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The EMBO Journal (2013) 32, 2421–2423
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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
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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
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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. Nature 498: 332–337
Gomelsky M (2011) cAMP, c-di-GMP, c-di-AMP and now cGMP:
bacteria use them all! Mol Microbiol 79: 562–565
Linder JU (2010) cGMP production in bacteria. Mol Cell Biochem
334: 215–219
Marden JN, Dong Q, Roychowdhury S, Berleman JE, Bauer CE
(2011) Cyclic GMP controls Rhodospirillum centenum cyst development. Mol Microbiol 79: 600–615
& 2013 European Molecular Biology Organization
Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C,
Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L,
Sonnhammer EL, Eddy SR, Bateman A, Finn RD (2012)
The Pfam protein families database. Nucleic Acids Res 40:
D290–D301
Römling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the
first 25 years of a universal bacterial second messenger. Microbiol
Mol Biol Rev 77: 1–52
Ryan RP, Fouhy Y, Lucey JF, Crossman LC, Spiro S, He YW, Zhang
LH, Heeb S, Camara M, Williams P, Dow JM (2006) Cell-cell
signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc Natl
Acad Sci USA 103: 6712–6717
Sunahara RK, Beuve A, Tesmer JJ, Sprang SR, Garbers DL, Gilman
AG (1998) Exchange of substrate and inhibitor specificities
between adenylyl and guanylyl cyclases. J Biol Chem 273:
16332–16338
Zhang X, Shi H, Wu J, Sun L, Chen C, Chen ZJ (2013) Cyclic GMPAMP containing mixed phosphodiester linkages is an endogenous
high-affinity ligand for STING. Mol Cell 51: 226–235
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