Calcium-Responsive Diguanylate Cyclase CasA Drives Cellulose-Dependent Biofilm Formation and Inhibits Motility in Vibrio fischeri.

The marine bacterium Vibrio fischeri colonizes its host, the Hawaiian bobtail squid, in a manner requiring both bacterial biofilm formation and motility. The decision to switch between sessile and motile states is often triggered by environmental signals and regulated by the widespread signaling molecule c-di-GMP. Calcium is an environmental signal previously shown to affect both biofilm formation and motility by V. fischeri. In this study, we investigated the link between calcium and c-di-GMP, determining that calcium increases intracellular c-di-GMP dependent on a specific diguanylate cyclase, calcium-sensing protein A (CasA). CasA is activated by calcium, dependent on residues in an N-terminal sensory domain, and synthesizes c-di-GMP through an enzymatic C-terminal domain. CasA is responsible for calcium-dependent inhibition of motility and activation of cellulose-dependent biofilm formation. Calcium regulates cellulose biofilms at the level of transcription, which also requires the transcription factor VpsR. Finally, the Vibrio cholerae CasA homolog, CdgK, is unable to complement CasA and may be inhibited by calcium. Collectively, these results identify CasA as a calcium-responsive regulator, linking an external signal to internal decisions governing behavior, and shed light on divergence between Vibrio spp. IMPORTANCE Biofilm formation and motility are often critical behaviors for bacteria to colonize a host organism. Vibrio fischeri is the exclusive colonizer of its host's symbiotic organ and requires both biofilm formation and motility to initiate successful colonization, providing a relatively simple model to explore complex behaviors. In this study, we determined how the environmental signal calcium alters bacterial behavior through production of the signaling molecule c-di-GMP. Calcium activates the diguanylate cyclase CasA to synthesize c-di-GMP, resulting in inhibition of motility and activation of cellulose production. These activities depend on residues in CasA's N-terminal sensory domain and C-terminal enzymatic domain. These findings thus identify calcium as a signal recognized by a specific diguanylate cyclase to control key bacterial phenotypes. Of note, CasA activity is seemingly inverse to that of the homologous V. cholerae protein, CdgK, providing insight into evolutionary divergence between closely related species.

Quorum Sensing and Cyclic di-GMP Exert Control Over Motility of Vibrio fischeri KB2B1.

Bacterial motility is critical for symbiotic colonization by Vibrio fischeri of its host, the squid Euprymna scolopes, facilitating movement from surface biofilms to spaces deep inside the symbiotic organ. While colonization has been studied traditionally using strain ES114, others, including KB2B1, can outcompete ES114 for colonization for a variety of reasons, including superior biofilm formation. We report here that KB2B1 also exhibits an unusual pattern of migration through a soft agar medium: whereas ES114 migrates rapidly and steadily, KB2B1 migrates slowly and then ceases migration. To better understand this phenomenon, we isolated and sequenced five motile KB2B1 suppressor mutants. One harbored a mutation in the gene for the cAMP receptor protein (crp); because this strain also exhibited a growth defect, it was not characterized further. Two other suppressors contained mutations in the quorum sensing pathway that controls bacterial bioluminescence in response to cell density, and two had mutations in the diguanylate cyclase (DGC) gene VF_1200. Subsequent analysis indicated that (1) the quorum sensing mutations shifted KB2B1 to a perceived low cell density state and (2) the high cell density state inhibited migration via the downstream regulator LitR. Similar to the initial point mutations, deletion of the VF_1200 DGC gene increased migration. Consistent with the possibility that production of the second messenger c-di-GMP inhibited the motility of KB2B1, reporter-based measurements of c-di-GMP revealed that KB2B1 produced higher levels of c-di-GMP than ES114, and overproduction of a c-di-GMP phosphodiesterase promoted migration of KB2B1. Finally, we assessed the role of viscosity in controlling the quorum sensing pathway using polyvinylpyrrolidone and found that viscosity increased light production of KB2B1 but not ES114. Together, our data indicate that while the two strains share regulators in common, they differ in the specifics of the regulatory control over downstream phenotypes such as motility.

