbmgene cluster that encodes biofilm matrix proteins. In-frame deletions of thevpsclusters and genes encoding matrix proteins drastically altered biofilm formation phenotypes. To determine which genes within thevpsgene clusters are required for biofilm formation and VPS synthesis, we generated in-frame deletion mutants for all thevpsgenes. Many of these mutants exhibited reduced capacity to produce VPS and biofilms. Infant mouse colonization assays revealed that mutants lacking eithervpsclusters orrbmA(encoding secreted matrix protein RbmA) exhibited a defect in intestinal colonization compared to the wild-type. Understanding the roles of the variousvpsgene products will aid in the biochemical characterization of the VPS biosynthetic pathway and elucidate howvpsgene products contribute to VPS biosynthesis, biofilm formation and virulence inV. cholerae.

The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189: 8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.

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-associated microbial communities known as biofilms. Mature biofilms rely on Vibrio polysaccharide to connect cells to each other and to a surface. We previously described a core regulatory network, which consists of two positive transcriptional regulators, VpsR and VpsT, and a negative transcriptional regulator HapR, that controls biofilm formation by regulating the expression of vps genes. In this study, we 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. We determined that VpsS acts through the HPT protein LuxU, which is involved in a quorum-sensing signal transduction network in V. cholerae . In vitro analysis 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 positively 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 .

Vibrio cholerae's capacity to cause outbreaks of cholera is linked to its survival and adaptability to changes in aquatic environments. One of the environmental conditions that can vary in V. cholerae's natural aquatic habitats is calcium (Ca(+2)). In this study, we investigated the response of V. cholerae to changes in extracellular Ca(2+) levels. Whole-genome expression profiling revealed that Ca(2+) decreased the expression of genes required for biofilm matrix production. Luria-Bertani (LB) medium supplemented with Ca(2+) (LBCa(2+)) caused V. cholerae to form biofilms with decreased thickness and increased roughness, as compared with biofilms formed in LB. Furthermore, addition of Ca(2+) led to dissolution in biofilms. Transcription of two genes encoding a two-component regulatory system pair, now termed calcium-regulated sensor (carS) and regulator (carR), was decreased in cells grown in LBCa(2+). Analysis of null and overexpression alleles of carS and carR revealed that expression of vps (Vibriopolysaccharide) genes and biofilm formation are negatively regulated by the CarRS two-component regulatory system. Through epistasis analysis we determined that CarR acts in parallel with HapR, the negative regulator of vps gene expression.

Indole has been proposed to act as an extracellular signal molecule influencing biofilm formation in a range of bacteria. For this study, the role of indole in Vibrio cholerae biofilm formation was examined. It was shown that indole activates genes involved in vibrio polysaccharide (VPS) production, which is essential for V. cholerae biofilm formation. In addition to activating these genes, it was determined using microarrays that indole influences the expression of many other genes, including those involved in motility, protozoan grazing resistance, iron utilization, and ion transport. A transposon mutagenesis screen revealed additional components of the indole-VPS regulatory circuitry. The indole signaling cascade includes the DksA protein along with known regulators of VPS production, VpsR and CdgA. A working model is presented in which global control of gene expression by indole is coordinated through 54 and associated transcriptional regulators. Bacterial cells synthesize myriad small organic molecules to signal and adapt to environmental, physiological, and population structure changes. These molecules include extracellular signals such as acyl homoserine lactones, butyrolactones, quinolones, a furanosyl borate diester, oligopeptides, 3-hydroxypalmitic acid methyl ester, and a hydroxyketone and intracellular signals including cyclic nucleotides and ppGpp (25; reviewed in reference 11). The phenotypic response to signaling compounds often involves traits that are beneficial under adverse conditions, such as biofilm formation, virulence, motility, bioluminescence, sporulation, competence, modification of carbon and energy utilization, and macromolecule biosynthesis.

Many pathogens in the environment can be transmitted to human populations and cause outbreaks and epidemics. Transmission is a multifactorial process influenced by the physiology of the pathogen as it exits its initial host, the mechanisms it uses for surviving outside the host, the physiology of the pathogen as it enters the next susceptible host and its ability to establish a successful infection. Few studies so far have focused on the processes responsible for modulating microbial survival in non-host environments and the transmission dynamics between infected and susceptible hosts, as well as the interplay between hosts. A better understanding of these mechanisms is thus necessary for predicting and preventing future outbreaks.

Vibrio cholerae , the causative agent of cholera, can undergo phenotypic variation generating rugose and smooth variants. The rugose variant forms corrugated colonies and well-developed biofilms and exhibits increased levels of resistance to several environmental stresses. Many of these phenotypes are mediated in part by increased expression of the vps genes, which are organized into vps- I and vps- II coding regions, separated by an intergenic region. In this study, we generated in-frame deletions of the five genes located in the vps intergenic region, termed rbmB to - F ( r ugosity and b iofilm structure m odulators B to F) in the rugose genetic background, and characterized the mutants for rugose colony development and biofilm formation. Deletion of rbmB , which encodes a protein with low sequence similarity to polysaccharide hydrolases, resulted in an increase in colony corrugation and accumulation of exopolysaccharides relative to the rugose variant. RbmC and its homolog Bap1 are predicted to encode proteins with carbohydrate-binding domains. The colonies of the rbmC bap1 double deletion mutant and bap1 single deletion mutant exhibited a decrease in colony corrugation. Furthermore, the rbmC bap1 double deletion mutant was unable to form biofilms at the air-liquid interface after 2 days, while the biofilms formed on solid surfaces detached readily. Although the colony morphology of rbmDEF mutants was similar to that of the rugose variant, their biofilm structure and cell aggregation phenotypes were different than those of the rugose variant. Taken together, these results indicate that vps intergenic region genes encode proteins that are involved in biofilm matrix production and maintenance of biofilm structure and stability.

In Vibrio cholerae , the second messenger 3′,5′-cyclic diguanylic acid (c-di-GMP) regulates several cellular processes, such as formation of corrugated colony morphology, biofilm formation, motility, and virulence factor production. Both synthesis and degradation of c-di-GMP in the cell are modulated by proteins containing GGDEF and/or EAL domains, which function as a diguanylate cyclase and a phosphodiesterase, respectively. The expression of two genes, cdgC and mbaA , which encode proteins harboring both GGDEF and EAL domains is higher in the rugose phase variant of V. cholerae than in the smooth variant. In this study, we carried out gene expression analysis to determine the genes regulated by CdgC in the rugose and smooth phase variants of V. cholerae . We determined that CdgC regulates expression of genes required for V. cholerae polysaccharide synthesis and of the transcriptional regulator genes vpsR , vpsT , and hapR . CdgC also regulates expression of genes involved in extracellular protein secretion, flagellar biosynthesis, and virulence factor production. We then compared the genes regulated by CdgC and by MbaA, during both exponential and stationary phases of growth, to elucidate processes regulated by them. Identification of the regulons of CdgC and MbaA revealed that the regulons overlap, but the timing of regulation exerted by CdgC and MbaA is different, suggesting the interplay and complexity of the c-di-GMP signal transduction pathways operating in V. cholerae .

Vibrio cholerae undergoes phenotypic variation that generates two morphologically different variants, termed smooth and rugose. The transcriptional profiles of the two variants differ greatly, and many of the differentially regulated genes are controlled by a complex regulatory circuitry that includes the transcriptional regulators VpsR, VpsT, and HapR. In this study, we identified the VpsT regulon and compared the VpsT and VpsR regulons to elucidate the contribution of each positive regulator to the rugose variant transcriptional profile and associated phenotypes. We have found that although the VpsT and VpsR regulons are very similar, the magnitude of the gene regulation accomplished by each regulator is different. We also determined that cdgA , which encodes a GGDEF domain protein, is partially responsible for the altered vps gene expression between the vpsT and vpsR mutants. Analysis of epistatic relationships among hapR , vpsT , and vpsR with respect to a whole-genome expression profile, colony morphology, and biofilm formation revealed that vpsR is epistatic to hapR and vpsT . Expression of virulence genes was increased in a vpsR hapR double mutant relative to a hapR mutant, suggesting that VpsR negatively regulates virulence gene expression in the hapR mutant. These results show that a complex regulatory interplay among VpsT, VpsR, HapR, and GGDEF/EAL family proteins controls transcription of the genes required for Vibrio polysaccharide and virulence factor production in V. cholerae .

Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins, and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this article, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa , and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation.

Two-component signal transduction systems (TCS) are used by bacteria to sense and respond to their environment. TCS are typically composed of a sensor histidine kinase (HK) and a response regulator (RR). The Vibrio cholerae genome encodes 52 RR, but the role of these RRs in V. cholerae pathogenesis is largely unknown. To identify RRs that control V. cholerae colonization, in-frame deletions of each RR were generated and the resulting mutants analyzed using an infant mouse intestine colonization assay. We found that 12 of the 52 RR were involved in intestinal colonization. Mutants lacking one previously uncharacterized RR, VCA0566 (renamed VxrB), displayed a significant colonization defect. Further experiments showed that VxrB phosphorylation state on the predicted conserved aspartate contributes to intestine colonization. The VxrB regulon was determined using whole genome expression analysis. It consists of several genes, including those genes that create the type VI secretion system (T6SS). We determined that VxrB is required for T6SS expression using several in vitro assays and bacterial killing assays, and furthermore that the T6SS is required for intestinal colonization. vxrB is encoded in a four gene operon and the other vxr operon members also modulate intestinal colonization. Lastly, though ΔvxrB exhibited a defect in single-strain intestinal colonization, the ΔvxrB strain did not show any in vitro growth defect. Overall, our work revealed that a small set of RRs is required for intestinal colonization and one of these regulators, VxrB affects colonization at least in part through its regulation of T6SS genes., Author Summary Pathogenic bacteria experience varying conditions during infection of human hosts and often use two-component signal transduction systems (TCSs) to monitor their environment. TCS consists of a histidine kinase (HK), which senses environmental signals, and a corresponding response regulator (RR), which mediates a cellular response. The genome of the human pathogen V. cholerae contains a multitude of genes encoding HKs and RRs proteins. In the present study, we systematically analyzed the role of each V. cholerae RR for its role in pathogenesis. We identified a previously uncharacterized RR, VxrB, as a new virulence factor. We demonstrated that VxrB controls expression of the type VI secretion system (T6SS), a virulence nanomachine that directly translocates effectors into bacterial or host cells, thereby facilitating colonization by competing with sister cells and intestinal microbiota. This study represents the first systematic analysis of the role of all RRs in V. cholerae pathogenesis and provides a foundation for understanding the signal transduction pathways controlling V. cholerae pathogenesis.

Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are associated 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 molecular mechanisms underlying this process and the signals that trigger biofilm assembly or dispersal have been areas of intense investigation over the past 20 years. In this Review, we discuss V. cholerae surface attachment, various matrix components and the regulatory networks controlling biofilm formation.

The 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.

Two-component systems play important roles in the physiology of many bacterial pathogens. Vibrio cholerae 's CarRS two-component regulatory system negatively regulates expression of vps ( Vibrio polysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of the almEFG genes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of the almEFG operon. Similarly to a carR mutant, strains lacking almE , almF , and almG exhibited enhanced polymyxin B sensitivity. We also observed that strains lacking almE or the almEFG operon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance in V. cholerae .

