OpaR Exerts a Dynamic Control over c-di-GMP Homeostasis and cpsA Expression in Vibrio parahaemolyticus through Its Regulation of ScrC and the Trigger Phosphodiesterase TpdA.

The second messenger cyclic dimeric GMP (c-di-GMP) plays a central role in controlling decision-making processes that are vitally important for the environmental survival of the human pathogen Vibrio parahaemolyticus. The mechanisms by which c-di-GMP levels and biofilm formation are dynamically controlled in V. parahaemolyticus are poorly understood. Here, we report the involvement of OpaR in controlling c-di-GMP metabolism and its effects on the expression of the trigger phosphodiesterase (PDE) TpdA and the biofilm-matrix related gene cpsA. Our results revealed that OpaR negatively modulates the expression of tpdA by maintaining a baseline level of c-di-GMP. The OpaR-regulated PDEs ScrC, ScrG, and VP0117 enable the upregulation of tpdA, to different degrees, in the absence of OpaR. We also found that TpdA plays the dominant role in c-di-GMP degradation under planktonic conditions compared to the other OpaR-regulated PDEs. In cells growing on solid medium, we observed that the role of the dominant c-di-GMP degrader alternates between ScrC and TpdA. We also report contrasting effects of the absence of OpaR on cpsA expression in cells growing on solid media compared to cells forming biofilms over glass. These results suggest that OpaR can act as a double-edged sword to control cpsA expression and perhaps biofilm development in response to poorly understood environmental factors. Finally, using an in-silico analysis, we indicate outlets of the OpaR regulatory module that can impact decision making during the motile-to-sessile transition in V. parahaemolyticus. IMPORTANCE The second messenger c-di-GMP is extensively used by bacterial cells to control crucial social adaptations such as biofilm formation. Here, we explore the role of the quorum-sensing regulator OpaR, from the human pathogen V. parahaemolyticus, on the dynamic control of c-di-GMP signaling and biofilm-matrix production. We found that OpaR is crucial to c-di-GMP homeostasis in cells growing on Lysogeny Broth agar and that the OpaR-regulated PDEs TpdA and ScrC alternate in the dominant role over time. Furthermore, OpaR plays contrasting roles in controlling the expression of the biofilm-related gene cpsA on different surfaces and growth conditions. This dual role has not been reported for orthologues of OpaR, such as HapR from Vibrio cholerae. It is important to investigate the origins and consequences of the differences in c-di-GMP signaling between closely and distantly related pathogens to better understand pathogenic bacterial behavior and its evolution.

The GGDEF-EAL protein CdgB from Azospirillum baldaniorum Sp245, is a dual function enzyme with potential polar localization.

Azospirillum baldaniorum Sp245, a plant growth-promoting rhizobacterium, can form biofilms through a process controlled by the second messenger cyclic diguanylate monophosphate (c-di-GMP). A. baldaniorum has a variety of proteins potentially involved in controlling the turnover of c-di-GMP many of which are coupled to sensory domains that could be involved in establishing a mutualistic relationship with the host. Here, we present in silico analysis and experimental characterization of the function of CdgB (AZOBR_p410089), a predicted MHYT-PAS-GGDEF-EAL multidomain protein from A. baldaniorum Sp245. When overproduced, CdgB behaves predominantly as a c-di-GMP phosphodiesterase (PDE) in A. baldaniorum Sp245. It inhibits biofilm formation and extracellular polymeric substances production and promotes swimming motility. However, a CdgB variant with a degenerate PDE domain behaves as diguanylate cyclase (DGC). This strongly suggest that CdgB is capable of dual activity. Variants with alterations in the DGC domain and the MHYT domain negatively affects extracellular polymeric substances production and induction of swimming motility. Surprisingly, we observed that overproduction of CdgB results in increased c-di-GMP accumulation in the heterologous host Escherichia coli, suggesting under certain conditions, the WT CdgB variant can behave predominantly as a DGC. Furthermore, we also demonstrated that CdgB is anchored to the cell membrane and localizes potentially to the cell poles. This localization is dependent on the presence of the MHYT domain. In summary, our results suggest that CdgB can provide versatility to signaling modules that control motile and sessile lifestyles in response to key environmental signals in A. baldaniorum.

CdgC, a Cyclic-di-GMP Diguanylate Cyclase of Azospirillum baldaniorum Is Involved in Internalization to Wheat Roots.

Azospirillum baldaniorum is a plant growth-promoting rhizobacterium (PGPR) capable of fixing nitrogen, the synthesis of several phytohormones including indole-acetic acid, and induction of plant defenses against phytopathogens. To establish a successful and prolonged bacteria-plant interaction, A. baldaniorum can form biofilms, bacterial communities embedded in a self-made matrix formed by extracellular polymeric substances which provide favorable conditions for survival. A key modulator of biofilm formation is the second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP), which is synthesized by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases. In this study, we analyzed the contribution of a previously uncharacterized diguanylate cyclase designated CdgC, to biofilm formation and bacterial-plant interaction dynamics. We showed that CdgC is capable of altering c-di-GMP levels in a heterologous host, strongly supporting its function as a DGC. The deletion of cdgC resulted in alterations in the three-dimensional structure of biofilms in a nitrogen-source dependent manner. CdgC was required for optimal colonization of wheat roots. Since we also observed that CdgC played an important role in exopolysaccharide production, we propose that this signaling protein activates a physiological response that results in the strong attachment of bacteria to the roots, ultimately contributing to an optimal bacterium-plant interaction. Our results demonstrate that the ubiquitous second messenger c-di-GMP is a key factor in promoting plant colonization by the PGPR A. baldaniorum by allowing proficient internalization in wheat roots. Understanding the molecular basis of PGPR-plant interactions will enable the design of better biotechnological strategies of agro-industrial interest.

A Trigger Phosphodiesterase Modulates the Global c-di-GMP Pool, Motility, and Biofilm Formation in Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells transit from free-swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic diguanylate monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase (PDE) is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called trigger phosphodiesterases, and hence we named the product of VP1881 TpdA, for trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while, its overexpression from an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible promoter showed a positive effect on both swimming and swarming motility and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes, as well as that of the biofilm-related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of these social behaviors. IMPORTANCE c-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, and hence not many examples of these type of signaling proteins have been reported in the literature. Here, we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.

Increased c-di-GMP Levels Lead to the Production of Alginates of High Molecular Mass in Azotobacter vinelandii.

Azotobacter vinelandii produces the linear exopolysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in A. vinelandii and Pseudomonas aeruginosa has several similarities but is regulated somewhat differently in the two microbes. Here, we show that the second messenger cyclic dimeric GMP (c-di-GMP) regulates the production and the molecular mass of alginate in A. vinelandii The hybrid protein MucG, containing conserved GGDEF and EAL domains and N-terminal HAMP and PAS domains, behaved as a c-di-GMP phosphodiesterase (PDE). This activity was found to negatively affect the amount and molecular mass of the polysaccharide formed. On the other hand, among the diguanylate cyclases (DGCs) present in A. vinelandii, AvGReg, a globin-coupled sensor (GCS) DGC that directly binds to oxygen, was identified as the main c-di-GMP-synthesizing contributor to alginate production. Overproduction of AvGReg in the parental strain phenocopied a ΔmucG strain with regard to alginate production and the molecular mass of the polymer. MucG was previously shown to prevent the synthesis of high-molecular-mass alginates in response to reduced oxygen transfer rates (OTRs). In this work, we show that cultures exposed to reduced OTRs accumulated higher levels of c-di-GMP; this finding strongly suggests that at least one of the molecular mechanisms involved in modulation of alginate production and molecular mass by oxygen depends on a c-di-GMP signaling module that includes the PAS domain-containing PDE MucG and the GCS DGC AvGReg.IMPORTANCE c-di-GMP has been widely recognized for its essential role in the production of exopolysaccharides in bacteria, such as alginate produced by Pseudomonas and Azotobacter spp. This study reveals that the levels of c-di-GMP also affect the physical properties of alginate, favoring the production of high-molecular-mass alginates in response to lower OTRs. This finding opens up new alternatives for the design of tailor-made alginates for biotechnological applications.

