Cyclic-di-GMP-binding CRP-like protein: a spectacular new role for a veteran signal transduction actor.

The note by Leduc and Roberts (14) in this issue of the Journal of Bacteriology sheds new light (i) on the mechanisms of action of the bacterial second messenger, cyclic dimeric GMP (c-di-GMP), and (ii) on the functions of the well-known family of transcription factors. The authors describe Clp from the plant pathogen Xanthomonas axonopodis pv. citri as a new c-di-GMP-binding protein. Surprisingly, Clp belongs to the CRP/CAP (cyclic AMP receptor protein or catabolite activator protein) family of transcription factors. To fully appreciate the implications of this finding, we will take a closer look at the veteran signal transduction actors (CRP-like proteins) and at their newly found calling (c-di-GMP binding). Let’s start with c-di-GMP. This cyclic dinucleotide was discovered over 20 years ago by Moshe Benziman and his colleagues at Hebrew University of Jerusalem (21, 22). However, until recently, c-di-GMP remained unknown to the majority of microbiologists. These days, c-di-GMP is at the height of its fame. No wonder—it has been shown to play key roles in a number of important decisions that bacteria make while adjusting their different lifestyles. One of the best-understood lifestyle changes affected by c-di-GMP involves a transition from the motile single-cellular state to the surface-attached multicellular state that can ultimately lead to biofilm formation. This transition has been explored most extensively in the Proteobacteria (reviewed in references 10 and 20). c-di-GMP has also been shown to affect bacterial virulence (reviewed in reference 26), development (18), cell cycle progression (5), cell differentiation (16), long-term survival (13), etc. There is little doubt that the number and diversity of phenomena found to be affected by c-di-GMP will increase as c-di-GMP continues to be “discovered” in new bacterial species. After all, c-di-GMP is just a messenger, and as such, it can be adapted to receive messages from various inputs and to deliver them to diverse outputs. The field of c-di-GMP-dependent signaling has experienced remarkable progress in recent years. We have learned a great deal about how c-di-GMP is made by diguanylate cyclases containing the GGDEF domain, how it is hydrolyzed by phosphodiesterases containing either the EAL or HD-GYP domain, how these enzymes work at the atomic resolution level, and how some of them are regulated by external factors. We have learned that c-di-GMP acts globally, at the whole-cell level, as well as locally, akin to signaling molecules in eukaryotes. We have learned about several kinds of protein domains and motifs that bind c-di-GMP (10, 20). However, we are just beginning to uncover the mechanisms through which c-di-GMP works, and that is where the contribution of Leduc and Roberts lies. Prior to the report by Leduc and Roberts, we knew about the following sites and protein domains involved in c-di-GMP binding (Fig. 1): (i) the PilZ (Pfam: PF07238) (6) domain (1, 24); (ii) the I site, an allosteric site for feedback inhibition present in the GGDEF-domain diguanylate cyclases (3), which is also present in the degenerate GGDEF domains that have lost enzymatic activity (15); (iii) the enzymatically inactive EAL domains, which retain ability to bind c-di-GMP but can no longer hydrolyze it (17); and (iv) the enzymatically inactive HD-GYP domains, which can bind c-di-GMP. The proteins belonging to the latter category have yet to be experimentally characterized, but their existence is readily predictable. These protein domains and binding sites are specific for c-di-GMP and can be deduced from sequence analysis, with at least some certainty. There is also a class of c-di-GMP-specific riboswitches, whose sequences are also identifiable, that control gene expression in a c-di-GMP-dependent manner (25). And then there are “unpredictable” c-di-GMP-binding proteins. The first such protein, FleQ from Pseudomonas aeruginosa, was discovered by Hickman and Harwood (11). FleQ is a transcriptional regulator that works with a -type factor. It represses expression of the flagellar biosynthesis genes and activates expression of the polysaccharide pel biosynthesis genes involved in biofilm formation. c-di-GMP decreases the affinity of FleQ to the pel promoter. The discovery of FleQ broke the orderly view of c-di-GMP-binding proteins that had just started to emerge. The paper by Leduc and Roberts is in some ways a sequel to the FleQ story. It describes a CRP-like transcription factor whose affinity to DNA is regulated by c-diGMP (Fig. 1). The CRP family proteins are expected to bind cAMP. The notion that Clp, which shares 45% sequence identity with the Escherichia coli CRP, binds and responds to a different nucleotide looks at first glance like “molecular treason.” However, a closer look reveals that the matter is not quite as dramatic as it first appears. CRP consists of two protein domains, the cNMP-binding domain (PF00027), involved in cyclic nucleotide binding, and the so-called Crp domain (PF00325), a DNA-binding domain specific to the CRP family. The cNMP-binding domain can bind cAMP, but it can also bind cGMP. Therefore, with respect to the ligand specificity of the cNMP-binding domain, a cAMP/cGMP dichotomy already exists. Furthermore, the * Mailing address: Department of Molecular Biology, University of Wyoming, Laramie, WY 82071. Phone: (307) 766-3522. Fax: (307) 766-3875. E-mail: [email protected]. Published ahead of print on 11 September 2009.