Differences in whole-genome expression patterns between the classical and El Tor biotypes of Vibrio cholerae O1 were determined under conditions that induce virulence gene expression in the classical biotype. A total of 524 genes (13.5% of the genome) were found to be differentially expressed in the two biotypes. The expression of genes encoding proteins required for biofilm formation, chemotaxis, and transport of amino acids, peptides, and iron was higher in the El Tor biotype. These gene expression differences may contribute to the enhanced survival capacity of the El Tor biotype in environmental reservoirs. The expression of genes encoding virulence factors was higher in the classical than in the El Tor biotype. In addition, the vieSAB genes, which were originally identified as regulators of ctxA transcription, were expressed at a fivefold higher level in the classical biotype. We determined the VieA regulon in both biotypes by transcriptome comparison of wild-type and vieA deletion mutant strains. VieA predominantly regulates gene expression in the classical biotype; 401 genes (10.3% of the genome), including those encoding proteins required for virulence, exopolysaccharide biosynthesis, and flagellum production as well as those regulated by σ E , are differentially expressed in the classical vieA deletion mutant. In contrast, only five genes were regulated by VieA in the El Tor biotype. A large fraction (20.8%) of the genes that are differentially expressed in the classical versus the El Tor biotype are controlled by VieA in the classical biotype. Thus, VieA is a major regulator of genes in the classical biotype under virulence gene-inducing conditions.

Vibrio cholerae , the causative agent of cholera, is a facultative human pathogen with intestinal and aquatic life cycles. The capacity of V. cholerae to recognize and respond to fluctuating parameters in its environment is critical to its survival. In many microorganisms, the second messenger, 3′,5′-cyclic diguanylic acid (c-di-GMP), is believed to be important for integrating environmental stimuli that affect cell physiology. Sequence analysis of the V. cholerae genome has revealed an abundance of genes encoding proteins with either GGDEF domains, EAL domains, or both, which are predicted to modulate cellular c-di-GMP concentrations. To elucidate the cellular processes controlled by c-di-GMP, whole-genome transcriptome responses of the El Tor and classical V. cholerae biotypes to increased c-di-GMP concentrations were determined. The results suggest that V. cholerae responds to an elevated level of c-di-GMP by increasing the transcription of the vps , eps , and msh genes and decreasing that of flagellar genes. The functions of other c-di-GMP-regulated genes in V. cholerae are yet to be identified.

Cyclic di-guanylic acid (c-diGMP) is a second messenger that modulates the cell surface properties of several microorganisms. Concentrations of c-diGMP in the cell are controlled by the opposing activities of diguanylate cyclases and phosphodiesterases, which are carried out by proteins harbouring GGDEF and EAL domains respectively. In this study, we report that the cellular levels of c-diGMP are higher in the Vibrio cholerae rugose variant compared with the smooth variant. Modulation of cellular c-diGMP levels by overexpressing proteins with GGDEF or EAL domains increased or decreased colony rugosity respectively. Several genes encoding proteins with either GGDEF or EAL domains are differentially expressed between the two V. cholerae variants. The generation and characterization of null mutants of these genes (cdgA-E, rocS and mbaA) revealed that rugose colony formation, exopolysaccharide production, motility and biofilm formation are controlled by their action. Furthermore, epistasis analysis suggested that cdgC, rocS and mbaA act in convergent pathways to regulate the phenotypic properties of the rugose and smooth variants, and are part of the VpsR, VpsT and HapR signal transduction pathway.

Phase variation between smooth and rugose colony variants of Vibrio cholerae is predicted to be important for the pathogen's survival in its natural aquatic ecosystems. The rugose variant forms corrugated colonies, exhibits increased levels of resistance to osmotic, acid, and oxidative stresses, and has an enhanced capacity to form biofilms. Many of these phenotypes are mediated in part by increased production of an exopolysaccharide termed VPS. In this study, we compared total protein profiles of the smooth and rugose variants using two-dimensional gel electrophoresis and identified one protein that is present at a higher level in the rugose variant. A mutation in the gene encoding this protein, which does not have any known homologs in the protein databases, causes cells to form biofilms that are more fragile and sensitive to sodium dodecyl sulfate than wild-type biofilms. The results indicate that the gene, termed rbmA ( r ugosity and b iofilm structure m odulator A), is required for rugose colony formation and biofilm structure integrity in V. cholerae . Transcription of rbmA is positively regulated by the response regulator VpsR but not VpsT.

