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The EMBO Journal (2013) 32, 2421–2423 www.embojournal.org Bacterial second messengers, cGMP and c-di-GMP, in a quest for regulatory dominance Mark Gomelsky1,* and Michael Y Galperin2,* 1 Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, USA and 2National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA. *Correspondence to: [email protected] or [email protected] The EMBO Journal (2013) 32, 2421–2423. doi:10.1038/emboj.2013.193; Published online 20 August 2013 Bacteria and eukaryotes differ in the organization of their key signal-transduction pathways but share certain signalling components, including cyclic nucleotide second messengers. In this issue, a paper by British, Irish and Taiwanese scientists (An et al, 2013) describes a signaltransduction pathway that regulates virulence and biofilm formation in the bacterial plant pathogen Xanthomonas campestris. Remarkably, this pathway involves a cascade of two nucleotide second messengers, with cyclic GMP (cGMP), a typically eukaryotic messenger, directly regulating synthesis of cyclic dimeric GMP (c-di-GMP), a ubiquitous bacterial messenger. This study broadens the scope of cGMP-regulated processes in bacteria, offers structural insights into cGMP binding by bacterial cGMP receptors, and expands the range of bacteria using cGMP in signal transduction. Such multi-level regulatory cascades may well function in other organisms. In higher eukaryotes, cAMP and cGMP are important second messengers that mediate effects of light, nitric oxide, hormones and other signals to regulate vision, muscle contraction, vasodilatory effects, sleep, memory and various other functions, see, for example, Beavo and Brunton (2002). Over the last half of a century, studies of cAMP- and cGMPrelated systems have been rewarded with six Nobel prizes in Physiology or Medicine and one in Chemistry. In all eukaryotes, synthesis of cAMP and cGMP is catalysed by similar enzymes that belong to the class III adenylate/ guanylate cyclase family (PF00211 in the Pfam database, cNMP phosphodiesterase O cNMP cNMP Pkinase cGMP cNMP N N H2N EUKARYOTES Ion channel N HN EUKARYOTES O BACTERIA BACTERIA O ? cNMP O PP2C P ? O OH Ion channel cNMP OH cNMP HTH cNMP GGDEF c-di-GMP BIOFILMS VIRULENCE Figure 1 Major cGMP receptors in bacterial and eukaryotic cells. The cGMP-specific cNMP-binding domains (PF00027 in Pfam) are present in both eukaryotes and bacteria. In eukaryotes, major cGMP receptors include protein kinases; cyclic nucleotide-gated ion channels and cNMP phosphodiesterases. In bacteria, the newly characterized cNMP–GGDEF domain fusion protein XC_0249 (UniProt entry Q4V037_XANC8) synthesizes c-di-GMP under the control of cGMP. Other potential cGMP targets include a likely cNMP-regulated ion channel BBta_5447 from Bradyrhizobium sp. (UniProt: A5EML8_BRASB, contains the PF00520 domain), protein serine phosphatase Cagg_2419 from Chloroflexus aggregans (UniProt: B8G3B9_CHLAD, contains the PP2C or SpoIIE domain, PF07228) and Rhodospirillum centenum transcriptional regulator RC1-3788 (Marden et al, 2011). & 2013 European Molecular Biology Organization The EMBO Journal VOL 32 | NO 18 | 2013 2421 cGMP receptors in bacterial and eukaryotic cells M Gomelsky and MY Galperin Punta et al, 2012) but differ in the number and specificity of the attached regulatory domains. The specificity of these cyclases towards their nucleotide substrates is determined by just a few residues in the substrate-binding pocket (Sunahara et al, 1998). The primary targets of cAMP and cGMP regulation are transmembrane ion channels, protein kinases and cNMP-dependent phosphodiesterases, many of which contain a cNMP-binding domain (PF00027), see Figure 1. In bacteria, studies of cyclic nucleotides gained fewer accolades but were no less exciting. Bacterial adenylate cyclases are far more diverse than eukaryotic ones, falling into four structurally distinct classes. Class III enzymes are the most widespread and often found in multiple copies per genome (see http://www.ncbi.nlm.nih.gov/Complete_ Genomes/SignalCensus.html). The best-known cAMP target in bacteria is CRP (cAMP receptor protein), a transcriptional regulator composed of an N-terminal cNMP-binding domain (PF00027) and a DNA-binding domain (PF00325). In contrast to widespread regulation by cAMP, involvement of cGMP in bacterial regulation has only recently been appreciated. The early reports of cGMP-mediated signalling in bacteria could not be confirmed and for a long time cGMP has been viewed as a nonfunctional by-product of adenylate cyclases (Linder, 2010). Only recent studies implicating cGMP in cyanobacterial adaptation to the UV stress (Cadoret et al, 2005) and in formation of cysts (dormant cells) in alphaproteobacteria (Marden et al, 2011) led to its recognition as a genuine bacterial second messenger (Gomelsky, 2011). In the last decade, attention of many bacteriologists has been focused on cyclic dinucleotide second messengers, that is, c-di-GMP and its recently discovered cousins, cyclic dimeric AMP (c-di-AMP) and the cyclic AMP-GMP hybrid (see Römling et al, 2013 for a recent review). Cyclic-di-GMP, found in representatives of all well-sampled bacterial phyla, controls a variety of processes, including production of exopolysaccharides, protein adhesins, pili and flagella, and cell differentiation. It plays a central role in the bacterial lifestyle switch from the motile state to the sessile, multicellular biofilm state, and is also involved in controlling virulence. In 2006, Ryan et al (2006) linked c-di-GMP to virulence and biofilm formation in X. campestris, the causative agent of black rot disease of cruciferous plants. This finding led to a series of studies aimed at characterizing the c-di-GMP targets and the signals that control the c-di-GMP levels in this pathogen. The impetus for the work published by these authors in the current issue of EMBO J (An et al, 2013) was the detection of cGMP in the X. campestris cell lysate. To identify the source of cGMP, the authors of the study analysed a transposon mutant library of X. campestris and found two mutants impaired in cGMP production. Both transposons resided in the XC_0250 gene, which encodes a class III nucleotidyl cyclase. Enzymatic analyses of the purified XC_0250 protein showed that it has a guanylate cyclase (but not adenylate cyclase) activity. Transcriptome analysis of the XC_0250 deletion mutant revealed multiple changes affecting expression of genes associated with motility and surface attachment, stress tolerance, virulence, transport, multidrug resistance, detoxification and signal transduction. Many of these features are typically associated with the c-di-GMP-mediated 2422 The EMBO Journal VOL 32 | NO 18 | 2013 regulation. Indeed, a mechanistic link from cGMP to c-diGMP has emerged from the analysis of the adjacent gene, XC_0249, which encodes a putative c-di-GMP synthase composed of a cNMP-binding domain (PF00027) linked to the diguanylate synthase GGDEF domain (PF00990). The authors overexpressed the cNMP-binding domain of XC_0249, purified it, and determined that it binds to cGMP with high affinity (Kd B0.3 mM), much better than cAMP (Kd B18 mM). Phenotypically, the XC_0250 and XC_0249 mutants were similarly impaired in biofilm formation and virulence. This suggested that they are part of the same signalling pathway, with XC_0249 acting as a downstream effector of XC_0250. Comparative transcriptome profiling of the XC_0250 and XC_0249 mutants revealed 450% commonly regulated genes, confirming this notion. The purified XC_0249 protein proved to have diguanylate cyclase activity that was upregulated by up to four-fold by cGMP. Thus, cGMP synthesized by XC_0250 interacts with the cNMP-binding domain of the diguanylate cyclase XC_0249 to stimulate c-di-GMP synthesis. Earlier, GGDEF domains have been seen linked to the primary sensors of light, oxygen, NO and cellular redox state (reviewed in Römling et al, 2013). GGDEF domains are also often found in two-component response regulators, suggesting the regulation of c-di-GMP synthesis by sensory histidine kinases. The report by An et al (2013) demonstrates a more complex, multi-layer cascade where c-di-GMP synthesis is controlled by another nucleotide second messenger cGMP (whose synthesis is likely controlled by yet another signal). How common are such cGMP–c-di-GMP cascades? The XC_0249-XC_0250 gene pair is