An experimentally evolved variant of RsmA confirms its central role in the control of Pseudomonas aeruginosa social motility

Bacteria can colonize a variety of different environments by modulating their gene regulation using two-component systems. The versatile opportunistic pathogen Pseudomonas aeruginosa has been studied for its capacity to adapt to a broad range of environmental conditions. The Gac/Rsm pathway is composed of the sensor kinase GacS, that detects environmental cues, and the response regulator GacA, that modulates the expression of a specific genes. This system, through the sRNA repressors RsmY and RsmZ, negatively controls the activity of the protein RsmA, which is centrally involved in the transition from chronic to acute infections by post-transcriptionally regulating several virulence functions. RsmA positively regulates swarming motility, a social surface behaviour. Through a poorly defined mechanism, RsmA is also indirectly regulated by HptB, and a Δ hptB mutant exhibits a severe swarming defect. Since a Δ hptB mutant retains all the known functions required for that type of motility, we used an experimental evolution approach to identify elements responsible for its swarming defect. After a few passages under swarming conditions, the defect of the Δ hptB mutant was rescued by the emergence of spontaneous single nucleotide substitutions in the gacA and rsmA genes. Since GacA indirectly represses RsmA activity, it was coherent that an inactivating mutation in gacA would compensate the Δ hptB swarming defect. However, the effect of the mutation in rsmA was unexpected since RsmA promotes swarming; indeed, using expression reporters, we found that the mutation that does not abolish its activity. Instead, using electrophoretic mobility shift assays and molecular simulations, we show that this variant of RsmA is actually less amenable to titration by its cognate repressor RsmY, supporting the other phenotypes observed for this mutant. These results confirm the central role of RsmA as a regulator of swarming motility in P. aeruginosa and identify residues crucial for RsmA function in social motility.