Genetic and biochemical analyses reveal direct interactions between LitR and genes important for Vibrio fischeri physiology, including biofilm production

Bacteria can link gene expression to population density to promote group behaviors using quorum sensing. Quorum sensing controls a multitude of bacterial processes such as virulence, motility, and biofilm formation. In Vibrio fischeri, the quorum sensing-dependent transcription factor LitR inhibits biofilm formation. A previous study showed that LitR modestly inhibits transcription of the bcs locus, which comprises the genes responsible for producing the cellulose polysaccharide. However, beyond that, the mechanism of LitR’s inhibitory effect on biofilm formation was unknown. Here, we find that LitR transcriptionally activates pdeV, which encodes a c-di-GMP phosphodiesterase that indirectly promotes cleavage of the large adhesive protein LapV from the surface of V. fischeri, leading to biofilm dispersal. LitR also induces transcription of the sensor kinase VF_A1016, which we determined to be important for biofilm inhibition. Like loss of LitR, loss of VF_A1016 modestly increased bcs transcription. Through chromatin immunoprecipitation sequencing (ChIP-seq), we found that LitR directly binds to the VF_A1016 and pdeV regulatory regions. In total, we identified 147 LitR binding sites in the genome and confirmed LitR’s transcriptional control over a subset of these putative regulatory targets. Of note, we determined that LitR induces transcription of the genes encoding the diguanylate cyclase VF_1200 and the glyoxylate shunt protein AceB and inhibits expression of the putative transcription factor TfoY. LitR also differentially regulates the response regulator ArcA in static and shaking conditions. These data define the mechanism of LitR regulation of genes involved in biofilm formation and the physiology of V. fischeri. Wild-type V. fischeri strain ES114, the ∆litR mutant, and the ∆qrr1 mutant were assessed after growth in LBS + 10 mM CaCl2 at 24°C to an OD600 of 1.0. The samples were then assessed by ChIP-seq using anti-LuxR (Vibrio campbellii) to pull down V. fischeri LitR. DNA bound to LitR were sequenced.