Nitric oxide inhibits biofilm formation by Vibrio fischeri via the nitric oxide sensor HnoX.

Nitric oxide (NO) is an important defense molecule secreted by the squid Euprymna scolopes and sensed by the bacterial symbiont, Vibrio fischeri, via the NO sensor HnoX. HnoX inhibits colonization through an unknown mechanism. The genomic location of hnoX adjacent to hahK, a recently identified positive regulator of biofilm formation, suggested that HnoX may inhibit colonization by controlling biofilm formation, a key early step in colonization. Indeed, the deletion of hnoX resulted in early biofilm formation in vitro, an effect that was dependent on HahK and its putative phosphotransfer residues. An allele of hnoX that encodes a protein with increased activity severely delayed wrinkled colony formation. Control occurred at the level of transcription of the syp genes, which produce the polysaccharide matrix component. The addition of NO abrogated biofilm formation and diminished syp transcription, effects that required HnoX. Finally, an hnoX mutant formed larger symbiotic biofilms. This work has thus uncovered a host-relevant signal controlling biofilm and a mechanism for the inhibition of biofilm formation by V. fischeri. The study of V. fischeri HnoX permits us to understand not only host-associated biofilm mechanisms, but also the function of HnoX domain proteins as regulators of important bacterial processes.

Vibrio fischeri Biofilm Formation Prevented by a Trio of Regulators.

Biofilms, complex communities of microorganisms surrounded by a self-produced matrix, facilitate attachment and provide protection to bacteria. A natural model used to study biofilm formation is the symbiosis between Vibrio fischeri and its host, the Hawaiian bobtail squid, Euprymna scolopes Host-relevant biofilm formation is a tightly regulated process and is observed in vitro only with strains that have been genetically manipulated to overexpress or disrupt specific regulators, primarily two-component signaling (TCS) regulators. These regulators control biofilm formation by dictating the production of the symbiosis polysaccharide (Syp-PS), the major component of the biofilm matrix. Control occurs both at and below the level of transcription of the syp genes, which are responsible for Syp-PS production. Here, we probed the roles of the two known negative regulators of biofilm formation, BinK and SypE, by generating double mutants. We also mapped and evaluated a point mutation using natural transformation and linkage analysis. We examined traditional biofilm formation phenotypes and established a new assay for evaluating the start of biofilm formation in the form of microscopic aggregates in shaking liquid cultures, in the absence of the known biofilm-inducing signal calcium. We found that wrinkled colony formation is negatively controlled not only by BinK and SypE but also by SypF. SypF is both required for and inhibitory to biofilm formation. Together, these data reveal that these three regulators are sufficient to prevent wild-type V. fischeri from forming biofilms under these conditions.IMPORTANCE Bacterial biofilms promote attachment to a variety of surfaces and protect the constituent bacteria from environmental stresses, including antimicrobials. Understanding the mechanisms by which biofilms form will promote our ability to resolve them when they occur in the context of an infection. In this study, we found that Vibrio fischeri tightly controls biofilm formation using three negative regulators; the presence of a single one of these regulators was sufficient to prevent full biofilm development, while disruption of all three permitted robust biofilm formation. This work increases our understanding of the functions of specific regulators and demonstrates the substantial negative control that one benign microbe exerts over biofilm formation, potentially to ensure that it occurs only under the appropriate conditions.

Tools for Rapid Genetic Engineering of Vibrio fischeri.

