Cyclic dimeric GMP, a novel bacterial second messenger: Enzymology of its turnover

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has come to the limelight as a result of recent advances in microbial genomics and increased interest in multicellular microbial behavior. Known for nearly 20 years as an activator of cellulose synthase in Gluconacetobacter xylinus, c-di-GMP is emerging as a novel second messenger, involved in regulation of bacterial adhesion, motility and virulence in a variety of bacteria. C-di-GMP synthesis and hydrolysis in G. xylinus had been associated with the proteins containing conserved protein domains GGDEF and EAL. However, many questions regarding c-di-GMP-dependent signaling remained unanswered. Among those, the key questions were the biochemistry of synthesis and degradation of c-di-GMP and the targets of c-di-GMP. To investigate the enzymology of c-di-GMP turnover, the proteins containing GGDEF domains from six representatives of diverse bacterial species were cloned, overexpressed in Escherichia coli and purified. They were shown to possess diguanylate cyclase (DGC) activity. This established that c-di-GMP synthesis is a wide-spread phenomenon in Bacteria. The individual GGDEF domains were shown to possess a low level DGC activity, which illustrates that the DGC activity is intrinsic to the GGDEF domain. Two factors, protein homodimerization and activation by the input signals from sensory domains, were required for full activity. To investigate biochemical activities of the EAL domain, the E. coli protein YahA and its individual EAL domain were purified and found to hydrolyze c-di-GMP. This provided the first biochemical evidence that EAL domain is sufficient for phosphodiesterase (PDE) activity. The PDE activity proved to be c-di-GMP specific, Mg2+ or Mn 2+-dependent, strongly inhibited by Ca2+ and optimal at alkaline pH. The immediate product of the c-diGMP hydrolysis was shown to be 5'-pGpG. Multiple amino acid sequence alignment of EAL domains from active PDEs and EAL domains from active DGCs containing both GGDEF and EAL domains revealed that the latter group corresponds to enzymatically inactive domains. This was based on the observation that EAL domains from DGCs contain substitutions in the conserved residues. An unusual bacteriophytochrome BphG1 from Rhodobacter sphaeroides, containing both GGDEF and EAL domains, has been characterized. Unlike known phytochromes, BphG1 does not have a kinase activity, instead, it possessed a PDE activity which was shown to be light-independent. Upon overexpression in E. coli, BphG1 was found to undergo partial cleavage into two species. The smaller species was identified as the EAL domain from BphG1 expressing PDE activity, while the larger species, lacking EAL, was found to possess photoactivated DGC activity. BphG1 appeared to be the first identified bifunctional enzyme. To investigate the mechanism of c-di-GMP action, the E. coli protein YcgR, containing newly discovered PilZ domain, was overexpressed and purified. It was found to bind c-diGMP in vitro tightly and specifically, with a Kd of 0.84 μM. The individual PilZ domain from YcgR also bound c-di-GMP, albeit with lesser affinity, indicating that PilZ is sufficient for binding. The site-directed mutagenesis performed on YcgR implicated the most conserved residues in the PilZ domain directly in c-di-GMP binding. YcgR is therefore the second identified c-di-GMP receptor after cellulose synthase. In enterobacteria, YcgR is involved in regulation of flagella-based motility in a c-di-GMP-dependent manner. This work provides significant insights into the biochemistry of c-di-GMP turnover and molecular mechanism of the novel bacterial second messenger c-di-GMP.