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 inhibits transcription (~1.4-fold) of the bcs locus, which comprises the genes responsible for producing the cellulose polysaccharide. However, beyond that, the mechanism of LitR-mediated inhibition of 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 gene for sensor kinase VF_A1016, which we determined to be important for biofilm inhibition. Like the loss of LitR, the loss of VF_A1016 increased bcs transcription (~1.6-fold). Through chromatin immunoprecipitation sequencing (ChIP-seq), we found that LitR directly binds to the VF_A1016 and pdeV regulatory region. In total, we identified 147 LitR-binding sites in the genome and confirmed transcriptional control over a subset of these putative regulatory targets. Specifically, 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. These data expand our understanding of LitR-mediated regulation of genes involved in biofilm formation and the physiology of V. fischeri.IMPORTANCEBacteria can coordinate their behaviors on a population level using quorum sensing, a process that results in altered gene regulation. In Vibrio fischeri, the quorum-sensing-regulated transcription factor LitR inhibits the formation of biofilms, communities of attached and protected bacteria, by diminishing the production of cellulose. Here, we determined that LitR controls additional known or putative biofilm factors. We also identified other possible targets of LitR regulation by high-throughput chromatin immunoprecipitation sequencing. This work furthers our understanding of the established connection between quorum sensing and biofilm formation in V. fischeri strain ES114. These findings also have the potential to translate to known pathways in other Vibrios where quorum sensing and biofilm production are linked.