Divide and conquer: the Pseudomonas aeruginosa two-component hybrid SagS enables biofilm formation and recalcitrance of biofilm cells to antimicrobial agents via distinct regulatory circuits

The opportunistic pathogen Pseudomonas aeruginosa forms antimicrobial resistant biofilms through sequential steps requiring several two-component regulatory systems. The sensor-regulator hybrid SagS plays a central role in biofilm development by enabling the switch from the planktonic to the biofilm mode of growth, and by facilitating the transition of biofilm cells to a highly tolerant state. However, the mechanism by which SagS accomplishes both functions is unknown. SagS harbours a periplasmic sensory HmsP, and phosphorelay HisKA and Rec domains. SagS domain was used as constructs and site-directed mutagenesis to elucidate how SagS performs its dual functions. It was demonstrated that HisKA-Rec and the phospho-signalling between SagS and BfiS contribute to the switch to the biofilm mode of growth, but not to the tolerant state. Instead, expression of SagS domain constructs harbouring HmsP rendered sagS biofilm cells as recalcitrant to antimicrobial agents as wild-type biofilms, likely by restoring BrlR production and cellular c-di-GMP levels to wild-type levels. Restoration of biofilm tolerance by HmsP was independent of biofilm biomass accumulation, RsmA, RsmYZ, HptB and BfiSR-downstream targets. Our findings thus suggest that SagS likely makes use of a divide-and-conquer mechanism to regulate its dual switch function, by activating two distinct regulatory networks via its individual domains.