The Pseudomonas aeruginosa Diguanylate Cyclase GcbA, a Homolog of P. fluorescens GcbA, Promotes Initial Attachment to Surfaces, but Not Biofilm Formation, via Regulation of Motility

Abstract

Cyclic di-GMP is a conserved signaling molecule regulating the transitions between motile and sessile modes of growth in a variety of bacterial species. Recent evidence suggests that Pseudomonas species harbor separate intracellular pools of c-di-GMP to control different phenotypic outputs associated with motility, attachment, and biofilm formation, with multiple diguanylate cyclases (DGCs) playing distinct roles in these processes, yet little is known about the potential conservation of functional DGCs across Pseudomonas species. In the present study, we demonstrate that the P. aeruginosa homolog of the P. fluorescens DGC GcbA involved in promoting biofilm formation via regulation of swimming motility likewise synthesizes c-di-GMP to regulate surface attachment via modulation of motility, however, without affecting subsequent biofilm formation. P. aeruginosa GcbA was found to regulate flagellum-driven motility by suppressing flagellar reversal rates in a manner independent of viscosity, surface hardness, and polysaccharide production. P. fluorescens GcbA was found to be functional in P. aeruginosa and was capable of restoring phenotypes associated with inactivation of gcbA in P. aeruginosa to wild-type levels. Motility and attachment of a gcbA mutant strain could be restored to wild-type levels via overexpression of the small regulatory RNA RsmZ. Furthermore, epistasis analysis revealed that while both contribute to the regulation of initial surface attachment and flagellum-driven motility, GcbA and the phosphodiesterase DipA act within different signaling networks to regulate these processes. Our findings expand the complexity of c-di-GMP signaling in the regulation of the motile-sessile switch by providing yet another potential link to the Gac/Rsm network and suggesting that distinct c-di-GMP-modulating signaling pathways can regulate a single phenotypic output.

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

“…This time point was chosen to ensure that the observed effects were not due to differences in the amounts of attached biomass, as inactivation of gcbA was previously demonstrated to affect attachment following 8 h but not 24 h of growth (39). Under the conditions tested, ⌬gcbA cells exhibited significantly reduced levels of BdlA processing relative to the levels in the wild-type PAO1 cells (Fig.…”

“…GcbA was recently shown to make a greater contribution to c-di-GMP levels in planktonic cells and those undergoing early attachment stages than it does in cells of developed biofilms, with GcbA not affecting the overall c-di-GMP levels of biofilm cells (39). Given that GcbA affected BdlA processing in established P. aeruginosa biofilms, we therefore asked whether BdlA cleavage is initiated during early biofilm formation and, if so, whether BdlA processing is GcbA dependent.…”

“…Complementation and overexpression were accomplished by conjugating into P. aeruginosa strains the pJN105 vector harboring the respective genes under the control of the arabinose-inducible P BAD promoter (45). In order to assess the native protein production levels of GcbA and BdlA, the chromosomal integration vectors pCTXgcbA-V5/His 6 and pCTX-bdlA-V5/His 6 (38,39), harboring constructs encoding the respective C-terminally V5/His 6 -tagged proteins under the control of the native promoters, were introduced into the indicated P. aeruginosa strains via conjugation. The resulting strains (PAO1::P bdlAbdlA-V5/His 6 , PAO1 ⌬gcbA::P bdlA -bdlA-V5/His 6 , PAO1::P gcbA -gcbA-V5/ His 6 , and PAO1 ⌬gcbA::P gcbA -gcbA-V5/His 6 ) contained chromosomally encoded gcbA-V5/His 6 or bdlA-V5/His 6 under the control of the respective native promoter at the attB site.…”

“…In contrast, while BdlA is detectable only in its intact form in planktonic cells, plasmid-borne overexpression of the gene PA4843 triggered BdlA cleavage in planktonic cells (38). PA4843 encodes the recently characterized active DGC GcbA (also identified as AdcA), which was found to be involved in the regulation of the transition from planktonic to surface-associated growth (39,40). Although BdlA plays a pivotal and central role in enabling P. aeruginosa biofilm dispersion in response to a variety of environmental cues, the system contributing to the elevation of c-di-GMP levels responsible for BdlA posttranslational modification and activation is currently not known.…”

Microbial Surface Colonization and Biofilm Development in Marine Environments

“…Cyclic di-GMP (c-di-GMP) is another key and ubiquitous second messenger molecule in prokaryotes, playing central roles in microbial signaling and adaptability (480). Despite the tremendous diversity of microbial components and processes and mechanisms that are involved in the switch from the planktonic to the sessile lifestyle, most bacteria examined to date employ c-di-GMP as the central regulator to control surface colonization and biofilm formation (480)(481)(482). For example, Ruegeria mobilis, a member of the MRC, employs the c-di-GMP regulatory pathway to modulate biofilm formation and antibiotic production (483).…”