Persistence of the opportunistic bacterial pathogen Vibrio cholerae in aquatic environments is the principal cause for seasonal occurrence of cholera epidemics. This causality has been explained by postulating that V. cholerae forms biofilms in association with animate and inanimate surfaces. Alternatively, it has been proposed that bacterial pathogens are an integral part of the natural microbial food web and thus their survival is constrained by protozoan predation. Here, we report that both explanations are interrelated. Our data show that biofilms are the protective agent enabling V. cholerae to survive protozoan grazing while their planktonic counterparts are eliminated. Grazing on planktonic V. cholerae was found to select for the biofilm-enhancing rugose phase variant, which is adapted to the surface-associated niche by the production of exopolymers. Interestingly, grazing resistance in V. cholerae biofilms was not attained by exopolymer production alone but was accomplished by the secretion of an antiprotozoal factor that inhibits protozoan feeding activity. We identified that the cell density-dependent regulator hapR controls the production of this factor in biofilms. The inhibitory effect of V. cholerae biofilms was found to be widespread among toxigenic and nontoxigenic isolates. Our results provide a mechanistic explanation for the adaptive advantage of surface-associated growth in the environmental persistence of V. cholerae and suggest an important contribution of protozoan predation in the selective enrichment of biofilm-forming strains in the out-of-host environment.

There is concern that biofilms could act as reservoirs of pathogenic organisms due to the growth advantages and the protection from conventional means for controlling bacterial growth that biofilms provide. Given the importance of waterborne, nosocomial, and food-borne diseases, the widespread ability of bacteria to form biofilms, and the resistance characteristics of biofilms, an attempt has been made to synthesize the prevalence, disease characteristics, and biofilm-forming capacity of several important pathogens to present a picture of the present and emerging threat of biofilms to human health. Researchers selected organisms that have documented prevalence in the environment and in locations that allow ingestion or inhalation, documented ability to form biofilms, and ability to cause disease. Such microbes include the waterborne pathogens Aeromonas spp., Legionella pneumophila, Mycobacterium spp., and Vibrio cholerae, and the food-borne pathogens Campylobacter spp., L. monocytogenes, and Salmonella spp. In summary, biofilms act as reservoirs of disease in a wide variety of habitats, including natural surface waters, groundwater, drinking water systems, food surfaces, and food industry surfaces. The presence of pathogenic bacteria in biofilms is clearly a challenge for protecting public health given the changing demographics of the world population, the increase in immunocompromised individuals, and the continued problems with water and food quality in the world.

Reversible phase variation between the rugose and smooth colony variants is predicted to be important for the survival of Vibrio cholerae in natural aquatic habitats. Microarray expression profiling studies of the rugose and smooth variants of the same strain led to the identification of 124 differentially regulated genes. Further expression profiling experiments showed how these genes are regulated by the VpsR and HapR transcription factors, which, respectively, positively and negatively regulate production of VPS(El Tor), a rugose-associated extracellular polysaccharide. The study of mutants of rpoN and rpoS demonstrated the effects of these alternative sigma factors on phase variation-specific gene expression. Bioinformatics analysis of these expression data shows that 'rugosity' and 'smoothness' are determined by a complex hierarchy of positive and negative regulators, which also affect the biofilm, surface hydrophobicity and motility phenotypes of the organism.

We show that Vibrio cholerae, the causative agent of cholera, use their flagella and mannose-sensitive hemagglutinin (MSHA) type IV pili synergistically to switch between two complementary motility states that together facilitate surface selection and attachment. Flagellar rotation counter-rotates the cell body, causing MSHA pili to have periodic mechanical contact with the surface for surface-skimming cells. Using tracking algorithms at 5 ms resolution we observe two motility behaviours: 'roaming', characterized by meandering trajectories, and 'orbiting', characterized by repetitive high-curvature orbits. We develop a hydrodynamic model showing that these phenotypes result from a nonlinear relationship between trajectory shape and frictional forces between pili and the surface: strong pili-surface interactions generate orbiting motion, increasing the local bacterial loiter time. Time-lapse imaging reveals how only orbiting mode cells can attach irreversibly and form microcolonies. These observations suggest that MSHA pili are crucial for surface selection, irreversible attachment, and ultimately microcolony formation.

