HsbA represses stationary phase biofilm formation in Pseudomonas 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 ΔhsbA mutant resumed biofilm formation after dispersal in late stationary phase and displayed increased pellicle formation at the medium-air interphase and 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 subcellular distribution of the three proteins. In spite of this, HsbA retained its ability to prevent biofilm formation regardless of its phosphorylation state. 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. While this pathway contributes to biofilm dispersal by denying de novo c-di-GMP synthesis during nutrient starvation, it may also enable quick restoration of the biofilm phenotype to colonize new sites or during biofilm maturation. HIGHLIGHTS Transcription of hsbAR-hptB is activated by the stringent response and RpoS. HptB and HsbR control the phosphorylation state of HsbA. HsbA prevents biofilm formation in stationary phase. HsbA inhibits the DGC activity of CfcR regardless of its phosphorylation state. Unphosphorylated HsbA elicits formation of a membrane-bound HsbA-CfcR-CfcA complex. Competing Interest Statement The authors have declared no competing interest. Footnotes Fig. 1 panel 1 modified. Supplementary Fig. S4 added Supplementary Fig. S15 added. Multiple text edits, including major edits in the Discussion