The Diguanylate Cyclase GcbA Facilitates Pseudomonas aeruginosa Biofilm Dispersion by Activating BdlA

Abstract

Biofilm dispersion is a highly regulated process that allows biofilm bacteria to respond to changing environmental conditions and to disseminate to new locations. The dispersion of biofilms formed by the opportunistic pathogen Pseudomonas aeruginosa is known to require a number of cyclic di-GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and the chemosensory protein BdlA, with BdlA playing a pivotal role in regulating PDE activity and enabling dispersion in response to a wide array of cues. BdlA is activated during biofilm growth via posttranslational modifications and nonprocessive cleavage in a manner that is dependent on elevated c-di-GMP levels. Here, we provide evidence that the diguanylate cyclase (DGC) GcbA contributes to the regulation of BdlA cleavage shortly after initial cellular attachment to surfaces and, thus, plays an essential role in allowing biofilm cells to disperse in response to increasing concentrations of a variety of substances, including carbohydrates, heavy metals, and nitric oxide. DGC activity of GcbA was required for its function, as a catalytically inactive variant could not rescue impaired BdlA processing or the dispersion-deficient phenotype of gcbA mutant biofilms to wild-type levels. While modulating BdlA cleavage during biofilm growth, GcbA itself was found to be subject to c-di-GMP-dependent and growth-mode-specific regulation. GcbA production was suppressed in mature wild-type biofilms and could be induced by reducing c-di-GMP levels via overexpression of genes encoding PDEs. Taken together, the present findings demonstrate that the regulatory functions of c-di-GMP-synthesizing DGCs expand beyond surface attachment and biofilm formation and illustrate a novel role for DGCs in the regulation of the reverse sessile-motile transition of dispersion.T he process of biofilm dispersion represents an important and highly regulated phenotypic switch that allows cells residing within a biofilm to respond to changing conditions outside and within biofilm structures and to evade environmental stresses. Dispersion occurs in response to a wide array of signals, with dispersion-inducing conditions ranging from directly sensed environmental cues to self-synthesized signaling molecules (1-6). Biofilm dispersion in various bacterial species, including Aeromonas hydrophila, Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens, is tightly linked to nutrient availability, with both elevation and reduction in available nutrients, such as carbon and nitrogen sources, having been previously reported to trigger the process (1, 2, 7-12). Additional environmental dispersion triggers include temperature changes (13), oxidative and nitrosative stress (4, 14, 15), oxygen limitation (16, 17), and iron availability (18). More recently, the accumulation of D-amino acids has been found to trigger dispersion of biofilms formed by Bacillus subtilis and to prevent biofilm formation by P. aeruginosa and Staphylococcus aureus, with various reports suggesting that the mechanism of action may b...

Escaping the biofilm in more than one way: desorption, detachment or dispersion

“…This is supported by the findings that dispersed cells differ significantly from biofilm cells with respect to gene expression, protein production, and posttranslational modification [2,9,44,47-49]. Compared to biofilm cells, dispersed cells are furthermore characterized by decreased levels of the intracellular signaling molecule c-di-GMP and increased production of matrix degrading enzymes [27,38-40,50,51]. While dispersion has often been described to coincide with increased motility of the escaping bacteria, several studies demonstrated that motility is not required for dispersion [42,44,52,53], suggesting that motility may be an indicator of dispersed cells returning to the planktonic mode of growth, but is not part of the mechanism enabling dispersion.…”

FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

“…8). MDS_0805 codes for a response regulator-GGDEF domain protein, homologous to GcbA, which was shown to facilitate P. aeruginosa biofilm dispersion by activating BdlA (31). Finally, in Pseudomonas brassicacearum and Pseudomonas fluorescens F113 strains, we found homologous operons that have in their promoter regions (upstream of PSEBR_a3747 and PSF113_1970) two sequences (GTCAAGGACTTGAT and GTCA TTTTGCTGAC) that resemble the FleQ consensus binding motif (see Fig.…”

What's on the Outside Matters: The Role of the Extracellular Polymeric Substance of Gram-negative Biofilms in Evading Host Immunity and as a Target for Therapeutic Intervention

“…Due to its pivotal importance in biofilm development, much of the work in this area has focused on the action of the second messenger cyclic di-GMP (see the accompanying background in the Valentini and Filloux review (93)). A recent study showed that the diguanylate cyclase GcbA mediated Pa biofilm dispersal via activation of the chemosensory protein BdlA (94). In a related line of investigation, a substituted fatty acid messenger, cis-2-decenoic acid (CDA), produced by Pa can disperse biofilms formed by a range of bacteria and even Candida albicans (95).…”