Downloaded from www.microbiologyresearch.org by IP: 54.159.222.200 On: Fri, 13 May 2016 03:11:13 The diguanylate cyclase YddV controls production of the exopolysaccharide poly-N-acetylglucosamine (PNAG) through regulation of the PNAG biosynthetic pgaABCD operon Letizia Tagliabue, 1 Davide Antoniani, 1 Anna Macia ˛g, 1 Paola Bocci, 2 Nadia Raffaelli 2 and Paolo Landini 1 Correspondence Paolo Landini paolo.landini@unimi.it Received 7 May 2010 Revised 21 June 2010 Accepted 23 June 2010 1 Department of Biomolecular Sciences and Biotechnology, Universita ` degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy 2 Department of Molecular Pathology and Innovative Therapies, Section of Biochemistry, Universita ` Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy 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. INTRODUCTION Most 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., 2007, 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 (Me ´ndez-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 host (Simm et al., Abbreviations: c-di-GMP, bis-(39,59)-cyclic di-guanylic acid; CF, Calcofluor; CR, Congo red; DGC, diguanylate cyclase; EPS, extracellular polysaccharides; PDE, c-di-GMP phosphodiesterase; PNAG, poly-N- acetylglucosamine; UTR, untranslated region. A supplementary figure, showing surface adhesion on polystyrene microtitre plates of strains carrying the pGEM-T Easy control vector, pAdrA, pYcdT and pYdaM, and two supplementary tables, showing the primers used in this work and the effects of DGC overexpression on cell motility and cell aggregation, are available with the online version of this paper. Microbiology (2010), 156, 2901–2911 DOI 10.1099/mic.0.041350-0 041350 G 2010 SGM Printed in Great Britain 2901