Here, we probe the response to calcium during growth on a surface and show that calcium influences the transcriptome and stimulates motility and virulence of Vibrio parahaemolyticus. Swarming (but not swimming) gene expression and motility were enhanced by calcium. Calcium also elevated transcription of one of the organism's two type III secretion systems (T3SS1 but not T3SS2) and heightened cytotoxicity toward host cells in coculture. Calcium stimulation of T3SS gene expression has not been reported before, although low calcium is an inducing signal for the T3SS of many organisms. EGTA was also found to increase T3SS1 gene expression and virulence; however, this was demonstrated to be the consequence of iron rather than calcium chelation. Ectopic expression of exsA, encoding the T3SS1 AraC-type regulator, was used to define the extent of the T3SS1 regulon and verify its coincident induction by calcium and EGTA. To begin to understand the regulatory mechanisms modulating the calcium response, a calcium-repressed, LysR-type transcription factor named CalR was identified and shown to repress swarming and T3SS1 gene expression. Swarming and T3SS1 gene expression were also demonstrated to be linked by LafK, a 54 -dependent regulator of swarming, and additionally connected by a negative-feedback loop on the swarming regulon propagated by ExsA. Thus, calcium and iron, two ions pertinent for a marine organism and pathogen, play a signaling role with global consequences on the regulation of gene sets that are relevant for surface colonization and infection.

The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP). In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ⌬scrABC and wild-type strains revealed expression differences with respect to ϳ100 genes. Elevated c-di-GMP repressed genes in the surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide. The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct, positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory module in the circuit. Cyclic dimeric GMP (c-di-GMP) plays a determining role in diverse adaptations of many bacteria, often by moderating switches between biofilm and planktonic lifestyles (reviewed in references 16 and 32). In Vibrio parahaemolyticus, this second messenger also participates in modulating choices; however, for this organism, we know that c-di-GMP plays a key role in influencing lifestyle decisions during growth on surfaces. In particular, it participates in the decision-making processes determining biofilm development or the profound differentiation events leading to swarming that occur during growth on surfaces. A high concentration of c-di-GMP impairs surface motility and promotes biofilm formation, whereas a smaller amount of this second messenger favors swarming. In part, the concentration of c-di-GMP that reciprocally influences swarming and capsule production is established by the membrane-bound ScrC enzyme, which contains functional GGDEF and EAL domains. Proteins with the GGDEF domain are responsible for the formation of c-di-GMP, whereas EAL or HD-GYP domain-containing proteins participate in the degradation of the second messenger (reviewed in references 12 and 18). During swarming, expression of the scrABC operon is upregulated. The enzyme ScrA produces the S signal, an autoinducer sign...

is a potent opportunistic human pathogen that contaminates the human food-chain by asymptomatically colonizing seafood. Expression of the 9-gene exopolysaccharide locus mediates surface adherence and is controlled by the secondary signaling molecule c-di-GMP and the regulator BrpT. Here, we show that c-di-GMP and BrpT also regulate the expression of an adjacent 5-gene cluster that includes the operon, and another VpsT-like transcriptional regulator, The expression of the 14 genes spanning the region increased with elevated intracellular c -di-GMP levels in a BrpT-dependent manner, save , which was positively regulated by c-di-GMP and repressed by BrpT. BrpS repressed expression and was required for rugose colony development. Mutation of its consensus WFSA c-di-GMP binding motif blocked these activities, suggesting that BrpS function is dependent on binding c-di-GMP. BrpT specifically bound the , and promoters and binding sites homologous to the VpsT binding site were identified upstream of and Transcription was initiated distal to , and a conserved RfaH-recruiting element and a potential Rho utilization () terminator site were identified within the 100 bp leader region, suggesting integration of early termination and operon polarity suppression into the regulation of transcription. The GC content and codon usage of the 16 kb region was 5.5% lower relative to the flanking DNA, suggesting its recent assimilation via horizontal transfer. Thus, architecturally, the region can be considered as an acquired biofilm and rugosity island that is subject to complex regulation. Biofilm and rugose colony formation are developmental programs that underpin the evolution of as a potent opportunistic human pathogen and successful environmental organism. Better understanding of the regulatory pathways governing theses phenotypes promote the development and implementation of strategies to mitigate food-chain contamination by this pathogen. c-di-GMP signaling is central to both pathways. We show that the molecule orchestrates the expression of 14 genes clustered in a 16 Kb segment of the genome that govern biofilm and rugose colony development. This region exhibits the hallmarks of horizontal transfer, suggesting complex regulatory control of a recently assimilated genetic island governing the colonization response of.

is an estuarine bacteria and potent opportunistic human pathogen. It enters the food-chain by asymptomatically colonizing a variety of marine organisms, most notably oysters. Expression of the -encoded extracellular polysaccharide, which enhances cell-surface adherence, is regulated by c-di-GMP and the activator BrpT. The and homologs, VpsT and CpsQ, directly bind c-di-GMP via a novel W[F/L/M][T/S]R motif and c-di-GMP binding is absolutely required for activity. Notably, BrpT belongs to a distinct subclass of VpsT-like regulators that harbor a proline in the third position of the c-di-GMP binding motif (WLPR) and the impact of this change on activity is unknown. We show that the locus is organized as two linked operons with BrpT specifically binding to promoters upstream of and Expression data and structural modelling suggested that BrpT might be less dependent on c-di-GMP binding for activity than VpsT or CpsQ. We show that the affinity of BrpT for c-di-GMP is low and that signal binding is not a requisite for BrpT function. Furthermore, a BrpT mutant engineered to carry a canonical WLTR motif (BrpT) bound c-di-GMP with high affinity and its activity was now c-di-GMP dependent. Conversely, introduction of the WLPR motif into VpsT suppressed its dependence on c-di-GMP for activity. This is the first demonstration of reduced dependence on signal-association for regulator function within this motif family. Thus, BrpT defines a new class of VpsT-like transcriptional regulators and the WLPR motif variant may similarly liberate the activity of other subclass members. A genome may encode nearly 100 proteins that make, break and bind c-di-GMP, underscoring its central role in the ecology of these bacteria. The activity of the biofilm regulators VpsT of and CpsQ of is regulated by the direct binding of c-di-GMP via a novel W[F/L/M][T/S]R motif. The homolog, BrpT, bears an unusual WLPR variant and remains active at low intracellular c-di-GMP levels. This suggests that the WLPR motif may also liberate the activity of other members of this subclass. A single point mutation at the 3 position of the motif was sufficient to moderate dependence on c-di-GMP binding for activator function, highlighting the simplicity with which complex bacterial signaling networks can be rewired.

The second messenger c-di-GMP is a key regulator of bacterial physiology. The V. vulnificus genome encodes nearly 100 proteins predicted to make, break, and bind c-di-GMP. However, relatively little is known regarding the environmental signals that regulate c-di-GMP levels and biofilm formation in V. vulnificus. Here, we identify calcium as a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers brp-mediated biofilm formation. We show that PAPS, a metabolic intermediate of the sulfate assimilation pathway, acts as a second messenger linking environmental calcium and sulfur source availability to the production of another intracellular second messenger (c-di-GMP) to regulate biofilm and rugose colony formation, developmental pathways that are associated with environmental persistence and efficient bivalve colonization by this potent human pathogen.

No abstract