AbstractIndividual cells of the bacteriumProteus mirabiliscan elongate up to 40-fold on surfaces before engaging in a cooperative surface-based motility termed swarming. How cells regulate this dramatic morphological remodeling remains an open question. In this paper, we move forward the understanding of this regulation by demonstrating thatP. mirabilisrequires the generffGfor swarmer cell elongation and subsequent swarm motility. TherffGgene encodes a protein homologous to the dTDP-glucose 4,6 dehydratase protein ofEscherichia coli, which contributes to Enterobacterial Common Antigen biosynthesis. Here we characterize therffGgene inP. mirabilis, demonstrating that it is required for the production of large lipopolysaccharide-linked moieties necessary for wild-type cell envelope integrity. We show that absence of therffGgene induces several stress-responsive pathways including those controlled by the transcriptional regulators RpoS, CaiF, and RcsB. We further show that inrffG-deficient cells, suppression of the Rcs phosphorelay, via loss of RcsB, is sufficient to induce cell elongation and swarm motility. However, loss of RcsB does not rescue cell envelope integrity defects and instead results in abnormally shaped cells, including cells producing more than two poles. We conclude that a RcsB-mediated response acts to suppress emergence of shape defects in cell envelope-compromised cells, suggesting an additional role for RcsB in maintaining cell morphology under stress conditions. We further propose that the composition of the cell envelope acts as a checkpoint before cells initiate swarmer cell elongation and motility.Importance statementP. mirabilisswarm motility has been implicated in pathogenesis. We have found that cells deploy multiple uncharacterized strategies to handle cell envelope stress beyond the Rcs phosphorelay when attempting to engage in swarm motility. While RcsB is known to directly inhibit the master transcriptional regulator for swarming, we have shown an additional role for RcsB in protecting cell morphology. These data support a growing appreciation that the Rcs phosphorelay is a multi-functional regulator of cell morphology in addition to its role in microbial stress responses. These data also strengthen the paradigm that outer membrane composition is a crucial checkpoint for modulating entry into swarm motility. Furthermore, therffG-dependent moieties provide a novel, attractive target for potential antimicrobials.
Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors
