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Nutrients such as amino acids play key roles in shaping the metabolism of microorganisms in natural environments and in host-pathogen interactions. Beyond taking part to cellular metabolism and to protein synthesis, amino acids are also signalling molecules able to influence group behaviour in microorganisms, such as biofilm formation. This lifestyle switch involves complex metabolic reprogramming controlled by local variation of the second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP). The intracellular levels of this dinucleotide are finely tuned by the opposite activity of dedicated diguanylate cyclases (GGDEF signature) and phosphodiesterases (EAL and HD-GYP signatures), which are usually allosterically controlled by a plethora of environmental and metabolic clues. Among the genes putatively involved in controlling c-di-GMP levels in P. aeruginosa, we found that the multidomain transmembrane protein PA0575, bearing the tandem signature GGDEF-EAL, is an L-arginine sensor able to hydrolyse c-di-GMP. Here, we investigate the basis of arginine recognition by integrating bioinformatics, molecular biophysics and microbiology. Although the role of nutrients such as L-arginine in controlling the cellular fate in P. aeruginosa (including biofilm, pathogenicity and virulence) is already well established, we identified the first L-arginine sensor able to link environment sensing, c-di-GMP signalling and biofilm formation in this bacterium.

Bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c-di-GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c-di-GMP-dependent response. Both the c-di-GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c-di-GMP-related processes. Here, we report a structure-function relationship study on the catalytic portion of the PA0575 protein from Pseudomonas aeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF-EAL portion is a GTP-dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide-dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis.

The nitrogen cycle pathways are responsible for the circulation of inorganic and organic N-containing molecules in nature. Among these pathways, those involving amino acids, N-oxides and in particular nitric oxide (NO) play strategic roles in the metabolism of microorganisms in natural environments and in host-pathogen interactions. Beyond their role in the N-cycle, amino acids and NO are also signalling molecules able to influence group behaviour in microorganisms and cell-cell communication in multicellular organisms, including humans. In this minireview, we summarise the role of these compounds in the homeostasis of the bacterial communities called biofilms, commonly found in environmental, industrial and medical settings. Biofilms are difficult to eradicate since they are highly resistant to antimicrobials and to the host immune system. We highlight the effect of amino acids such as glutamate, glutamine and arginine and of NO on the signalling pathways involved in the metabolism of 3',5'-cyclic diguanylic acid (c-di-GMP), a master regulator of motility, attachment and group behaviour in bacteria. The study of the metabolic routes involving these N-containing compounds represents an attractive topic to identify targets for biofilm control in both natural and medical settings.

One of the most important signals involved in controlling biofilm formation is represented by the intracellular second messenger 3',5'-cyclic diguanylic acid (c-di-GMP). Since the pathways involved in c-di-GMP biosynthesis and breakdown are found only in bacteria, targeting c-di-GMP metabolism represents an attractive strategy for the development of biofilm-disrupting drugs. Here, we present the workflow required to perform a structure-based design of inhibitors of diguanylate cyclases, the enzymes responsible for c-di-GMP biosynthesis. Downstream of the virtual screening process, detailed in the first part of the chapter, we report the step-by-step protocols required to test the positive hits in vitro and to validate their selectivity, thus minimizing possible off-target effects.