Cyclic di-GMP-Mediated Regulation of Extracellular Mannuronan C-5 Epimerases Is Essential for Cyst Formation in Azotobacter vinelandii.

The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCEA. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.

Reciprocal c-di-GMP signaling: Incomplete flagellum biogenesis triggers c-di-GMP signaling pathways that promote biofilm formation.

The assembly status of the V. cholerae flagellum regulates biofilm formation, suggesting that the bacterium senses a lack of movement to commit to a sessile lifestyle. Motility and biofilm formation are inversely regulated by the second messenger molecule cyclic dimeric guanosine monophosphate (c-di-GMP). Therefore, we sought to define the flagellum-associated c-di-GMP-mediated signaling pathways that regulate the transition from a motile to a sessile state. Here we report that elimination of the flagellum, via loss of the FlaA flagellin, results in a flagellum-dependent biofilm regulatory (FDBR) response, which elevates cellular c-di-GMP levels, increases biofilm gene expression, and enhances biofilm formation. The strength of the FDBR response is linked with status of the flagellar stator: it can be reversed by deletion of the T ring component MotX, and reduced by mutations altering either the Na+ binding ability of the stator or the Na+ motive force. Absence of the stator also results in reduction of mannose-sensitive hemagglutinin (MSHA) pilus levels on the cell surface, suggesting interconnectivity of signal transduction pathways involved in biofilm formation. Strains lacking flagellar rotor components similarly launched an FDBR response, however this was independent of the status of assembly of the flagellar stator. We found that the FDBR response requires at least three specific diguanylate cyclases that contribute to increased c-di-GMP levels, and propose that activation of biofilm formation during this response relies on c-di-GMP-dependent activation of positive regulators of biofilm production. Together our results dissect how flagellum assembly activates c-di-GMP signaling circuits, and how V. cholerae utilizes these signals to transition from a motile to a sessile state.

A Novel OmpR-Type Response Regulator Controls Multiple Stages of the Rhizobium etli - Phaseolus vulgaris N2-Fixing Symbiosis.

OmpR, is one of the best characterized response regulators families, which includes transcriptional regulators with a variety of physiological roles including the control of symbiotic nitrogen fixation (SNF). The Rhizobium etli CE3 genome encodes 18 OmpR-type regulators; the function of the majority of these regulators during the SNF in common bean, remains elusive. In this work, we demonstrated that a R. etli mutant strain lacking the OmpR-type regulator RetPC57 (ΔRetPC57), formed less nodules when used as inoculum for common bean. Furthermore, we observed reduced expression level of bacterial genes involved in Nod Factors production (nodA and nodB) and of plant early-nodulation genes (NSP2, NIN, NF-YA and ENOD40), in plants inoculated with ΔRetPC57. RetPC57 also contributes to the appropriate expression of genes which products are part of the multidrug efflux pumps family (MDR). Interestingly, nodules elicited by ΔRetPC57 showed increased expression of genes relevant for Carbon/Nitrogen nodule metabolism (PEPC and GOGAT) and ΔRetPC57 bacteroids showed higher nitrogen fixation activity as well as increased expression of key genes directly involved in SNF (hfixL, fixKf, fnrN, fixN, nifA and nifH). Taken together, our data show that the previously uncharacterized regulator RetPC57 is a key player in the development of the R. etli - P. vulgaris symbiosis.

A Conserved Regulatory Circuit Controls Large Adhesins in Vibrio cholerae.

The dinucleotide second messenger c-di-GMP has emerged as a central regulator of reversible cell attachment during bacterial biofilm formation. A prominent cell adhesion mechanism first identified in pseudomonads combines two c-di-GMP-mediated processes: transcription of a large adhesin and its cell surface display via posttranslational proteolytic control. Here, we characterize an orthologous c-di-GMP effector system and show that it is operational in Vibrio cholerae, where it regulates two distinct classes of adhesins. Through structural analyses, we reveal a conserved autoinhibition mechanism of the c-di-GMP receptor that controls adhesin proteolysis and present a structure of a c-di-GMP-bound receptor module. We further establish functionality of the periplasmic protease controlled by the receptor against the two adhesins. Finally, transcription and functional assays identify physiological roles of both c-di-GMP-regulated adhesins in surface attachment and biofilm formation. Together, our studies highlight the conservation of a highly efficient signaling effector circuit for the control of cell surface adhesin expression and its versatility by revealing strain-specific variations.IMPORTANCEVibrio cholerae, the causative agent of the diarrheal disease cholera, benefits from a sessile biofilm lifestyle that enhances survival outside the host but also contributes to host colonization and infectivity. The bacterial second messenger c-di-GMP has been identified as a central regulator of biofilm formation, including in V. cholerae; however, our understanding of the pathways that contribute to this process is incomplete. Here, we define a conserved signaling system that controls the stability of large adhesion proteins at the cell surface of V. cholerae, which are important for cell attachment and biofilm formation. Insight into the regulatory circuit underlying biofilm formation may inform targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments.

Functional Specialization in Vibrio cholerae Diguanylate Cyclases: Distinct Modes of Motility Suppression and c-di-GMP Production.

Vibrio cholerae biofilm formation and associated motility suppression are correlated with increased concentrations of cyclic diguanylate monophosphate (c-di-GMP), which are in turn driven by increased levels and/or activity of diguanylate cyclases (DGCs). To further our understanding of how c-di-GMP modulators in V. cholerae individually and collectively influence motility with cellular resolution, we determined how DGCs CdgD and CdgH impact intracellular c-di-GMP levels, motility, and biofilm formation. Our results indicated that CdgH strongly influences swim speed distributions; cells in which cdgH was deleted had higher average swim speeds than wild-type cells. Furthermore, our results suggest that CdgD, rather than CdgH, is the dominant DGC responsible for postattachment c-di-GMP production in biofilms. Lipopolysaccharide (LPS) biosynthesis genes were found to be extragenic bypass suppressors of the motility phenotypes of strains ΔcdgD and ΔcdgH We compared the motility regulation mechanism of the DGCs with that of Gmd, an LPS O-antigen biosynthesis protein, and discovered that comodulation of c-di-GMP levels by these motility effectors can be positively or negatively cooperative rather than simply additive. Taken together, these results suggest that different environmental and metabolic inputs orchestrate DGC responses of V. cholerae via c-di-GMP production and motility modulation.IMPORTANCE Cyclic diguanylate monophosphate (c-di-GMP) is a broadly conserved bacterial signaling molecule that affects motility, biofilm formation, and virulence. Although it has been known that high intracellular concentrations of c-di-GMP correlate with motility suppression and biofilm formation, how the 53 predicted c-di-GMP modulators in Vibrio cholerae collectively influence motility is not understood in detail. Here we used a combination of plate assays and single-cell tracking methods to correlate motility and biofilm formation outcomes with specific enzymes involved in c-di-GMP synthesis in Vibrio cholerae, the causative agent of the disease cholera.

NtrC Adds a New Node to the Complex Regulatory Network of Biofilm Formation and vps Expression in Vibrio cholerae.

The biofilm growth mode is important in both the intestinal and environmental phases of the Vibrio cholerae life cycle. Regulation of biofilm formation involves several transcriptional regulators and alternative sigma factors. One such factor is the alternative sigma factor RpoN, which positively regulates biofilm formation. RpoN requires bacterial enhancer-binding proteins (bEBPs) to initiate transcription. The V. cholerae genome encodes seven bEBPs (LuxO, VC1522, VC1926 [DctD-1], FlrC, NtrC, VCA0142 [DctD-2], and PgtA) that belong to the NtrC family of response regulators (RRs) of two-component regulatory systems. The contribution of these regulators to biofilm formation is not well understood. In this study, we analyzed biofilm formation and the regulation of vpsL expression by RpoN activators. Mutants lacking NtrC had increased biofilm formation and vpsL expression. NtrC negatively regulates the expression of core regulators of biofilm formation (vpsR, vpsT, and hapR). NtrC from V. cholerae supported growth and activated glnA expression when nitrogen availability was limited. However, the repressive activity of NtrC toward vpsL expression was not affected by the nitrogen sources present. This study unveils the role of NtrC as a regulator of vps expression and biofilm formation in V. choleraeIMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, contributing to both environmental survival and transmission to a human host. Identifying key regulators of V. cholerae biofilm formation is necessary to fully understand how this important growth mode is modulated in response to various signals encountered in the environment and the host. In this study, we characterized the role of RRs that function as coactivators of RpoN in regulating biofilm formation and identified new components in the V. cholerae biofilm regulatory circuitry.

