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

Abstract

We previously showed the isolation of biofilmpersistent Pseudomonas putida mutants that fail to undergo biofilm dispersal upon entry in stationary phase. Two such mutants were found to bear insertions in PP0914, encoding a GGDEF/EAL domain protein with high similarity to Pseudomon asaeruginosa BifA. Here we show the phenotypic characterization of a ΔbifA mutant in P. putida KT2442.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 ΔbifA mutant correlates with a general increase in cyclic diguanylate (c-di-GMP) levels, Congo Red-binding exopolyaccharide 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 ΔbifA 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.

Trophic regulation of autoaggregation in Pseudomonas taiwanensis VLB120

“…The scenario that a GGDEF domain that is responsible for c-di-GMP synthesis and an EAL domain that degrades c-di-GMP are arranged in tandem in the same enzyme is very common, but frequently, one of the two domains is catalytically inactive (Roemling et al 2013). PVLB_04710 shows high similarity to BifA of P. aeruginosa and P. putida (81 and 96 % amino acid identity, respectively), which were shown to possess solely phosphodiesterase activity (Jiménez-Fernández et al 2015;Kuchma et al 2007Kuchma et al , 2010. Additionally, PVLB_04710 carries a highly conserved EAL motif and an altered GGDQF motif which supports the classification of PVLB_04710 as a phosphodiesterase.…”

“…The P. aeruginosa ΔbifA mutant developed WR colonies, showing an increased extracellular polysaccharide production, hyperbiofilm formation and a defect in swarming motility; however, autoaggregation was not reported (Kuchma et al 2007). P. putida ΔbifA displayed increased biofilm and pellicle formation, enhanced exopolysaccharide production and autoaggregated in liquid culture (Jiménez-Fernández et al 2015). Several of these characteristics like the WR morphology, the elevated exopolysaccharides and the autoaggregation are identical to the behaviour of the here-described mutants; however, a carbon source dependency of the aggregative behaviour has never been revealed before.…”

“…To elucidate transcriptional cross links between the five genes which were separately deactivated in the five respective aggregating mutants, reverse transcription quantitative real-time PCR (RT-qPCR) was performed. Irrespective of the mutation, the transcription of a gene encoding a putative phosphodiesterase that is highly similar to the phosphodiesterase BifA of other pseudomonads (Jiménez-Fernández et al 2015;Kuchma et al 2007Kuchma et al , 2010Martinez-Granero et al 2014), degrading c-di-GMP, was decreased or even deactivated in all mutants. To support these findings, overexpression of this gene completely prevented autoaggregation in all mutants.…”

“…During the past two decades, c-diElectronic supplementary material The online version of this article (doi:10.1007/s00253-015-7006-2) contains supplementary material, which is available to authorized users. GMP has been described in numerous studies to be a ubiquitous regulator in biofilms (Roemling et al 2013) and was also suggested to play a key role during aggregate formation (Jiménez-Fernández et al 2015;Klebensberger et al 2007;Starkey et al 2009). Besides this global regulator, the composition of the lipopolysaccharide (LPS) has been shown to play an important part in aggregate formation.…”

Adaptation Genomics of a Small-Colony Variant in a Pseudomonas chlororaphis 30-84 Biofilm

“…We hypothesized that these mutations either affect c-di-GMP levels (e.g., reduce pools to be more similar to wild-type levels) or directly affect phenotypes controlled by c-di-GMP levels, e.g., type IV pili or extracellular polymeric substance production (13,15,35). The phenotypic revertant characterized in this study had a point mutation in the gene encoding a well-studied PDE, bifA (33,36). Hypothetically, a mutation that increases the catalytic activity of the PDE bifA could counteract the effects of the yfiR mutation by breaking down the c-di-GMP that accumulated in the cells as a result of the unregulated catalytic activity of yfiN (tpbB).…”