Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile.

International audienceThe catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (cre(CD) motif), that is, 'RRGAAAANGTTTTCWW'. Binding of purified CcpA protein to 19 target cre(CD) sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators

Spx mediates oxidative stress regulation of the methionine sulfoxide reductases operon in Bacillus subtilis

<p>Abstract</p> <p>Background</p> <p>All aerobically grown living cells are exposed to oxidative damage by reactive oxygen species (ROS). A major damage by ROS to proteins is caused by covalent modifications of methionine residues giving methionine sulfoxide (Met-SO). Methionine sulfoxide reductases are enzymes able to regenerate methionine and restore protein function after oxidative damage.</p> <p>Results</p> <p>We characterized the methionine sulfoxide reductase genes <it>msrA </it>and <it>msrB </it>in <it>Bacillus subtilis</it>, forming an operon transcribed from a single sigma A-dependent promoter. The <it>msrAB </it>operon was specifically induced by oxidative stress caused by paraquat (PQ) but not by H₂O₂. Spx, a global oxidative stress regulator in <it>B. subtilis</it>, is primarily responsible for this PQ-specific induction of <it>msrAB </it>expression. In support of this finding, an <it>spx </it>deletion mutant is extremely sensitive to PQ, and increased expression of <it>msrA </it>was identified in a <it>clpX </it>mutant in which Spx accumulated. However, the Spx effect was also visible under conditions where the protein did not accumulate (PQ treatment), suggesting a specific molecular effect at the level of the Spx protein. Indeed, the CXXC motif of Spx was found essential for its function in the PQ-specific induction of <it>msrAB </it>expression. PQ caused a modification of Spx requiring at least one of the cysteines of the CXXC motif of Spx. The PQ modified form of Spx showed a dynamic change <it>in vivo</it>.</p> <p>Conclusion</p> <p>The Spx mediated PQ-specific regulation pathway of the <it>msrAB </it>operon in <it>B. subtilis </it>is reported. Our results suggest that PQ induced the expression of <it>msrAB </it>partially through an oxidation on Spx via modification of its CXXC motif.</p

Global regulation of gene expression in response to cysteine availability in Clostridium perfringens

<p>Abstract</p> <p>Background</p> <p>Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In <it>Clostridium perfringens</it>, the two-component system, VirR/VirS, controls the expression of the <it>ubiG </it>operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the <it>C. perfringens </it>virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail.</p> <p>Results</p> <p>We first performed a tentative reconstruction of sulfur metabolism in <it>C. perfringens </it>and correlated these data with the growth of strain 13 in the presence of various sulfur sources. Surprisingly, <it>C. perfringens </it>can convert cysteine to methionine by an atypical still uncharacterized pathway. We further compared the expression profiles of strain 13 after growth in the presence of cystine or homocysteine that corresponds to conditions of cysteine depletion. Among the 177 genes differentially expressed, we found genes involved in sulfur metabolism and controlled by premature termination of transcription via a cysteine specific T-box system (<it>cysK</it>-<it>cysE</it>, <it>cysP1 </it>and <it>cysP2</it>) or an S-box riboswitch (<it>metK </it>and <it>metT</it>). We also showed that the <it>ubiG </it>operon was submitted to a triple regulation by cysteine availability via a T-box system, by the VirR/VirS system via the VR-RNA and by the VirX regulatory RNA.</p> <p>In addition, we found that expression of <it>pfoA </it>(theta-toxin), <it>nagL </it>(one of the five genes encoding hyaluronidases) and genes involved in the maintenance of cell redox status was differentially expressed in response to cysteine availability. Finally, we showed that the expression of genes involved in [Fe-S] clusters biogenesis and of the <it>ldh </it>gene encoding the lactate dehydrogenase was induced during cysteine limitation.</p> <p>Conclusion</p> <p>Several key functions for the cellular physiology of this anaerobic bacterium were controlled in response to cysteine availability. While most of the genes involved in sulfur metabolism are regulated by premature termination of transcription, other still uncharacterized mechanisms of regulation participated in the induction of gene expression during cysteine starvation.</p

