Vibrio fischeri: a model for host-associated biofilm formation.

Multicellular communities of adherent bacteria known as biofilms are often detrimental in the context of a human host, making it important to study their formation and dispersal, especially in animal models. One such model is the symbiosis between the squid Euprymna scolopes and the bacterium Vibrio fischeri. Juvenile squid hatch aposymbiotically and selectively acquire their symbiont from natural seawater containing diverse environmental microbes. Successful pairing is facilitated by ciliary movements that direct bacteria to quiet zones on the surface of the squid's symbiotic light organ where V. fischeri forms a small aggregate or biofilm. Subsequently, the bacteria disperse from that aggregate to enter the organ, ultimately reaching and colonizing deep crypt spaces. Although transient, aggregate formation is critical for optimal colonization and is tightly controlled. In vitro studies have identified a variety of polysaccharides and proteins that comprise the extracellular matrix. Some of the most well-characterized matrix factors include the symbiosis polysaccharide (SYP), cellulose polysaccharide, and LapV adhesin. In this review, we discuss these components, their regulation, and other less understood V. fischeri biofilm contributors. We also highlight what is currently known about dispersal from these aggregates and host cues that may promote it. Finally, we briefly describe discoveries gleaned from the study of other V. fischeri isolates. By unraveling the complexities involved in V. fischeri's control over matrix components, we may begin to understand how the host environment triggers transient biofilm formation and dispersal to promote this unique symbiotic relationship.

Genetic Analysis Reveals a Requirement for the Hybrid Sensor Kinase RscS in para-Aminobenzoic Acid/Calcium-Induced Biofilm Formation by Vibrio fischeri.

The marine bacterium Vibrio fischeri initiates symbiotic colonization of its squid host, Euprymna scolopes, by forming and dispersing from a biofilm dependent on the symbiosis polysaccharide locus (syp). Historically, genetic manipulation of V. fischeri was needed to visualize syp-dependent biofilm formation in vitro, but recently, we discovered that the combination of two small molecules, para-aminobenzoic acid (pABA) and calcium, was sufficient to induce wild-type strain ES114 to form biofilms. Here, we determined that these syp-dependent biofilms were reliant on the positive syp regulator RscS, since the loss of this sensor kinase abrogated biofilm formation and syp transcription. These results were of particular note because loss of RscS, a key colonization factor, exerts little to no effect on biofilm formation under other genetic and medium conditions. The biofilm defect could be complemented by wild-type RscS and by an RscS chimera that contains the N-terminal domains of RscS fused to the C-terminal HPT domain of SypF, the downstream sensor kinase. It could not be complemented by derivatives that lacked the periplasmic sensory domain or contained a mutation in the conserved site of phosphorylation, H412, suggesting that these cues promote signaling through RscS. Lastly, pABA and/or calcium was able to induce biofilm formation when rscS was introduced into a heterologous system. Taken together, these data suggest that RscS is responsible for recognizing pABA and calcium, or downstream consequences of those cues, to induce biofilm formation. This study thus provides insight into signals and regulators that promote biofilm formation by V. fischeri. IMPORTANCE Bacterial biofilms are common in a variety of environments. Infectious biofilms formed in the human body are notoriously hard to treat due to a biofilm's intrinsic resistance to antibiotics. Bacteria must integrate signals from the environment to build and sustain a biofilm and often use sensor kinases that sense an external signal, which triggers a signaling cascade to elicit a response. However, identifying the signals that kinases sense remains a challenging area of investigation. Here, we determine that a hybrid sensor kinase, RscS, is crucial for Vibrio fischeri to recognize para-aminobenzoic acid and calcium as cues to induce biofilm formation. This study thus advances our understanding of the signal transduction pathways leading to biofilm formation.

Mutational Analysis of Vibrio fischeri c-di-GMP-Modulating Genes Reveals Complex Regulation of Motility.

