HsbA represses stationary phase biofilm formation inPseudomonas putida

Abstract

Pseudomonas putida biofilm growth is associated to nutrient-sufficient conditions and biofilm dispersal is induced by nutrient starvation, signaled by the stringent response-associated nucleotide alarmone (p)ppGpp. We have used transcriptomic analysis to show that (p)ppGpp regulates the hsbAR-hptB gene cluster, encoding components of a phosphorelay pathway and an anti-σ factor antagonist, and cfcR, encoding a response regulator with diguanylate cyclase (DGC) activity. Transcription of hsbAR-hptB and cfcR is RpoS-dependent and induced by stationary phase and the stringent response. A ∆hsbA mutant resumed biofilm formation after dispersal in late stationary phase, stimulated liquid-air biofilm formation and increased Congo Red adsorption. All these phenotypes were traced down to increased c-di-GMP levels in stationary phase dependent on the activity of CfcR and its cognate sensor kinase, CfcA. HsbA was reversibly phosphorylated by the combined action of HptB and HsbR HsbA phosphorylation conditioned its interaction with CfcR and CfcA and the intracellular distribution of the three proteins, but did not greatly influence biofilm regulation. Our results support a model in which HsbA forms a complex with CfcR to inhibit its DGC activity regardless of its phosphorylation state. Upon HsbA dephosphorylation, this complex is recruited to the cell membrane by CfcA to strengthen the inhibitory effect. This system contributes to biofilm dispersal by denying biofilm formation, but allows quick onset of de novo biofilm formation in response to physiological cues that release HsbA inhibition to restore CfcR DGC activity.