A single point mutation is sufficient to drive <i>syp</i> -dependent biofilm formation and promote colonization by <i>Vibrio fischeri</i>

Symbiotic colonization by <i>Vibrio fischeri</i> relies on the <i>syp</i> gene cluster, which encodes proteins predicted to synthesize and export a polysaccharide, SYP, that functions in cell-cell adherence. In strain ES114, four two-component sensor kinase/phosphatases, including central regulator SypF and the nitric oxide/HnoX-controlled HahK, dictate the activities of two response regulators, SypG and SypE, which in turn control SYP production. Here, we report that a single nucleotide change (C/A) upstream of the <i>hnoX-hahK</i> operon caused a substantial 80-fold increase in its transcription. While a search for negative regulators yielded Zur (zinc uptake regulator), loss of Zur only modestly (approximately threefold) increased transcription. We found instead that the C/A change engendered a new transcriptional start site. Furthermore, the C/A change was sufficient to robustly promote <i>syp</i>-dependent biofilm formation dependent on HahK and SypG but only partially dependent on the central regulator SypF. Rather, the residual biofilm formation in the absence of SypF relied on the luminescence regulator LuxU. Consistent with its ability to produce <i>syp</i>-dependent biofilms, a Δ<i>sypF</i> mutant that carried the C/A-<i>hahK</i> allele outcompeted its Δ<i>sypF</i> parent for squid colonization. Finally, bioinformatic analyses of the <i>hnoX</i> promoter region in various <i>V. fischeri</i> isolates revealed that most contained G or C nucleotides lacking in ES114, indicating an evolutionary divergence between different isolates. Together, these findings uncover the ability of HahK to signal through both SypF and LuxU to induce <i>syp</i>-dependent biofilm formation and host colonization, thus advancing our understanding of the regulators that control <i>syp</i>-dependent biofilm formation by <i>V. fischeri</i>.IMPORTANCEBiofilms promote the attachment of bacteria to each other and to surfaces. For <i>Vibrio fischeri</i>, biofilm formation dependent on the symbiosis polysaccharide (<i>syp</i>) locus promotes colonization of its symbiotic host. Multiple two-component regulators, including the central sensor kinase SypF and nitric oxide/HnoX-controlled sensor kinase HahK, induce SYP production. Here, we identify a C/A change in the <i>hnoX-hahK</i> regulatory region that substantially increases its transcription and SYP-dependent biofilm formation. We further determined that HahK signals through both SypF and the luminescence regulator LuxU to promote biofilm formation and host colonization. Our findings thus provide insight into the regulatory crossover between two major pathways, quorum sensing-controlled luminescence and biofilm formation, in <i>V. fischeri</i>.