A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels

Abstract

Pseudomonas aeruginosa causes chronic biofilm infections, and its ability to attach to surfaces and other cells is important for biofilm formation and maintenance. Mutations in a gene called wspF, part of a putative chemosensory signal-transduction operon, have been shown to result in cell aggregation and altered colony morphology. The WspF phenotypes depend on the presence of WspR, which is a member of a family of signal transduction proteins known as response regulators. It is likely that the effect of the wspF mutation is to cause constitutive activation of WspR by phosphorylation. WspR contains a GGDEF domain known to catalyze formation of a cytoplasmic signaling molecule cyclic diguanylate (c-diGMP). We determined that purified WspR catalyzed the formation of c-diGMP in vitro and phosphorylation stimulated this activity. We observed increased cellular levels of c-diGMP and increased biofilm formation in a wspF mutant. Expression of a protein predicted to catalyze degradation of c-diGMP reversed the phenotypes of a wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-diGMP is necessary for biofilm formation. A transcriptome analysis showed that expression levels of at least 560 genes were affected by a wspF deletion. The psl and pel operons, which are involved in exopolysaccharide production and biofilm formation, were expressed at high levels in a wspF mutant. Together, the data suggest that the wsp signal transduction pathway regulates biofilm formation through modulation of cyclic diguanylate levels.Pseudomonas aeruginosa ͉ GGDEF domain ͉ exopolysaccharide ͉ EAL domain B iofilms are surface-associated multicellular communities encased in a self-produced extracellular matrix. Existence in a biofilm provides many advantages over a planktonic (single-cell) existence, including increased resistance to predation and antimicrobial agents (1-4). Biofilms of P. aeruginosa cause chronic infections of humans with underlying predispositions (5).A number of genes involved in adherence and aggregation of P. aeruginosa, properties that are thought to be important during biofilm formation, have been identified (6-10). A mutation in one such gene, wspF, has been shown to result in increased cell aggregation and a wrinkled colony morphology (7). wspF is part of a gene cluster predicted to encode a signal transduction system similar to that which regulates swimming-mediated chemotaxis in bacteria (7). The WspF protein is homologous to CheB, a methylesterase involved in adaptation to chemotactic stimuli. Chemotaxis signal transduction systems also include response regulators called CheY. The response regulator of the Wsp chemosensory system, WspR, is a CheY homolog that contains the conserved GGDEF domain (7). These domains have been implicated in the formation of the intracellular signaling molecule cyclic diguanylate (c-diGMP) (11-15).c-diGMP was first identified in Gluconacetobacter xylinus, where it regulates production of cellulose through modulation of cellulose synthase a...

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…WspR is part of the Wsp chemosensory signal transduction system, which operates to produce c-di-GMP as its effector output function. The Wsp system is homologous to chemotaxis systems and includes a membrane-bound receptor protein, WspA, and a response regulator GGDEF protein, WspR, that catalyzes c-di-GMP synthesis when phosphorylated and upon contact with surfaces (61). In addition to controlling the adhesiveness of P. aeruginosa upon surface contact, WspR is also linked to regulation of the chaperone usher pathway (cup) genes that encode putative fimbrial adhesins.…”

“…Mutations in a gene called wspF, part of the Wsp chemosensory signal-transduction operon, have been shown to result in cell aggregation and altered colony morphology, a phenotype which correlated with increased cellular levels of c-di-GMP and increased biofilm formation. Transcriptomic analysis suggested that the wspF mutant phenotype can be attributed to increased expression of the pel and psl operons, which were among at least 560 genes with affected expression in this strain (61). Expression of a protein predicted to catalyze degradation of c-di-GMP (e.g., PvrR) reversed the phenotype of a ⌬wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-di-GMP is necessary for biofilm formation (42,61).…”

“…Transcriptomic analysis suggested that the wspF mutant phenotype can be attributed to increased expression of the pel and psl operons, which were among at least 560 genes with affected expression in this strain (61). Expression of a protein predicted to catalyze degradation of c-di-GMP (e.g., PvrR) reversed the phenotype of a ⌬wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-di-GMP is necessary for biofilm formation (42,61). A mutant screen in Yersinia pestis resulted in the identification of three c-di-GMP-modulating proteins playing a role in attachment and biofilm formation, including the DGC HmsT and the PDE HmsP, which inversely regulate biofilm formation by controlling the synthesis of the polysaccharide poly-␤-1,6-N-acetylglucosamine, both in vitro and in the foregut of its flea vector, as well as y3730, encoding a functional DGC (13,148).…”

The structure–function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain

“…Although the biological pathways through which c-di-GMP regulates persistence, cell aggregation and the switch to the commensal lifestyle are currently poorly understood, the proteins responsible for c-di-GMP synthesis and degradation have been determined and, at least partially, characterized. Bacterial diguanylate cyclase (DGC) activity is found in GGDEF domain-containing proteins (Paul et al, 2004;Ryjenkov et al, 2005;Hickman et al, 2005;Kulesekara et al, 2006), whilst EAL domain-containing proteins have been shown to have c-di-GMP-specific phosphodiesterase (PDE) activity (Bobrov et al, 2005;Christen et al, 2005;Schmidt et al, 2005;Kulesekara et al, 2006).…”

“…For example, PleD controls the onset of motility during the C. crescentus cell cycle (Aldridge et al, 2003;Paul et al, 2004). In Pseudomonas aeruginosa, autoaggregation is controlled by the GGDEF response regulator WspR (D'Argenio et al, 2002;Hickman et al, 2005), and twitching motility by FimX, which contains GGDEF and EAL domains (Huang et al, 2003;Kazmierczak et al, 2006). Additional examples of the effects of GGDEF domains on cell attachment and motility include ScrC regulation of attachment factors in Vibrio parahaemolyticus (Boles & McCarter, 2002), control of curli fimbriae by AdrA in S. typhimurium (Simm et al, 2004), and RocS effects on V. cholerae motility (Rashid et al, 2003).…”