RtmR is a membrane-embedded RRM family RNA-binding protein

The animal symbiont Vibrio fischeri has served as a model organism for molecular processes underlying bacterial group behaviors, including quorum sensing, and biofilm development. Here, using a genetic approach to identify negative regulators of biofilm formation in V. fischeri, we identified a membrane-bound RNA-binding protein, RtmR (VF_2432), that acts as an inhibitor of the symbiosis polysaccharide (SYP) biofilm. Membrane localization of the protein seems to be required for protein stability or expression, as truncation of the transmembrane helices led to an inability to detect the protein. The conserved RNP1 and RNP2 motifs in RtmR's cytoplasmic RNA recognition motif (RRM) domain are required for function, and we demonstrate binding to RNA substrates. Identification of RtmR RNA ligands was conducted with an individual-nucleotide resolution crosslinking and immunoprecipitation sequencing (iiCLIP-seq) approach that revealed a large interactome. One transcript identified was that of the biofilm regulatory histidine kinase RscS. We found that RtmR biofilm inhibition depends on RscS activity, and that RtmR negatively regulates levels of RscS. Overall, this work characterizes a novel type of bacterial RNA-binding protein.IMPORTANCEBacterial RNA-binding proteins (RBPs) perform key functions to regulate stress responses and development. Bacterial RBPs, including the RNA chaperones Hfq and ProQ, the global regulator CsrA, and the cold shock proteins (Csps), have been extensively studied, although additional classes of RBPs have been predicted by bioinformatic methods, including those carrying an RNA recognition motif (RRM) domain. This work expands on recent studies of RRM domain proteins in bacteria to characterize a membrane-bound RRM protein that regulates bacterial biofilm development. Given our rapidly expanding knowledge regarding the role of RNA-binding proteins in bacterial molecular biology, this work contributes a new class of membrane-bound regulators with homologs in human pathogens and marine symbionts.