Subcellular location characteristics of the Pseudomonas aeruginosa GGDEF protein, WspR, indicate that it produces cyclic‐di‐GMP in response to growth on surfaces

Abstract

SummaryThe Pseudomonas aeruginosa Wsp signal transduction system produces cyclic-di-GMP (c-di-GMP), an intracellular messenger that stimulates biofilm formation and suppresses motility. The Wsp system is homologous to chemotaxis systems and includes a membrane-bound receptor protein, WspA, and a response regulator GGDEF protein, WspR, that catalyses c-di-GMP synthesis when phosphorylated. We found that the subcellular distributions of fluorescent protein-tagged WspA and WspR differed markedly from their chemotaxis counterparts. WspA-YFP formed patches in cells whereas WspR-YFP was dispersed when unphosphorylated and formed bright cytoplasmic clusters when phosphorylated. WspR formed clusters in cells of a DwspF mutant, a genetic background that causes constitutive phosphorylation of WspR, but was dispersed in cells of a wspA mutant, a genetic background necessary for WspR phosphorylation. In addition, WspR mutated at Asp70, its predicted site of phosphorylation, did not form clusters. C-di-GMP synthesis was not required for cluster formation. WspR-YFP was dispersed in liquid-grown wild-type cells, but formed clusters that sometimes appeared and disappeared over the course of a few minutes in cells grown on an agar surface. Our results suggest that the compartmentalized production of c-di-GMP in response to a stimulus associated with growth on a surface is an important functional characteristic of the Wsp system.

Quantification of high-specificity cyclic diguanylate signaling

“…This differential activity could be attributable to a number of factors, including growth on a surface, density of the culture, oxygen concentration, or degree of agitation. Surface activation of a DGC has been described previously because the DGC WspR from P. aeruginosa forms active clusters when grown on a solid surface (25). This observation further highlights the importance of quantifying c-di-GMP and phenotypic output from identical growth conditions.…”

Self-produced exopolysaccharide is a signal that stimulates biofilm formation inPseudomonas aeruginosa

“…3A). WspR, which is involved in c-di-GMP synthesis in response to surface contact (15,17), had no role in the Psl response (Fig. S3).…”

“…We initially targeted our analysis to three diguanylate cyclases that have been strongly linked to P. aeruginosa biofilm formation: SadC, SiaD, and WspR (15,16,28). Using directed mutagenesis, we demonstrated that SadC and SiaD both contribute to Pslinduced c-di-GMP production (Fig.…”

“…However, few specific instances of environmental signals that modulate the activity of these enzymes have been identified. In P. aeruginosa, the diguanylate cyclases SiaD and WspR are activated upon exposure to SDS or contact with solid surfaces, respectively (15)(16)(17). In Escherichia coli, the activity of the DosCP cyclase/phosphodiesterase pair is modulated by oxygen availability (18), whereas diffusible signal factor was shown to activate c-di-GMP phosphodiesterase activity through the two-component regulatory system RpfG/C in Xanthomonas campestris (19).…”

Global Regulatory Pathways and Cross-talk ControlPseudomonas aeruginosaEnvironmental Lifestyle and Virulence Phenotype

“…WspR consists of a CheYlike phospho-receiver domain and a GGDEF domain that has DGC activity in vivo and in vitro (D'Argenio, 2002;Hickman, 2005;Güvener, 2007). WspR is a response regulator encoded by the wsp operon (wspABCDEFR), which comprises a multi-component chemosensory system with homology to the well-characterized chemotaxis (Che) pathway of E. coli (Ridgway, 1977;D'Argenio, 2002;Güvener, 2007).…”