Vibrio cholerae biofilms contain exopolysaccharide and three matrix proteins RbmA, RbmC and Bap1. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. VrrA is a conserved, 140-nt sRNA of V. cholerae, whose expression is controlled by sigma factor sigma(E). In this study, we demonstrate that VrrA negatively regulates rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation is not stringently dependent on the RNA chaperone protein Hfq. These results point to VrrA as a molecular link between the sigma(E)-regulon and biofilm formation in V. cholerae. In addition, VrrA represents the first example of direct regulation of sRNA on biofilm matrix component, by-passing global master regulators.

The physiological role of cyclic di-GMP (c-di-GMP) in Vibrio cholerae has emerged from the analysis of phenotypes of mutants unable to produce GGDEF/EAL/HD-GYP proteins as well as from phenotypes of strains overproducing GGDEF/EAL/HD-GYP proteins. In V. cholerae, c-di-GMP is involved in the regulation of multiple cellular processes including biofilm formation, motility, and virulence. This chapter first introduces the processes regulated by c-di-GMP signaling, discusses what is currently known about the involvement of specific GGDEF/EAL/HD-GYP proteins in these processes, and then summarizes our current understanding of regulation of GGDEF/EAL/HD-GYP genes. In V. cholerae, similar to other organisms, c-di-GMP controls a motile-to-sessile lifestyle switch and negatively regulates motility. The importance of c-di-GMP in V. cholerae biology has been conclusively shown in studies involving VieA (VC1652). The VieA response regulator harbors an EAL domain and has phosphodiesterase (PDE) activity. A set of GGDEF/EAL/HD-GYP proteins produce a phenotype only when they are overproduced. Studies in other organisms have indicated that c-di- GMP can regulate motility at least at three different levels: transcriptional, posttranscriptional, and functional. cdgF overexpression also affected biofilm formation. Cells with increased c-di-GMP levels exhibited enhanced vps, vpsT, and vpsR expression and, consequently, formed thicker and more developed biofilms than those with wild-type levels of c-di-GMP. The role of Plz proteins in intestinal colonization was analyzed using an infant mouse colonization assay. Strains harboring a plzB deletion or a mutated version of the PilZ domain exhibited a decrease in colonization.

International audience; The ability to form biofilms is important for environmental survival, transmission, and infectivity of Vibrio cholerae, the causative agent of cholera in humans. To form biofilms, V. cholerae produces an extracellular matrix composed of proteins, nucleic acids and a glycoconjugate, termed Vibrio exopolysaccharide (VPS). Here, we present the data on isolation and characterization of the polysaccharide part of the VPS (VPS-PS), which has the following structure: -4)-α-GulpNAcAGly3OAc-(1-4)-β-D-Glcp-(1-4)-α-Glcp-(1-4)-α-D-Galp-(1- where α-D-Glc is partially (,20%) replaced with α-D-GlcNAc. α-GulNAcAGly is an amide between 2-acetamido-2-deoxy-aguluronic acid and glycine. Apparently, the polysaccharide is bound to a yet unidentified component, which gives it high viscosity and completely suppresses any NMR signals belonging to the sugar chains of the VPS. The only reliable method to remove this component at present is a treatment of the whole glycoconjugate with concentrated hydrochloric acid.

The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPS ETr ) 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 homologue of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic background yielded a nonreverting smooth-colony morphotype that produced no detectable EPS ETr and did not form an architecturally mature biofilm. Analysis of two genes, vpsA and vpsL , within the vps cluster of EPS ETr 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 positive regulator of vpsA and vpsL and thus acts to positively regulate EPS ETr production and biofilm formation.

Decoding a Second-Messenger's Message Biofilms are aggregates of bacteria on a surface often associated with increased resistance to antibiotics and stress. In Vibrio cholerae , the bacterial species that causes cholera, biofilm formation is promoted by the bacterial second-messenger cyclic diguanylate (c-di-GMP) and involves the transcription regulator, VpsT. Krasteva et al. (p. 866 ) show that VpsT is itself a receptor for c-di-GMP and that binding of the small signaling molecule promotes VpsT dimerization, which is required for DNA recognition and transcriptional regulation. As well as activating components of the biofilm pathway, VpsT also down-regulates motility genes in a c-di-GMP–dependent manner.