Reconstruction and analysis of the genetic and metabolic regulatory networks of the central metabolism of Bacillus subtilis

International audienceBACKGROUND: Few genome-scale models of organisms focus on the regulatory networks and none of them integrates all known levels of regulation. In particular, the regulations involving metabolite pools are often neglected. However, metabolite pools link the metabolic to the genetic network through genetic regulations, including those involving effectors of transcription factors or riboswitches. Consequently, they play pivotal roles in the global organization of the genetic and metabolic regulatory networks. RESULTS: We report the manually curated reconstruction of the genetic and metabolic regulatory networks of the central metabolism of Bacillus subtilis (transcriptional, translational and post-translational regulations and modulation of enzymatic activities). We provide a systematic graphic representation of regulations of each metabolic pathway based on the central role of metabolites in regulation. We show that the complex regulatory network of B. subtilis can be decomposed as sets of locally regulated modules, which are coordinated by global regulators. CONCLUSION: This work reveals the strong involvement of metabolite pools in the general regulation of the metabolic network. Breaking the metabolic network down into modules based on the control of metabolite pools reveals the functional organization of the genetic and metabolic regulatory networks of B. subtilis

Clostridioides difficile para-Cresol Production Is Induced by the Precursor para-Hydroxyphenylacetate.

Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para-cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para-hydroxyphenylacetate (p-HPA) to produce p-cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA, and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p-HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P1, P2, and P?54; only the P1 promoter was responsible for both basal and p-HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p-HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p-HPA in minimal media. We show that induction of the hpdBCA operon in response to p-HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-N13-CTTTTT) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p-HPA, which drives the production of p-cresol. This provides insights into the regulatory control of p-cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis.IMPORTANCE Clostridioides difficile infection results from antibiotic-associated dysbiosis. para-Cresol, a phenolic compound produced by C. difficile, selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p-HPA to p-cresol, is upregulated in response to elevated exogenous p-HPA, with induction occurring between >0.1 and ?0.25?mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p-HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p-HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p-HPA was required for p-cresol production. Identifying regulatory controls of p-cresol production will provide novel therapeutic targets to prevent p-cresol production, reducing C. difficile's competitive advantage

The Pleiotropic CymR Regulator of Staphylococcus aureus Plays an Important Role in Virulence and Stress Response

We have characterized a novel pleiotropic role for CymR, the master regulator of cysteine metabolism. We show here that CymR plays an important role both in stress response and virulence of Staphylococcus aureus. Genes involved in detoxification processes, including oxidative stress response and metal ion homeostasis, were differentially expressed in a ΔcymR mutant. Deletion of cymR resulted in increased sensitivity to hydrogen peroxide-, disulfide-, tellurite- and copper-induced stresses. Estimation of metabolite pools suggests that this heightened sensitivity could be the result of profound metabolic changes in the ΔcymR mutant, with an increase in the intracellular cysteine pool and hydrogen sulfide formation. Since resistance to oxidative stress within the host organism is important for pathogen survival, we investigated the role of CymR during the infectious process. Our results indicate that the deletion of cymR promotes survival of S. aureus inside macrophages, whereas virulence of the ΔcymR mutant is highly impaired in mice. These data indicate that CymR plays a major role in virulence and adaptation of S. aureus for survival within the host

Caracterisation des genes de l'operon levanase de Bacillus subtilis et etude de la regulation de leur expression

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

The Regulatory Networks That Control Clostridium difficile Toxin Synthesis

The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exotoxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of toxins that are produced during host infection. This observation strengthened the idea that the regulation of toxin synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and toxin synthesis strongly suggests that toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection

Caractérisation de gènes impliqués dans le métabolisme du soufre chez Bacillus subtilis

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF