Rrp1 is the sole c-di-GMP producing protein (diguanylate cyclase) of Borrelia burgdorferi. To test the hypothesis that Rrp1 regulates critical processes involved in the transmission of spirochetes between ticks and mammals, an rrp1 deletion mutant (B31-Δrrp1) and a strain that constitutively produces elevated levels of Rrp1 (B31-OV) were constructed. The strains were assessed for progression through the enzootic cycle using an Ixodes tick/C3H-HeJ mouse model and tick immersion feeding methods. B31-Δrrp1 infected mice as efficiently as wild type but had altered motility, decreased chemotactic responses to N-acetylglucosamine (NAG) and attenuated ability to disseminate or colonize distal organs. While this strain infected mice, it was not able to survive in ticks. In contrast, the B31-OV displayed normal motility patterns and chemotactic responses but was non-infectious in mice. Using immersion feeding techniques we demonstrate that B31-OV can establish a population in ticks and survive exposure to a natural bloodmeal. The results presented here indicate Rrp1, and by extension, c-di-GMP, are not required for murine infection, but are required for the successful establishment of a productive population of B. burgdorferi in ticks. These analyses provide significant new insight into the genetic regulatory mechanisms

In Gram-negative bacteria, production of bis-(3',5')-cyclic diguanylic acid (c-di-GMP) by diguanylate cyclases (DGCs) is the main trigger for production of extracellular polysaccharides and for biofilm formation. Mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation, thus making DGCs interesting targets for new antimicrobial agents with anti-biofilm activity. In this report, we describe a strategy for the screening for DGC inhibitors consisting of a combination of three microbiological assays. The primary assay utilizes an Escherichia coli strain overexpressing the adrA gene, encoding the DGC protein AdrA, and relies on detection of AdrA-dependent cellulose production as red colony phenotype on solid medium supplemented with the dye Congo red (CR). Presence of DGC inhibitors blocking AdrA activity would result in a white phenotype on CR medium. The CR assay can be performed in 96-well microtiter plates, making it suitable for high-throughput screenings. To confirm specific inhibition of c-di-GMP biosynthesis, chemical compounds positive in the CR assay are tested for their ability to inhibit biofilm formation and in a reporter gene assay which monitors expression of curli-encoding genes as a function of DGC activity. Screening of a chemical library using the described approach allowed us to identify sulfathiazole, an antimetabolite drug, as an inhibitor of c-di-GMP biosynthesis. Sulfathiazole probably affects c-di-GMP biosynthesis in an indirect fashion rather than by binding to DGCs; however, sulfathiazole represents the first example of drug able to affect biofilm formation by interfering with c-di-GMP metabolism.

In Gram-negative bacteria, production of the signal molecule c-di-GMP by diguanylate cyclases (DGCs) is a key trigger for biofilm formation, which, in turn, is often required for the development of chronic bacterial infections. Thus, DGCs represent interesting targets for new chemotherapeutic drugs with anti-biofilm activity. We searched for inhibitors of the WspR protein, a Pseudomonas aeruginosa DGC involved in biofilm formation and production of virulence factors, using a set of microbiological assays developed in an Escherichia coli strain expressing the wspR gene. We found that azathioprine, an immunosuppressive drug used in the treatment of Crohn's disease, was able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells. However, in vitro enzymatic assays ruled out direct inhibition of WspR DGC activity either by azathioprine or by its metabolic derivative 2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an enzyme involved in purine biosynthesis, which suggests that inhibition of c-di-GMP biosynthesis by azathioprine may be due to perturbation of intracellular nucleotide pools. Consistent with this hypothesis, WspR activity is abolished in an E. coli purH mutant strain, unable to produce AICAR transformylase. Despite its effect on WspR, azathioprine failed to prevent biofilm formation by P. aeruginosa; however, it affected production of extracellular structures in E. coli clinical isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in this bacterium. Our results indicate that azathioprine can prevent biofilm formation in E. coli through inhibition of c-di-GMP biosynthesis and suggest that such inhibition might contribute to its anti-inflammatory activity in Crohn's disease.

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...

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