Mechanisms of cyclic-di-GMP signaling : insight into the biochemistry, signal transduction and regulation of the bacterial second messenger cyclic-di-GMP

Bacteria are able to switch between two mutually exclusive lifestyles, motile single cells and sedentary multicellular communities that colonize surfaces. These behavioral changes contribute to an increased fitness in structured environments and are controlled by the ubiquitous bacterial second messenger c-di-GMP. In response to changing environments, fluctuating levels of c-di- GMP antagonistically affect motility and virulence of single, planktonic cells as well as cell surface adhesins and persistence of sedentary, multicellular communities. The cellular levels of c-di-GMP are controlled by opposing enzymatic activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), which represent two large families of output domains found in bacterial one- and two-component systems. The present work investigates structural, functional and regulatory aspects of diguanylate cyclases and phosphodiesterases, and explores their role in signal transduction processes transmitting environmental stimuli into a range of different cellular functions. Furthermore we report the isolation and characterization of novel components of the cdi- GMP signaling network mediating its output functions. In (Christen et al. 2005, JBC 280:30829-30837), we report the finding that the c-di-GMP specific phosphodiesterase activity resides in the widespread EAL domain. By analyzing the enzymatic reaction products and investigating the substrate specificity of wild type and various mutant enzymes, we demonstrate that a single EAL domain itself catalyzes, in a Mg2+ dependent manner, the cleavage of the second messenger c-di-GMP into the linear dinucleotide pGpG. Furthermore, we report the discovery that in a GGDEF-EAL protein a catalytic inactive GGDEF domain can bind GTP and in response allosterically activates the EAL domain. Thus we conclude that GGDEF domains can have either catalytic or regulatory function and suggest, that the cellular GTP pool may serve as an input signal into c-di-GMP-mediated signal transduction. In (Christen & Christen et al. 2006, JBC 281:32015-32024), we describe an important novel feature of GGDEF proteins, which produce the ubiquitous bacterial signaling molecule c-di-GMP. This paper reports the results of in depth structure-function analysis of an allosteric feedback inhibition mechanism that generally acts to regulate diguanylate cyclase activities in bacteria. The mechanism involves binding of the second messenger product, c-di-GMP at an inhibition site (Isite) that is coupled via a conserved beta-strand to the active site (A-site) of the enzyme. The study involves an array of biochemical and genetic techniques applied on various diguanylate cyclases to establish the sequence determinants of the I-site as well as the in vivo physiological relevance of Isite function. Allosteric product inhibition of diguanylate cyclases turns out to have fundamental functional and physiological implications, including threshold setting for c-di-GMP production by particular GGDEF proteins, which can contribute to precision, robustness, noise reduction and accelerated kinetics of c-di-GMP signaling. The definition of the I-site binding pocket provides an entry point into unraveling the molecular mechanisms of ligand-protein interactions involved in c-di- GMP signaling, and makes DGCs a valuable target for drug design to develop new strategies against biofilm-related diseases. In (Christen & Christen et al. 2007, PNAS), we enlighten the signal transduction mechanism of the bacterial second messenger c-di-GMP and demonstrate the existence of diguanylate receptor proteins mediating its output functions. We report the biochemical purification of c-di-GMP receptor proteins from C. crescentus crude extract and describe their physiological role in c-di-GMP dependent repression of cell motility. A multitude of biochemical, genetic and NMR experiments was used to characterize these effector proteins and homologs from S. enterica and P. aeruginosa down to molecular level. In particular we used [33P] c-di-GMP UV cross linking studies to demonstrate that these receptors specifically bind c-di-GMP in the sub micromolar range and, in combination with NMR spectrometry, to elicit determinants for c-di-GMP binding. Furthermore, we performed genetic suppressor analysis and epistasis experiments with receptor deletion and pointmutants, to corroborate that the identified diguanlyate receptors from C. crescentus act in vivo downstream of the second messenger c-di-GMP. We further report the isolation and characterization of a C. crescentus adenylosuccinate synthetase (PurA, CC3103), an enzyme of the purine biosynthesis pathway that has high affinity for c-di-GMP. Using recombinant purified PurA for kinetic and ligand binding studies, we show that c-di-GMP is a potent inhibitor of PurA activity. Initial rate kinetics revealed that c-di-GMP inhibition is competitive with respect to GTP and noncompetitive with respect to IMP. These findings suggest a role for c-di-GMP as regulator of the cellular nucleotide pool. We propose that c-di-GMP inhibits the first step of the de novo biosynthesis of AMP and by that directs IMP toward guanine biosynthesis, thereby preventing the drainage of the guanine pool.