conserved in xanthomonads, suggesting that the regulation by cGMP is widespread among these plant pathogens that affect many important crops. Chemicals that inhibit the cGMP–c-di-GMP cascade may be useful in confronting plant diseases caused by xanthomonads. The XC_0249-XC_0250 gene pair is also found in Stenotrophomonas maltophilia, a xanthomonad associated with respiratory and urinary infections, particularly in immunocompromised patients. Further, proteins with the cNMP-binding–GGDEF domain architecture (same as in XC_0249) are encoded by more than 50 other bacteria, which suggests that the cGMP–c-di-GMP regulatory hierarchy may be even more common. An et al, (2013) report the crystal structure of the cNMPbinding domain of XC_0249 with bound cGMP (PDB entry 4KG1), which provides insights into the distribution of cGMPbinding domains in bacteria. The domain structure shows that the cGMP-binding pocket in XC_0249 is similar to the cAMP- and cGMP-binding pockets of other cNMP-binding domains. The residues involved in binding ribose, phosphate and the guanine imidazole ring are mostly conserved, as are hydrophobic residues that pack against the base (see Figure 4 in An et al, 2013). In contrast, residues that interact with the guanine ring are different from those interacting with adenine in the cAMP-specific domains. As expected, mutations in the cGMP-binding residues seen in the structure impaired cGMP binding and downstream signalling. A database search for the sequence motif involved in cGMP binding in XC_0249 retrieves a number of bacterial CPR-like transcriptional regulators, as well as PP2C-type protein phosphatases and ion channels, mostly from Chloroflexi and Deltaproteobacteria. & 2013 European Molecular Biology Organization cGMP receptors in bacterial and eukaryotic cells M Gomelsky and MY Galperin Thus, based on the results of An et al (2013), cGMP may exist in bacteria that have not been suspect otherwise, and it may regulate new kinds of protein targets (Figure 1). Finally, this work can be seen as yet another example of the ongoing expansion of eukaryotic and bacterial cyclic monoand dinucleotide signalling cascades. This year brought the discovery of the first eukaryotic cyclic dinucleotide second messenger, cyclic GMP-AMP hybrid molecule (cGAMP). It is synthesized in response to the presence of DNA in the cytosol and signals through the same stimulator of interferon genes, STING, as do bacterial second messengers, c-di-GMP and c-di-AMP. The presence of either of these cyclic dinucleotide second messengers in the cytoplasm is interpreted by eukaryotic cells as a sign of bacterial or viral invasion and triggers innate immune response (Civril et al, 2013; Zhang et al, 2013). Thus, cGMP that was once considered exclusively eukaryotic second messenger is no longer just eukaryotic, and c-di-NMPs that were once considered exclusively bacterial are no longer exclusively bacterial. These recent developments make the world of cyclic mono- and dinucleotide second messengers more fascinating than ever. Acknowledgements MG is supported by the National Science Foundation (MCB 1052575), National Institutes of Health (R21CA167862) and Cooperative State Research Education Extension Service Grant via Agricultural Experiment Station. MYG is supported by the NIH Intramural Research Program at the National Library of Medicine. Conflict of interest The authors declare that they have no conflict of interest. References An S-Q, Chin K-H, Febrer M, McCarthy Y, Yang J-G, Liu C-L, Swarbreck D, Rogers J, Dow JM, Chou S-H, Ryan RP (2013) A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis. EMBO J 32: 2430–2438 Beavo JA, Brunton LL (2002) Cyclic nucleotide research—still expanding after half a century. Nat Rev Mol Cell Biol 3: 710–718 Cadoret JC, Rousseau B, Perewoska I, Sicora C, Cheregi O, Vass I, Houmard J (2005) Cyclic nucleotides, the photosynthetic apparatus and response to a UV-B stress in the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem 280: 33935–33944 Civril F, Deimling T, de Oliveira Mann CC, Ablasser A, Moldt M, Witte G, Hornung V, Hopfner KP (2013) Structural mechanism of cytosolic DNA sensing by cGAS. 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