The protein Clp from Xanthomonas axonopodis pv. citri regulates pathogenesis and is a member of the CRP (cyclic AMP receptor protein) superfamily. We show that unlike the DNA-binding activity of other members of this family, the DNA-binding activity of Clp is allosterically inhibited by its effector and that cyclic di-GMP serves as that effector at physiological concentrations.Xanthomonas is a genus of bacteria whose members cause disease in a wide variety of commercially important plants throughout the world. The transcription factor Clp (for CRP [cyclic AMP receptor protein]-like protein) regulates the expression of approximately 300 genes involved in pathogenesis in xanthomonads (5). Clp is a homologue (45% sequence identity) of the model transcription factor CRP of Escherichia coli. The six amino acids in CRP that contact cAMP (10) are largely conserved in Xanthomonas axonopodis pv. citri Clp, which suggested that Clp activity might be regulated by some nucleotide.The paradigm for the effector response in the CRP family is activation of DNA binding upon effector binding. However, previous studies using X. campestris pv. campestris Clp have demonstrated that the protein is competent to bind target DNA with high affinity in the absence of any effector (8). This suggests that effector binding by Clp might cause inhibition, which would represent the first reported case of negative allosteric regulation of a CRP superfamily member. Neither adenylate cyclase nor cAMP has been found experimentally in X. campestris pv. campestris (2), and BLAST searches for cya homologues in the sequenced Xanthomonas genomes have also been negative. We therefore hypothesized that Clp is allosterically inhibited by a nucleotide other than cAMP.The signaling network controlling virulence in the xanthomonads is largely encoded in the rpf cluster (for regulation of pathogenicity factors), including RpfG, a phosphodiesterase that degrades the signaling molecule cyclic di-GMP (c-di-GMP) (9). In X. campestris pv. campestris, deletion of either rpfG or clp significantly decreases expression of virulence-associated functions (5). The deletion of ravR, which encodes another c-di-GMP-specific phosphodiesterase in X. campestris, has effects very similar to those of deletion of rpfG, and an rpfG ravR mutant is even more severely affected (4). Collectively, these results suggest that both c-di-GMP and Clp have roles in virulence regulation and that these roles are linked in some way. Based on physiology, we hypothesized that c-di-GMP was the most obvious candidate as a Clp inhibitor.In order to test our hypothesis in vitro, Clp (Xac0483) was PCR amplified from X. axonopodis pv. citri 306 genomic DNA using Pfu Turbo polymerase (Stratagene) with the addition of a C-terminal six-His tag and cloned into the SacI and SmaI sites of pEXT20. His-tagged X. axonopodis pv. citri Clp protein was overexpressed and purified as described previously for CRP (12). Protein purity was Ͼ90%, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data no...

CooA is a CO-sensing protein that activates the transcription of genes encoding the CO-oxidation (coo) regulon, whose polypeptide products are required for utilizing CO as an energy source in Rhodospirillum rubrum. CooA binds to a position overlapping the ؊35 element of the P cooF promoter, similar to the arrangement of class II CRP (cAMP receptor protein)-and FNR (fumarate and nitrate reductase activator protein)-dependent promoters when expressed in Escherichia coli. Gain-of-function CooA variants were isolated in E. coli following mutagenesis of the portion of cooA encoding the effectorbinding domain. Some of the mutations affect regions of CooA that are homologous to the activating regions (AR2 and AR3) previously identified in CRP and FNR, whereas others affect residues that lie in a region of CooA between AR2 and AR3. These CooA variants are comparable to wild-type (WT) CooA in DNA binding affinity in response to CO but differ in transcription activation, presumably because of altered interactions with E. coli RNA polymerase. Based on predictions of similarity to CRP and FNR, loss-of-function CooA variants were obtained in the AR2 and AR3 regions that have minimal transcriptional activity, yet have WT-like DNA binding affinities in response to CO. This study demonstrates that WT CooA contains AR2-and AR3-like surfaces that are required for optimal transcription activation.Rhodospirillum rubrum is a photosynthetic bacterium capable of oxidizing carbon monoxide (CO) to CO 2 with concomitant evolution of H 2 , which is coupled to energy generation (1). The components of the CO-oxidizing (coo) regulon, which are produced only in the presence of exogenous CO, are encoded by two operons, and their expression is controlled by CooA, a transcriptional activator that binds CO under reducing conditions and is itself expressed constitutively from an adjacent operon (2). The structural components of the CO-oxidizing system include CooS (CO dehydrogenase), which oxidizes CO to CO 2 , CooF, a CooS-associated iron-sulfur protein that donates reducing equivalents to CooH, a CO-tolerant hydrogenase (3-5).CooA is a heme-containing protein that belongs to the cAMP receptor protein (CRP 1 (6)) and the fumarate and nitrate reductase activator protein (FNR (7)) superfamily of transcriptional activator proteins. All three proteins are homodimers when competent to bind DNA, and the structures of CooA and CRP reveal effector-binding and DNA-binding domains in each monomer. The structure of CooA in the reduced (Fe II ) form has been solved recently (8) and revealed that the general folding topology of CO-free CooA and cAMP-bound CRP (9) were similar (see Fig. 1 below). In the case of CRP, only the effector (cAMP)-bound structure has been solved (9), and for CooA, only the effector (CO)-free structure is known (8). Significant differences were observed in the positions of the DNA-binding domains of the two proteins. However, because both proteins bind to similar DNA sequences (2), it is assumed that the DNAbinding regions of the effe...