Molecular mechanisms of polar flagella assembly and biofilm development in Pseudomonas putida

Abstract

Life cycles of most bacteria are characterized by the alternation of a single cell-based planktonic stage and a sessile stage during which they develop highly structured communities associated to surfaces known as biofilms. Biofilm development proceeds through distinct stages of adhesion, proliferation, and maturation, and concludes with programmed biofilm dispersal in response to physiological or environmental conditions. For many bacteria, transition to the planktonic lifestyle implies de novo assembly of one or several flagella, complex proteinaceous appendages that allow motility in liquid and semi-solid media. Pseudomonas putida is a Gram-negative rhizosphere-colonizing bacterium that carries a tuft of 3-6 flagella at one single pole. A single region in the P. putida genome, the flagellar cluster, encodes at least 63 genes potentially involved in the biogenesis and regulation of the flagellar system. In this work, we clarify the transcriptional organization of the flagellar cluster and define the complete regulon of the flagellar sigma factor FliA. After each cell division, the flagellar apparatus and its associated chemotaxis machinery are newly synthesized and correctly positioned at the cell pole to ensure motility of both daughter cells. We have shown that the polar landmark protein FlhF determines polar location of the flagellar proteins, while FleN regulates the flagella number. In addition, ParC and ParP coordinate polar anchoring of the chemotaxis proteins. Our findings revealed that the polar organizer FimV is a peptidoglycan-binding protein linked to the site of division that mediates recruitment of both machineries. Thus, FimV is essential to polar anchoring of FleN and ParC and prevents stochastic dissociation of FlhF from the cell pole. We have also identified a novel function for FleN and FimV in preventing premature initiation of flagellar assembly. On the other hand, biofilms of P. putida undergo rapid dispersal in response to nutritional stress, mediated by the nucleotide signalling molecule (p)ppGpp. Through transcriptomic analysis, we have identified that (p)ppGpp induces the expression of cfcR, encoding a two-component system response regulator with diguanylate cyclase activity, and the hsbAR-hptB operon encoding a phosphorelay pathway and an anti-sigma factor antagonist. We have unveiled that HsbA prevents biofilm formation during stationary phase by inhibiting c-di-GMP production by CfcR. Our results support that HsbA phosphorylation controls the association of an HsbA-CfcR complex with the transmembrane sensor histidine kinase CfcA.