Characterisation of p-nitrophenylglycerol-resistant Proteus mirabilis super-swarming mutants

p-Nitrophenylglycerol (PNPG) inhibits the co-ordinately regulated activitie s of swarming behaviour and virulence factor expression in Proteus mirabili s. The inhibitory action of PNPG was investigated by the isolation of Tn5 i nsertion mutants that could swarm, albeit with much reduced ability, in the presence of PNPG. The mutants exhibited a super-swarming phenotype in the absence of PNPG; i.e., they migrated further in a given time than did the w ild-type cells. Cloning and sequence analysis of the mutants indicated that Tn5 was inserted into the rsbA gene, which may encode a membrane sensor hi stidine kinase of the bacterial two-component signalling system. In the abs ence of PNPG, the mutants exhibited several swarming-related phenotypes tha t were different from those of the wild type; they initiated swarming earli er and had a less conspicuous consolidation phase, they differentiated earl ier and maintained a differentiated state for longer, they started to expre ss virulence factors earlier and maintained high expression levels of these factors for longer, and they had higher cell invasion ability than the wil d type. These mutant phenotypes could be complemented by a plasmid-borne co py of rsbA. Together, these data suggest that RsbA may act as a repressor o f swarming and virulence factor expression. In the presence of PNPG, these rsbA-mutated mutants could still swarm, differentiate and express virulence factors, whereas the wild type could not, suggesting that PNPG may target RsbA or RsbA-regulated pathways to exert its inhibitory effect. Together, t hese data reveal a novel mechanism through which bacteria may negatively re gulate swarming differentiation and virulence factor expression and identif y a potential target of PNPG action.