Cyclic dimeric GMP (c-di-GMP) has emerged as a ubiquitous second messenger in bacteria that controls the transition from the free-living, motile lifestyle to the biofilm lifestyle (5, 12, 19). Production and degradation of c-di-GMP are controlled by proteins containing the GGDEF and EAL or HD-GYP domains, respectively. The GGDEF domain harbors intrinsic diguanylate cyclase (DGC) activity, and the EAL or HD-GYP domain has c-di-GMP phosphodiesterase (PDE) activity (4, 21-23). c-di-GMP was initially identified in Gluconacetobacter xylinus as an allosteric activator of cellulose synthase (20). Recent studies have shown that c-di-GMP modulates cell differentiation, biofilm formation, motility, and virulence in a diverse group of microorganisms (5, 12, 19). Our understanding of the biological processes and target molecules controlled by c-di-GMP signaling is increasing. However, at present, we have a limited understanding of the mechanisms through which c-di-GMP affects biofilm formation, mechanisms by which the activity of GGDEF/EAL proteins are regulated, and how multiple GGDEF and EAL domain proteins contribute to cellular c-di-GMP levels. In this issue, a new study by Ferreira et al. (8) is beginning to shed light on these questions in Vibrio parahaemolyticus, a facultative human pathogen responsible for the most common Vibrio-associated, seafood-borne gastroenteritis.

During the transition from a free-swimming, single-cell lifestyle to a sessile, multicellular state called a biofilm, bacteria produce and secrete an extracellular matrix comprised of nucleic acids, exopolysaccharides, and adhesion proteins. The Vibrio cholerae biofilm matrix contains three major protein components, RbmA, Bap1, and RbmC, which are unique to Vibrio cholerae and appear to support biofilm formation at particular steps in the process. Here, we focus on RbmA, a structural protein with an unknown fold. RbmA participates in the early cell-cell adhesion events and is found throughout the biofilm where it localizes to cell-cell contact sites. We determined crystal structures of RbmA and revealed that the protein folds into tandem fibronectin type III (FnIII) folds. The protein is dimeric in solution and in crystals, with the dimer interface displaying a surface groove that is lined with several positively charged residues. Structure-guided mutagenesis studies establish a crucial role for this surface patch for RbmA function. On the basis of the structure, we hypothesize that RbmA serves as a tether by maintaining flexible linkages between cells and the extracellular matrix.

Biofilm formation is a major cause of bacterial persistence in nosocomial infections, leading to extended treatment times and increased rates of morbidity and mortality. Despite this, there are currently no biofilm inhibitors approved for clinical use. The synthesis and biological evaluation of a library of amino alcohol quinolines as lead compounds for the disruption of biofilm formation in Vibrio cholerae is now reported. Application of selective metal-halogen exchange chemistry installed both stereocenters in one step, to afford a simpler scaffold than the initial lead molecule, with an EC5010 μM.

A Chalmydomonas reinhardtii adenosine triphosphate (ATP) sulfurylase cDNA clone (pATS1) was selected by complementing a mutation in the ATP sulfurylase gene (cysD) of Escherichia coli. E. coli cysD strains harboring pATS1 grow on medium containing sulfate as the sole sulfur source and exhibit ATP sulfurylase activity. The amino acid sequence of the C. reinhardtii ATP sulfurylase, derived from the nucleotide sequence of the complementing gene (ATS1), is 25 to 40% identical to that of ATP sulfurylases in other eukaryotic organisms and has a putative transit peptide at its amino terminus. ATP sulfurylase mRNA was present when cells were grown in sulfur-replete medium, but accumulated to higher levels when the cells were exposed to sulfur-limiting conditions. Furthermore, sulfur-stress-induced accumulation of the ATS1 transcript was reduced in a strain defective in SAC1, a gene that is critical for acclimation to sulfur-limited growth.

The sac1 mutant of Chlamydomonas reinhardtii is aberrant in most of the normal responses to sulfur limitation; it cannot synthesize arylsulfatase, does not take up sulfate as rapidly as wild-type cells, and does not synthesize periplasmic proteins that normally accumulate during sulfur-limited growth. Here, we show that the sac1 mutant dies much more rapidly than wild-type cells during sulfur deprivation; this emphasizes the vital role of the acclimation process. The loss of viability of the sac1 mutant during sulfur deprivation is only observed in the light and is mostly inhibited by DCMU. During sulfur-stress, wild-type cells, but not the sac1 mutant, downregulate photosynthesis. Thus, death of the sac1 mutant during sulfur deprivation is probably a consequence of its inability to downregulate photosynthesis. Furthermore, since SAC1 is necessary for the downregulation of photosynthesis, the process must be highly controlled and not simply the result of a general decrease in protein synthesis due to sulfur limitation. Genomic and cDNA copies of the SAC1 gene have been cloned. The deduced amino acid sequence of Sac1 is similar to an Escherichia coli gene that may involved in the response of E.coli to nutrient deprivation.

Monotrichous bacteria on surfaces exhibit complex spinning movements. Such spinning motility is often a part of the surface detachment launch sequence of these cells. To understand the impact of spinning motility on bacterial surface interactions, we develop a hydrodynamic model of a surface-bound bacterium, which reproduces behaviours that we observe in Pseudomonas aeruginosa , Shewanella oneidensis and Vibrio cholerae , and provides a detailed dictionary for connecting observed spinning behaviour to bacteria–surface interactions. Our findings indicate that the fraction of the flagellar filament adhered to the surface, the rotation torque of this appendage, the flexibility of the flagellar hook and the shape of the bacterial cell dictate the likelihood that a microbe will detach and the optimum orientation that it should have during detachment. These findings are important for understanding species-specific reversible attachment, the key transition event between the planktonic and biofilm lifestyle for motile, rod-shaped organisms.

Supporting Table S1, Bacterial Strains and Plasmids, does not appear. Please view Table S1 here: Click here for additional data file.(1.4M, tif) The legend for Figure 9 is: Bacterial strains and plasmids used in this work.

Vibrio cholerae inhabits aquatic environments and colonizes the human digestive tract to cause the disease cholera. In these environments, V. cholerae copes with fluctuations in salinity and osmolarity by producing and transporting small, organic, highly soluble molecules called compatible solutes, which counteract extracellular osmotic pressure. Currently, it is unclear how V. cholerae regulates the expression of genes important for the biosynthesis or transport of compatible solutes in response to changing salinity or osmolarity conditions. Through a genome-wide transcriptional analysis of the salinity response of V. cholerae, we identified a transcriptional regulator we name CosR for compatible solute regulator. The expression of cosR is regulated by ionic strength and not osmolarity. A transcriptome analysis of a ΔcosR mutant revealed that CosR represses genes involved in ectoine biosynthesis and compatible solute transport in a salinity-dependent manner. When grown in salinities similar to estuarine environments, CosR activates biofilm formation and represses motility independently of its function as an ectoine regulator. This is the first study to characterize a compatible solute regulator in V. cholerae and couples the regulation of osmotic tolerance with biofilm formation and motility.

We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.

Cyclic dimeric guanosine monophosphate (c-di-GMP) is a common, bacterial second messenger that regulates diverse cellular processes in bacteria. Opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) control c-di-GMP homeostasis in the cell. Many microbes have a large number of genes encoding DGCs and PDEs that are predicted to be part of c-di-GMP signaling networks. Other building blocks of these networks are c-di-GMP receptors which sense the cellular levels of the dinucleotide. C-di-GMP receptors form a more diverse family, including various transcription factors, PilZ domains, degenerate DGCs or PDEs, and riboswitches. Recent studies revealing the molecular basis of c-di-GMP signaling mechanisms enhanced our understanding of how this molecule controls downstream biological processes and how c-di-GMP signaling specificity is achieved.