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

Abstract

uses biofilm formation to promote symbiotic colonization of its squid host, Control over biofilm formation is exerted at the level of transcription of the symbiosis polysaccharide () 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 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-dependent and bacterial cellulose synthesis ()-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 cells, the coordinate production of two polysaccharides involved in distinct biofilm behaviors, and a new regulator that contributes to control over these processes. 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. forms biofilms to colonize its symbiotic host, making this organism useful for studying biofilms. Biofilm formation depends on the 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 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.

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

“…Recent work revealed that the sensor kinase HahK promotes biofilm formation by V. fischeri (Tischler et al ., ). Because hahK is located downstream of hnoX , and because the genes for HnoX/HahK regulatory partners are typically co‐transcribed (Iyer et al ., ), we hypothesized that HahK is negatively controlled by HnoX.…”

“…B). When grown with shaking in the presence of exogenous calcium, biofilm‐competent cells can form macroscopic cell clumping (Tischler et al ., ) and/or microscopic cell aggregates (Thompson et al ., ). There was no consistent difference in the timing of macroscopic (Fig.…”

“…) (Yip et al ., ; Hussa et al ., ; ; Norsworthy and Visick, ; Thompson et al . (); Tischler et al ., ). In addition, two negative regulators have been identified that regulate transcription of the syp locus (Brooks et al ., ; Pankey et al ., ; Thompson et al ., ; Tischler et al ., ), and one that posttranscriptionally regulates Syp‐PS production (Morris et al ., ; Morris and Visick, ; ).…”

“…Recently, calcium was identified as a signal that controls host‐relevant biofilm formation by V. fischeri (Tischler et al ., ). While calcium is plentiful in seawater, the relevance of this signal, if any, to squid colonization is as‐yet unknown.…”

Should they stay or should they go? Nitric oxide and the clash of regulators governing Vibrio fischeri biofilm formation

“…The fact that Syp biofilms are essentially not made by wild‐type grown in standard culture media has proven an experimental obstacle in this and other regards. Recently, manipulation of salt and nutrient conditions has led to increased sypA transcription in culture (Marsden et al ., ), and calcium in particular is syp ‐inducing (Tischler et al ., ). However, neither of these studies has yet pointed to a clearly documented symbiotic cue, and two critical open questions remain.…”