The Pseudomonas putida flhA-flhF-fleN-fliA cluster encodes a component of the flagellar export gate and three regulatory elements potentially involved in flagellar biogenesis and other functions. Here we show that these four genes form an operon, whose transcription is driven from the upstream PflhA promoter. A second promoter, PflhF, provides additional transcription of the three distal genes. PflhA and PflhF are σN-dependent, activated by the flagellar regulator FleQ, and negatively regulated by FleN. Motility, surface adhesion and colonization defects of a transposon insertion mutant in flhF revealed transcriptional polarity on fleN and fliA, as the former was required for strong surface adhesion and biofilm formation, and the latter was required for flagellar synthesis. On the other hand, FlhF and FleN were necessary to attain proper flagellar location and number for a fully functional flagellar complement. FleN, along with FleQ and the second messenger c-di-GMP differentially regulated transcription of lapA and the bcs operon, encoding a large adhesion protein and cellulose synthase. FleQ positively regulated the PlapA promoter and activation was antagonized by FleN and c-di-GMP. PbcsD was negatively regulated by FleQ and FleN, and repression was antagonized by c-di-GMP. FleN promoted FleQ binding to both PlapA and PbcsD in vitro, while c-di-GMP antagonized interaction with PbcsD and stimulated interaction with PlapA. A single FleQ binding site in PlapA was critical to activation in vivo. Our results suggest that FleQ, FleN and c-di-GMP cooperate to coordinate the regulation of flagellar motility and biofilm development.

Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida.