Little is known about cyclic AMP (cAMP) function in Mycobacterium tuberculosis, despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins. M. tuberculosis Rv3676, which we have designated CRP Mt , is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRP Mt 's interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRP Mt Tuberculosis (TB) remains a serious global health problem that is growing at an estimated rate of 3% per year (49). This TB epidemic is exacerbated by an unexplained synergy with human immunodeficiency virus and steadily increasing rates of drug resistance that are a by-product of lengthy treatment regimens (15,20). A better understanding of Mycobacterium tuberculosis biology is needed to improve treatment and develop a more effective vaccine. A key area of interest is how M. tuberculosis senses and responds to the environments it encounters during host infection.Cyclic AMP (cAMP) is a critical signaling molecule in many bacterial and eukaryotic cells. The role of cAMP signal transduction in mediating catabolite repression has been well characterized in Escherichia coli, and this forms the paradigm for cAMP-mediated gene regulation in prokaryotes (7,10,11,16,33,36). A class I adenylate cyclase (AC) in E. coli catalyzes the synthesis of cAMP, which then transduces the signal by binding cAMP receptor protein (CRP) and activating it as a transcription factor (18). cAMP signaling is also critical for virulence in a diverse range of pathogens, including yeast, fungi, bacteria, and parasites (3,12,19,25,37,38,42,50,70). In some cases, cAMP regulates virulence genes within the pathogen (3, 38, 42). For example, CRP-cAMP signaling is essential for virulence in Salmonella enterica serovar Typhimurium (17) and has recently been shown to control virulence-associated type III secretion systems in Pseudomonas aeruginosa and Yersinia enterocolitica (50, 70).The M. tuberculosis genome contains 15 putative class III adenylate cyclase genes (46). The activity of at least 10 of these cyclases has been confirmed with biochemical assays (13,26,40,41,61,64), making it likely that cAMP contributes substantially to signal transduction in M. tuberculosis. We recently identified the first cAMP-regulated genes in M. tuberculosis by using an exogenous cAMP culture model (24). Some of these genes are upregulated during intracellular growth in macrophages (29), suggesting that cAMP signaling may be important to M. tuberculosis during its interaction with the host. This observation is intriguing in light of a previous study that reported elevated levels of cAMP in macrophages that showed an impairment of phagosome-lysosome fusion upon infection with Mycobacterium microti (44).The mechanism of cAMP-mediated gene regulation in M. tuberculosis has not been explored. We previously reported that the M. tuberculosis Rv3676 protein belongs to a superfamily of proteins that contain both cAMP binding and helix-tur...

We have analyzed the cyclic nucleotide (cNMP)-binding protein and nucleotide cyclase superfamilies using Bayesian computational methods of protein family identification and classification. In addition to the known cNMP-binding proteins (cNMP-dependent kinases, cNMP-gated channels, cAMP-guanine nucleotide exchange factors, and bacterial cAMP-dependent transcription factors), new functional groups of cNMP-binding proteins were identified, including putative ABC-transporter subunits, translocases, and esterases. Classification of the nucleotide cyclases revealed subtle differences in sequence conservation of the active site that distinguish the five classes of cyclases: the multicellular eukaryotic adenylyl cyclases, the eukaryotic receptor-type guanylyl cyclases, the eukaryotic soluble guanylyl cyclases, the unicellular eukaryotic and prokaryotic adenylyl cyclases, and the putative prokaryotic guanylyl cyclases. Phylogenetic distribution of the cNMP-binding proteins and cyclases was analyzed, with particular attention to the 22 complete archaeal and eubacterial genome sequences. Mycobacterium tuberculosis H37Rv and Synechocystis PCC6803 were each found to encode several more putative cNMP-binding proteins than other prokaryotes; many of these proteins are of unknown function. M. tuberculosis also encodes several more putative nucleotide cyclases than other prokaryotic species.Signal transduction pathways control many critical cellular processes, including chemotaxis, differentiation, proliferation, and apoptosis. For example, signal transduction pathways are necessary for bacterial pathogens to sense and respond to host environments, cellular differentiation during embryogenesis, conductance of nerve impulses, and cell cycle control. Disruption of these pathways can result in neoplasia, arteriosclerosis, neurological and developmental abnormalities, and cell death. The most common mechanisms of signal transduction include the phosphorylation or dephosphorylation of effector proteins by kinases and phosphatases, respectively, and the production of second messengers. Cyclic nucleotides were first recognized as second messengers 40 years ago. Such diverse molecules as (p)ppGpp, Ca 2+ , inositol triphosphate, and diacylglycerol have also been recognized as second messengers since then.The cyclic nucleotides adenosine 3Ј,5Ј-cyclic monophosphate (cAMP) and guanosine 3Ј,5Ј-cyclic monophosphate (cGMP) are key universal second messengers, mediating cellular functions in organisms as phylogenetically diverse as Escherichia coli and Homo sapiens. Intracellular concentrations of cyclic nucleotides (cNMPs) are controlled by regulation of their relative rates of synthesis, excretion, and degradation (Botsford and Harman 1992;). The nucleotide cyclases (adenylyl and guanylyl cyclase), the cNMP phosphodiesterases, and the cyclic nucleotide effector proteins (cNMP-binding proteins) have been particularly intense areas of signal transduction research, providing detailed studies of these proteins (for reviews, see Kolb et

The availability of the complete genome sequence for Shewanella oneidensis MR-1 has permitted a comprehensive characterization of the ferric uptake regulator (Fur) modulon in this dissimilatory metal-reducing bacterium. We have employed targeted gene mutagenesis, DNA microarrays, proteomic analysis using liquid chromatography-mass spectrometry, and computational motif discovery tools to define the S. oneidensis Fur regulon. Using this integrated approach, we identified nine probable operons (containing 24 genes) and 15 individual open reading frames (ORFs), either with unknown functions or encoding products annotated as transport or binding proteins, that are predicted to be direct targets of Fur-mediated repression. This study suggested, for the first time, possible roles for four operons and eight ORFs with unknown functions in iron metabolism or iron transport-related functions. Proteomic analysis clearly identified a number of transporters, binding proteins, and receptors related to iron uptake that were up-regulated in response to a fur deletion and verified the expression of nine genes originally annotated as pseudogenes. Comparison of the transcriptome and proteome data revealed strong correlation for genes shown to be undergoing large changes at the transcript level. A number of genes encoding components of the electron transport system were also differentially expressed in a fur deletion mutant. The gene omcA (SO1779), which encodes a decaheme cytochrome c, exhibited significant decreases in both mRNA and protein abundance in the fur mutant and possessed a strong candidate Fur-binding site in its upstream region, thus suggesting that omcA may be a direct target of Fur activation.