Detection of the bacterial second messenger cyclic-di-GMP and evaluation of diguanylate cyclise function by microbiological assays suitable for High-Throughtput-Screening of biofilm inhibitors

Biofilms are microbial communities embedded in a self-produced polymeric matrix, mainly composed by extracellular polysaccharides, which confers resistance to environmental stresses and tolerance to antibiotic treatments. In Gram negative bacteria, production of the cyclic-di-GMP (c-di-GMP) signal molecule is the main trigger for exopolysaccharides (EPS) and adhesion factors\u2019 production: indeed, mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation. In this report, we describe a simple microbiological assay to detect c-di-GMP biosynthesis by bacterial diguanylate cyclase enzymes (DGC\u2019s). The assay relies on expression of a DGC-encoding gene from a multicopy plasmid and detection of c-di-GMP-dependent extracellular factors on agar medium supplemented with Congo red (CR), a dye which binds amyloid fibers and polysaccharides. The CR assay can be performed in 96-well microtiter plates, making it suitable for High-Throughput Screening for chemical inhibitors of c-di-GMP biosynthesis. In addition, it can be used for functional studies of diguanylate cyclases and for the elucidation of their role in production of extracellular structures. We have used the CR assay to assess the effects of five different DGC-encoding genes: adrA, ycdT, ydaM and yddV from Escherichia coli and wspR from Pseudomonas aeruginosa, in a set of E. coli strains deficient in production of EPS and/or adhesion factors. Results of the CR assays suggest that, even when expressed ectopically from a multicopy plasmid, DGC\u2019s affect production of cell surface-associated factors in a specific manner. Different effects of the various DGC\u2019s on production of extracellular structures, cell motility and gene expression could be confirmed by specific assay