The symbiont Vibrio fischeri uses motility to colonize its host. In numerous bacterial species, motility is negatively controlled by cyclic-di-GMP (c-di-GMP), which is produced by diguanylate cyclases (DGCs) with GGDEF domains and degraded by phosphodiesterases with either EAL or HD-GYP domains. To begin to decode the functions of the 50 Vibrio fischeri genes with GGDEF, EAL, and/or HD-GYP domains, we deleted each gene and assessed each mutant's migration through tryptone broth salt (TBS) soft agar medium containing or lacking magnesium (Mg) and calcium (Ca), which are known to influence V. fischeri motility. We identified 6, 13, and 16 mutants with altered migration in TBS-Mg, TBS, and TBS-Ca soft agar, respectively, a result that underscores the importance of medium conditions in assessing gene function. A biosensor-based assay revealed that Mg and Ca affected c-di-GMP levels negatively and positively, respectively; the severe decrease in c-di-GMP caused by Mg addition correlates with its strong positive impact on bacterial migration. A mutant defective for VF_0494, a homolog of V. cholerae rocS, exhibited a severe defect in migration across all conditions. Motility of a VF_1603 VF_2480 double mutant was also severely defective and could be restored by expression of "c-di-GMP-blind" alleles of master flagellar regulator flrA. Together, this work sheds light on the genes and conditions that influence c-di-GMP-mediated control over motility in V. fischeri and provides a foundation for (i) assessing roles of putative c-di-GMP-binding proteins, (ii) evaluating other c-di-GMP-dependent phenotypes in V. fischeri, (iii) uncovering potential redundancy, and (iv) deciphering signal transduction mechanisms. IMPORTANCE Critical bacterial processes, including motility, are influenced by c-di-GMP, which is controlled by environment-responsive synthetic and degradative enzymes. Because bacteria such as Vibrio fischeri use motility to colonize their hosts, understanding the roles of c-di-GMP-modulating enzymes in controlling motility has the potential to inform on microbe-host interactions. We leveraged recent advances in genetic manipulation to generate 50 mutants defective for putative c-di-GMP synthetic and degradative enzymes. We then assessed the consequences on motility, manipulating levels of magnesium and calcium, which inversely influenced motility and levels of c-di-GMP. Distinct subsets of the 50 genes were required under the different conditions. Our data thus provide needed insight into the functions of these enzymes and environmental factors that influence them.

sRNA chaperone Hfq controls bioluminescence and other phenotypes through Qrr1-dependent and -independent mechanisms in Vibrio fischeri.

Colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation, motility, and bioluminescence. Previous work has demonstrated an inhibitory role for the small RNA (sRNA) Qrr1 in quorum-induced bioluminescence of V. fischeri, but the contribution of the corresponding sRNA chaperone, Hfq, was not examined. We thus hypothesized that V. fischeri Hfq similarly functions to inhibit bacterial bioluminescence as well as regulate other key steps of symbiosis, including bacterial biofilm formation and motility. Surprisingly, deletion of hfq increased luminescence of V. fischeri beyond what was observed for the loss of qrr1 sRNA. Epistasis experiments revealed that, while Hfq contributes to the Qrr1-dependent regulation of light production, it also functions independently of Qrr1 and its downstream target, LitR. This Hfq-dependent, Qrr1-independent regulation of bioluminescence is also independent of the major repressor of light production in V. fischeri, ArcA. We further determined that Hfq is required for full motility of V. fischeri in a mechanism that partially depends on the Qrr1/LitR regulators. Finally, Hfq also appears to function in the control of biofilm formation: loss of Hfq delayed the timing and diminished the extent of wrinkled colony development, but did not eliminate the production of SYP-polysaccharide-dependent cohesive colonies. Furthermore, loss of Hfq enhanced production of cellulose and resulted in increased Congo red binding. Together, these findings point to Hfq as an important regulator of multiple phenotypes relevant to symbiosis between V. fischeri and its squid host.