The c‐di‐GMP phosphodiesterase BifA regulates biofilm development in Pseudomonas putida

Summary We previously showed the isolation of biofilm‐persistent P seudomonas putida mutants that fail to undergo biofilm dispersal upon entry in stationary phase. Two such mutants were found to bear insertions in PP 0914, encoding a GGDEF/EAL domain protein with high similarity to P seudomonas aeruginosa BifA . Here we show the phenotypic characterization of a Δ bifA mutant in P . putida KT 2442. This mutant displayed increased biofilm and pellicle formation, cell aggregation in liquid medium and decreased starvation‐induced biofilm dispersal relative to the wild type. Unlike its P . aeruginosa counterpart, P . putida BifA did not affect swarming motility. The hyperadherent phenotype of the Δ bifA mutant correlates with a general increase in cyclic diguanylate ( c‐di‐GMP ) levels, C ongo Red‐binding exopolysaccharide production and transcription of the adhesin‐encoding lapA gene. Integrity of the EAL motif and a modified GGDEF motif (altered to GGDQF ) were crucial for BifA activity, and c‐di‐GMP depletion by overexpression of a heterologous c‐di‐GMP phosphodiesterase in the Δ bifA mutant restored wild‐type biofilm dispersal and lapA expression. Our results indicate that BifA is a phosphodiesterase involved in the regulation of the c‐di‐GMP pool and required for the generation of the low c‐di‐GMP signal that triggers starvation‐induced biofilm dispersal.