MicroReview: C-di-GMP: the dawning of a novel bacterial signaling system C-di-GMP is emerging as a novel global second messenger. Recent findings show involvement in cell metabolism and biological processes in the cell. GGDEF domain protein involved in c-di-GMP synthesis. EAL domain protein involved in c-di-GMP degradation. These two proteins affect cell differentiation, multicellular behavior, and interactions between microorganisms and their eukaryotic hosts. GGDEF and EAL domain found across many branches of the phylogenetic tree of bacteria. 1 GGDEF domain has ½ GTP binding catalytic site, so dimer of 2 GGDEF form active diguanylate cyclase activity (DGC). In E. coli, S. enterica, Pseudomonas fluorescence, activated GGDEF domain proteins and resulting elevated c-di-GMP concentrations favour the production of adhesive matrix components. Also, GGDEF protein is found to play a role in stimulation of motility in E. coli. In C. crescentus, the GGDEF domain protein named PleD controls cell morphology. Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di- guanylate cyclase output domain Background Developmental transitions depend on adaptation in levels and arrangement of proteins. Relies on transcriptional regulators Establishment of gradients through the compartmentalization of signaling complexes. Two component regulatory system Sensor kinase Soluble response regulator C. crescentus has obligate developmental transition that allows switch between sessile, adhesive, and motile cell during cell cycle. Cell poles are continuously remodeled during cell differentiation. In the predivisional cell, a pili being assembled at one pole while the opposite pole consists of a stalk (an adhesive organelle). Purpose of study Investigate the function and regulation of the PleD response regulator in C.crescentus polar development. Investigate whether polar kinases DivJ and PleC involved in modulating the phosphorylation state of PleD. Propose PleD contains an intrinsic nucleotide cyclase activity that converts two molecules of GTP into cycli diguanylic acid (c-di-GMP). DivJ and PleC directly control PleD phosphyrylation Previous studies established a role of polar kinases DivJ and PleC in PleD control (Aldridge et. Al. 2003). Use in vitro phosphorylation assays. The PleD response regulator localizes to the stalked pole PleD is a soluble cytoplasmic protein. DivJ and PleC are sensor kinases membrane bound. Hypothesis: PleD might perform its regulatory function at the pole. Test PleD perform its regulatory function locally Analyzed the subcellular distribution of PleD during the C. crescentus cell cycle. A PleD-GFP fusion was introduced into mutant pleD strain. Analysis showed that most stalked and predivisional cells have PleD-GFP concentrated at the stalked pole. Absent from the flagellated swarmer pole - Data for the insertion into mutant pleD strain was not shown in the table. What is the spatial distribution of PleD-GFP during the cell cycle? Perform time lapse fluorescence microscopy with isolated swarmer cells of WT strain. First, PleD protein evenly distributed within C. crescentus swarmer cells. As time passes then it concentrates at the emerging stalked pole during the swarmer-to- stalked cell differentiation. With increasing time signal at the stalked pole increases in strengths. Localization of activated PleD PleD might exist in two different forms. Test whether phosphorylation of PleD is required for dynamic localization. Fused GFP to an inactive mutant PleD at position 53. Mutated site is Aspartic acid phosphoryl acceptor Immunoblot analysis showed PleD- D53N-GFP mutant was distributed evenly in all cells. Also, PleD-GFP in absence of DivJ and PleC kinases failed to localize at the pole So phosphorylation is important in sequestering PleD to the pole. Test to distinguish between: Phosphorylation might constitute the targeting signal PleD prefers binding to the cell pole in its active confirmation Analyzed constitutively active PleD mutant protein, PleD* D53N (lacks the phosphoryl acceptor site @ position 53). PleD* D53N –GFP localizes almost exclusively to the pole in WT and divJ, pleC double mutant. Analysis of PleDGG368DE-GFP fusion protein, which lacks an active C-terminal output domain. Results: localization of PleD at the pole. Conclusion: An activated conformation of PleD, rather than the PleD readout itself, is required for polar sequestration of the regulator. What is the output signal of PleD? We know now PleD accumulates at the old pole of the cell only in its activated state. We know from genetic data (Aldridge et. Al. 2003) phosphorylated PleD is required for the differentiation of a flagellated into a stalked pole. We know that PleD could coordinate the developmental events involved in pole remodeling. Past research (Ross et. Al. 1991, Tal et. Al. 1998) showed a link between: GGDEF domain Synthesis of cyclic di-GMP To examine possibilities that the PleD output domain harbors di-guanylate cyclase activity. Performed a biochemical assay to test PleD’s ability to convert GTP into c-di-GMP. Extracts of CB15N ∆pleD showed no activity. 5 10 15 20 30 minutes To demonstrate that PleD was responsible for the activity. PleD with a C-terminal His-tag was overexpressed in E. Coli. 30 45 60 300 seconds Confirm indeed it is cyclic GMP The reaction product of PleD and GTP was analyzed by mass spectrometry. Exactly matches the molecular weight of c-di-GMP C-di-GMP Competing? When synthesized c-di-GMP was added to the reaction mix to a similar concentration of the GTP substrate. Each reaction mix contained: 25µg PleD 100µM of GTP Suggest that c-di-GMP competes with GTP for the binding site. Does PleD function as a phosphodiesterase (PDE)? C-di-GMP concentration was measured quantatively by High- pressured Liquid Chromatography (HPLC) after incubation with purified PleD-His6 protein for several hours. HPLC analysis of Reaction Products Reactants 2 minute intervals This suggests that PleD has a di- guanylate cyclase activity but lacks phosphodiesterase activity. The di-guanylate cyclase activity constitutes as the signaling output of the PleD response regulator. Cyclases activity is GTP specific Unlabeled nucleotides Formation of c-di-GMP Structure of PleD response regulator suggests: Receiver Domain (N-terminal) information input Regulatory output domain (C-terminal) contains GGDEF domain Test whether the guanylate cyclase activity is indeed localized in the GGDEF domain. Determined activity of WT PleD through: Examining the activities of two mutant proteins in the GGDEF domain. Mutant alleles pleD∆368-372 (lacking the entire GGDEF motif) pleDGG368DE (2 Gly replaced by Asp and Glu) Results showed both mutant proteins lacked di-guanylate cyclase activity in vitro. Thus, requires an intact GGDEF output domain. Discussion Activated confirmation of the protein provides the information for polar localization. Not, phosphorylation itself. Two different mechanisms can be envisioned to explain the coupling between activity and polar localization of PleD. PleD could auto-catalytically controls its own subcellular positioning Pole recognition might be restricted to the activated form of PleD. Observations of inactive PleDD53N-GFP Present throughout the cell Observations of GFP fused to PleDGG368DE, unable to generate c-di-GMP. Present at the pole Better favors the 2nd mechanism. More conclusions GGDEF domain of PleD posseses DGC, cyclase activity but not PDE activity. GGDEF domains represent a signal output of a complex bacterial signal transduction network, in which leads to the production of c-di-GMP. GGDEF domain and different sensory inputs Presence of a large number of potential DGCs in single bacterial species raises the question of how the output specificity of parallel signaling pathways might be achieved?