The Bioactive Lipid (S)-Sebastenoic Acid Impacts Motility and Dispersion in Vibrio cholerae.

Although Gram-negative bacterial pathogens continue to impart a substantial burden on global healthcare systems, much remains to be understood about aspects of basic physiology in these organisms. In recent years, cyclic-diguanylate (c-di-GMP) has emerged as a key regulator of a number of important processes related to pathogenicity, including biofilm formation, motility and virulence. In an effort to discover chemical genetic probes for studying V. cholerae we have developed a new motility-based high-throughput screen to identify compounds that modulate c-di-GMP levels. Using this new screening platform, we tested a library of microbially-derived marine natural products extracts, leading to the discovery of the bioactive lipid (S)-sebastenoic acid. Evaluation of the effect of this new compound on bacterial motility, vpsL expression and biofilm formation implied that (S)-sebastenoic acid may alter phenotypes associated to c-di-GMP signaling in V. cholerae.

The ins and outs of cyclic di-GMP signaling in Vibrio cholerae.

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 led towards mechanistic understanding of c-di-GMP regulatory circuits in V. cholerae.

Expanding the regulatory network that controls nitrogen fixation in Sinorhizobium meliloti: elucidating the role of the two-component system hFixL-FxkR.

In Sinorhizobium meliloti, nitrogen fixation is regulated in response to oxygen concentration through the FixL-FixJ two-component system (TCS). Besides this conserved TCS, the field isolate SM11 also encodes the hFixL-FxkR TCS, which is responsible for the microoxic response in Rhizobium etli. Through genetic and physiological assays, we evaluated the role of the hFixL-FxkR TCS in S. meliloti SM11. Our results revealed that this regulatory system activates the expression of a fixKf orthologue (fixKa), in response to low oxygen concentration. Null mutations in either hFixL or FxkR promote upregulation of fixK1, a direct target of FixJ. Furthermore, the absence of this TCS translates into higher nitrogen fixation values as well as higher expression of fixN1 in nodules. Individual mutations in each of the fixK-like regulators encoded in the S. meliloti SM11 genome do not completely restrict fixN1 or fixN2 expression, pointing towards redundancy among these regulators. Both copies of fixN are necessary to achieve optimal levels of nitrogen fixation. This work provides evidence that the hFixL-FxkR TCS is activated in response to low oxygen concentration in S. meliloti SM11 and that it negatively regulates the expression of fixK1, fixN1 and nitrogen fixation.

C-di-GMP Regulates Motile to Sessile Transition by Modulating MshA Pili Biogenesis and Near-Surface Motility Behavior in Vibrio cholerae.

In many bacteria, including Vibrio cholerae, cyclic dimeric guanosine monophosphate (c-di-GMP) controls the motile to biofilm life style switch. Yet, little is known about how this occurs. In this study, we report that changes in c-di-GMP concentration impact the biosynthesis of the MshA pili, resulting in altered motility and biofilm phenotypes in V. cholerae. Previously, we reported that cdgJ encodes a c-di-GMP phosphodiesterase and a ΔcdgJ mutant has reduced motility and enhanced biofilm formation. Here we show that loss of the genes required for the mannose-sensitive hemagglutinin (MshA) pilus biogenesis restores motility in the ΔcdgJ mutant. Mutations of the predicted ATPase proteins mshE or pilT, responsible for polymerizing and depolymerizing MshA pili, impair near surface motility behavior and initial surface attachment dynamics. A ΔcdgJ mutant has enhanced surface attachment, while the ΔcdgJmshA mutant phenocopies the high motility and low attachment phenotypes observed in a ΔmshA strain. Elevated concentrations of c-di-GMP enhance surface MshA pilus production. MshE, but not PilT binds c-di-GMP directly, establishing a mechanism for c-di-GMP signaling input in MshA pilus production. Collectively, our results suggest that the dynamic nature of the MshA pilus established by the assembly and disassembly of pilin subunits is essential for transition from the motile to sessile lifestyle and that c-di-GMP affects MshA pilus assembly and function through direct interactions with the MshE ATPase.