Vibrio fischeri is used as a model for a number of processes, including symbiosis, quorum sensing, bioluminescence, and biofilm formation. Many of these studies depend on generating deletion mutants and complementing them. Engineering such strains, however, is a time-consuming, multistep process that relies on cloning and subcloning. Here, we describe a set of tools that can be used to rapidly engineer deletions and insertions in the V. fischeri chromosome without cloning. We developed a uniform approach for generating deletions using PCR splicing by overlap extension (SOEing) with antibiotic cassettes flanked by standardized linker sequences. PCR SOEing of the cassettes to sequences up- and downstream of the target gene generates a DNA product that can be directly introduced by natural transformation. Selection for the introduced antibiotic resistance marker yields the deletion of interest in a single step. Because these cassettes also contain FRT (FLP recognition target) sequences flanking the resistance marker, Flp recombinase can be used to generate an unmarked, in-frame deletion. We developed a similar methodology and tools for the rapid insertion of specific genes at a benign site in the chromosome for purposes such as complementation. Finally, we generated derivatives of these tools to facilitate different applications, such as inducible gene expression and assessing protein production. We demonstrated the utility of these tools by deleting and inserting genes known or predicted to be involved in motility. While developed for V. fischeri strain ES114, we anticipate that these tools can be adapted for use in other V. fischeri strains and, potentially, other microbes.IMPORTANCEVibrio fischeri is a model organism for studying a variety of important processes, including symbiosis, biofilm formation, and quorum sensing. To facilitate investigation of these biological mechanisms, we developed approaches for rapidly generating deletions and insertions and demonstrated their utility using two genes of interest. The ease, consistency, and speed of the engineering is facilitated by a set of antibiotic resistance cassettes with common linker sequences that can be amplified by PCR with universal primers and fused to adjacent sequences using splicing by overlap extension and then introduced directly into V. fischeri, eliminating the need for cloning and plasmid conjugation. The antibiotic cassettes are flanked by FRT sequences, permitting their removal using Flp recombinase. We augmented these basic tools with a family of constructs for different applications. We anticipate that these tools will greatly accelerate mechanistic studies of biological processes in V. fischeri and potentially other Vibrio species.

Discovery of Calcium as a Biofilm-Promoting Signal for Vibrio fischeri Reveals New Phenotypes and Underlying Regulatory Complexity.

Vibrio fischeri uses biofilm formation to promote symbiotic colonization of its squid host, Euprymna scolopes Control over biofilm formation is exerted at the level of transcription of the symbiosis polysaccharide (syp) locus by a complex set of two-component regulators. Biofilm formation can be induced by overproduction of the sensor kinase RscS, which requires the activities of the hybrid sensor kinase SypF and the response regulator SypG and is negatively regulated by the sensor kinase BinK. Here, we identify calcium as a signal that promotes biofilm formation by biofilm-competent strains under conditions in which biofilms are not typically observed (growth with shaking). This was true for RscS-overproducing cells as well as for strains in which only the negative regulator binK was deleted. The latter results provided, for the first time, an opportunity to induce and evaluate biofilm formation without regulator overexpression. Using these conditions, we determined that calcium induces both syp-dependent and bacterial cellulose synthesis (bcs)-dependent biofilms at the level of transcription of these loci. The calcium-induced biofilms were dependent on SypF, but SypF's Hpt domain was sufficient for biofilm formation. These data suggested the involvement of another sensor kinase(s) and led to the discovery that both RscS and a previously uncharacterized sensor kinase, HahK, functioned in this pathway. Together, the data presented here reveal both a new signal and biofilm phenotype produced by V. fischeri cells, the coordinate production of two polysaccharides involved in distinct biofilm behaviors, and a new regulator that contributes to control over these processes.IMPORTANCE Biofilms, or communities of surface-attached microorganisms adherent via a matrix that typically includes polysaccharides, are highly resistant to environmental stresses and are thus problematic in the clinic and important to study. Vibrio fischeri forms biofilms to colonize its symbiotic host, making this organism useful for studying biofilms. Biofilm formation depends on the syp polysaccharide locus and its regulators. Here, we identify a signal, calcium, that induces both SYP-PS and cellulose-dependent biofilms. We also identify a new syp regulator, the sensor kinase HahK, and discover a mutant phenotype for the sensor kinase RscS. This work thus reveals a specific biofilm-inducing signal that coordinately controls two polysaccharides, identifies a new regulator, and clarifies the regulatory control over biofilm formation by V. fischeri.