The diguanylate cyclase YddV controls production of the exopolysaccharide poly-N-acetylglucosamine (PNAG) through regulation of the PNAG biosynthetic pgaABCD operon

Abstract

In Gram-negative bacteria, production of adhesion factors and extracellular polysaccharides (EPS) is promoted by the activity of diguanylate cyclases (DGCs), a class of enzymes able to catalyse the synthesis of the signal molecule bis-(39,59)-cyclic di-guanylic acid (c-di-GMP). In this report we show that in Escherichia coli, overexpression of the YddV protein, but not of other DGCs such as AdrA and YcdT, induces the production of the EPS poly-N-acetylglucosamine (PNAG) by stimulating expression of pgaABCD, the PNAG-biosynthetic operon. Stimulation of PNAG production and activation of pgaABCD expression by the YddV protein are abolished by inactivation of its GGDEF motif, responsible for DGC activity. Consistent with the effects of YddV overexpression, inactivation of the yddV gene negatively affects pgaABCD transcription and PNAG-mediated biofilm formation. pgaABCD regulation by the yddV gene also takes place in a mutant carrying a partial deletion of the csrA gene, which encodes the main regulator of pgaABCD expression, suggesting that YddV does not regulate pgaABCD through modulation of CsrA activity. Our results demonstrate that PNAG production does not simply respond to intracellular c-di-GMP concentration, but specifically requires the DGC activity of the YddV protein, thus supporting the notion that in E. coli, c-di-GMP biosynthesis by a given DGC protein triggers regulatory events that lead to activation of specific sets of EPS biosynthetic genes or proteins. INTRODUCTIONMost bacteria are able to switch between two different 'lifestyles': single cells (planktonic mode) and biofilm, i.e. a sessile microbial community. Biofilm and planktonic cells differ significantly in their physiology, in their gene expression pattern and even in their morphology. In particular, biofilm cells are characterized by production of adhesion factors and extracellular polysaccharides (EPS), resistance to environmental stresses, and lower sensitivity to antibiotics compared with planktonic cells (Costerton et al., 1995;Anderl et al., 2000;Harrison et al., 2007Harrison et al., , 2009).Transition from planktonic cells to biofilm is regulated by environmental and physiological cues, relayed to the bacterial cell by signal molecules or 'second messengers'. A second messenger, bis-(39,59)-cyclic diguanylic acid, better known as cyclic-di-GMP (c-di-GMP), plays a pivotal role in biofilm formation and maintenance by stimulating production of EPS and adhesion factors (Ross et al., 1991;Simm et al., 2004;Kader et al., 2006;Weber et al., 2006). In addition, c-di-GMP biosynthesis affects important cellular processes, such as morphological differentiation and cell replication in Caulobacter crescentus (Paul et al., 2004), cell motility (Méndez-Ortiz et al., 2006; Jonas et al., 2008) and virulence factor production (Kulasakara et al., 2006;Hammer & Bassler, 2009). In Enterobacteria, c-di-GMP seems to be involved in regulation of adhesion factors, such as curli and cellulose, important for adaptation and survival outside the warm-blooded hos...

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…Two DGCs, DosC (YddV) and YdeH, are required for PAG-dependent biofilm formation. While DosC affects transcription of the PAG biosynthesis operon, pgaABCD, the activity of YdeH stabilizes the PAG biosynthesis protein PgaD posttranscriptionally (266,271). The molecular mechanism of PAG activation by c-di-GMP has recently been elucidated (271a).…”

Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes

“…atrosepticum could be induced to flocculate through c-di-GMP-dependent production of the exopolysaccharide adhesin poly-b-1,6-N-acetyl-D-glucosamine (PNAG) (Itoh et al, 2008;Pérez-Mendoza et al, 2011b;Tagliabue et al, 2010). PNAG (and the associated flocculation phenotype) was expressed upon induction of a heterologous, constitutively active PleD, revealing that Pe.…”

N-Acetylglucosamine-dependent biofilm formation in Pectobacterium atrosepticum is cryptic and activated by elevated c-di-GMP levels

“…However, the unaltered levels of bgalactosidase activity observed (with a transcriptional lacZ fusion to the pga promoter following perturbations of ppGpp or c-di-GMP levels) argue in favour of posttranscriptional regulation (Boehm et al, 2009). By contrast, Tagliabue et al (2010) have shown that c-di-GMP positively upregulates transcript levels of the E. coli pgaABCD operon. Furthermore, those authors demonstrate that PGA production does not simply respond to intracellular c-di-GMP concentration, but specifically requires the DGC activity of the YddV protein and is not affected by the overexpression of two other active DGCs, AdrA and YcdT (Tagliabue et al, 2010).…”

“…By contrast, Tagliabue et al (2010) have shown that c-di-GMP positively upregulates transcript levels of the E. coli pgaABCD operon. Furthermore, those authors demonstrate that PGA production does not simply respond to intracellular c-di-GMP concentration, but specifically requires the DGC activity of the YddV protein and is not affected by the overexpression of two other active DGCs, AdrA and YcdT (Tagliabue et al, 2010).…”

“…CsrA has also been associated with c-di-GMP metabolism in E. coli, in which this protein regulates the expression of several GGDEF proteins at the post-transcriptional level (Jonas et al, 2008). The regulation of PGA by c-di-GMP is not well defined, and recently c-di-GMP has been proposed to control expression of the E. coli pgaABCD operon at posttranscriptional and post-translational levels (Boehm et al, 2009;Tagliabue et al, 2010). A role for c-di-GMP in the regulation of PGA synthesis by the hmsHFRS operon of the animal pathogen Yersinia pestis has also been described (Bobrov et al, 2008(Bobrov et al, , 2011Kirillina et al, 2004).…”