Living in the matrix: assembly and control of Vibrio cholerae biofilms.

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.

Polymyxin B resistance and biofilm formation in Vibrio cholerae are controlled by the response regulator CarR.

Two-component systems play important roles in the physiology of many bacterial pathogens. Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression 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.

Identification and characterization of VpsR and VpsT binding sites in Vibrio cholerae.

UNLABELLED: The ability to form biofilms is critical for environmental survival and transmission of Vibrio cholerae, a facultative human pathogen responsible for the disease cholera. Biofilm formation is controlled by several transcriptional regulators and alternative sigma factors. In this study, we report that the two main positive regulators of biofilm formation, VpsR and VpsT, bind to nonoverlapping target sequences in the regulatory region of vpsL in vitro. VpsR binds to a proximal site (the R1 box) as well as a distal site (the R2 box) with respect to the transcriptional start site identified upstream of vpsL. The VpsT binding site (the T box) is located between the R1 and R2 boxes. While mutations in the T and R boxes resulted in a decrease in vpsL expression, deletion of the T and R2 boxes resulted in an increase in vpsL expression. Analysis of the role of H-NS in vpsL expression revealed that deletion of hns resulted in enhanced vpsL expression. The level of vpsL expression was higher in an hns vpsT double mutant than in the parental strain but lower than that in an hns mutant. In silico analysis of the regulatory regions of the VpsR and VpsT targets resulted in the identification of conserved recognition motifs for VpsR and VpsT and revealed that operons involved in biofilm formation and vpsT are coregulated by VpsR and VpsT. Furthermore, a comparative genomics analysis revealed substantial variability in the promoter region of the vpsT and vpsL genes among extant V. cholerae isolates, suggesting that regulation of biofilm formation is under active selection. IMPORTANCE: Vibrio cholerae causes cholera and is a natural inhabitant of aquatic environments. One critical factor that is important for environmental survival and transmission of V. cholerae is the microbe's ability to form biofilms, which are surface-associated communities encased in a matrix composed of the exopolysaccharide VPS (Vibrio polysaccharide), 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 the vpsL gene, determined the target sequences recognized by VpsT and VpsR, and analyzed their distribution and conservation patterns in multiple V. cholerae isolates. 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.

Transcript profiling of common bean nodules subjected to oxidative stress.

Several environmental stresses generate high amounts of reactive oxygen species (ROS) in plant cells, resulting in oxidative stress. Symbiotic nitrogen fixation (SNF) in the legume-rhizobia symbiosis is sensitive to damage from oxidative stress. Active nodules of the common bean (Phaseolus vulgaris) exposed to the herbicide paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride hydrate), which stimulates ROS accumulation, exhibited reduced nitrogenase activity and ureide content. We analyzed the global gene response of nodules subjected to oxidative stress using the Bean Custom Array 90K, which includes probes from 30,000 expressed sequence tags (ESTs). A total of 4280 ESTs were differentially expressed in stressed bean nodules; of these, 2218 were repressed. Based on Gene Ontology analysis, these genes were grouped into 42 different biological process categories. Analysis with the PathExpress bioinformatic tool, adapted for bean, identified five significantly repressed metabolic pathways related to carbon/nitrogen metabolism, which is crucial for nodule function. Quantitative reverse transcription (qRT)-PCR analysis of transcription factor (TF) gene expression showed that 67 TF genes were differentially expressed in nodules exposed to oxidative stress. Putative cis-elements recognized by highly responsive TF were detected in promoter regions of oxidative stress regulated genes. The expression of oxidative stress responsive genes and of genes important for SNF in bacteroids analyzed in stressed nodules revealed that these conditions elicited a transcriptional response.