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

André, Gaëlle; Haudecoeur, Elise; Monot, Marc; Ohtani, Kaori; Shimizu, Tohru; Dupuy, Bruno; Martin‐Verstraete, Isabelle

doi:10.1186/1471-2180-10-234

Cited by 21 publications

(44 citation statements)

References 63 publications

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“…Escherichia coli strain NEB 10-beta (BioLabs) and E. coli strain HB101(RP4) were used, respectively, for cloning and as a donor strain for C. difficile conjugation experiments. C. difficile strains were grown anaerobically (5% H 2 , 5% CO 2 , 90% N 2 ) in PY medium (20 (12). After 9 h of cell growth, 15 mM acetate or 10 mM pyruvate, Na 2 S, or formate was added to the PY medium.…”

Section: Methodsmentioning

confidence: 99%

“…Links between bacterial virulence and cysteine metabolism have been described in several pathogenic bacteria. In Clostridium perfringens and Bordetella pertussis, toxin synthesis is regulated in response to cysteine availability (20,21). Additionally, genes involved in sulfur metabolism are induced when Mycobacterium tuberculosis, Yersinia ruckeri, Staphylococcus aureus, and Nesseiria meningitidis interact with human cells (22)(23)(24).…”

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Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

et al. 2016

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The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H 2 S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601. Clostridium difficile is a Gram-positive spore-forming obligate anaerobe and the major cause of nosocomial diarrhea associated with antibiotic therapy. The symptoms of C. difficile infection (CDI) vary from mild diarrhea to life-threatening pseudomembranous colitis, a severe form of CDI (1). Virulent C. difficile strains produce two large toxins: an enterotoxin (TcdA) and a cytotoxin (TcdB). The tcdA and tcdB genes are clustered within a single chromosomal region, called the pathogenicity locus (PaLoc), with three accessory genes: tcdR, tcdE, and tcdC. The expression of the toxin genes is controlled through the coordinated action of the alternative sigma factor TcdR and its antagonist factor, TcdC (2-4). The tcdE gene encodes a holin-like protein that is required for toxin release (5).The spectrum of diseases caused by C. difficile depends on host factors and, for the severe forms, on the level of toxins produced, suggesting that the regulation of toxin synthesis is a critical determinant of C. difficile pathogenicity (6). Toxin production starts when C. difficile cultures enter the stationary growth phase (7) and is modulated in response to various environmental signals. Exposure to subinhibitory ...

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Section: Methodsmentioning

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Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

et al. 2016

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“…RNA was labeled with either Cy3 or Cy5 fluorescent dye (GE Healthcare, Little Chalfont, United Kingdom) using a SuperScript Indirect cDNA labeling kit (Invitrogen) as previously described (4). The cDNAs were then mixed with Cy3 or Cy5 dyes, incubated for 1 h at room temperature in the dark, and purified on SNAP columns (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile

et al. 2011

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Toxin synthesis in Clostridium difficile increases as cells enter into stationary phase. We first compared the expression profiles of strain 630E during exponential growth and at the onset of stationary phase and showed that genes involved in sporulation, cellular division, and motility, as well as carbon and amino acid metabolism, were differentially expressed under these conditions. We inactivated the sigH gene, which encodes an alternative sigma factor involved in the transition to post-exponential phase in Bacillus subtilis. Then, we compared the expression profiles of strain 630E and the sigH mutant after 10 h of growth. About 60% of the genes that were differentially expressed between exponential and stationary phases, including genes involved in motility, sporulation, and metabolism, were regulated by SigH, which thus appears to be a key regulator of the transition phase in C. difficile. SigH positively controls several genes required for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The spo0A and CD2492 genes, encoding the master regulator of sporulation and one of its associated kinases, and the spoIIA operon were transcribed from a SigH-dependent promoter. The expression of tcdA and tcdB, encoding the toxins, and of tcdR, encoding the sigma factor required for toxin production, increased in a sigH mutant. Finally, SigH regulates the expression of genes encoding surface-associated proteins, such as the Cwp66 adhesin, the S-layer precursor, and the flagellum components. Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting a SigH consensus in their promoter regions are good candidates for direct SigH targets.

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“…IscR is a [2Fe-2S] cluster-containing transcriptional regulator encoded by the first gene of the iscRSUA-hscBA-fdx operon that regulates both ISC and SUF systems in E. coli and other Gram-negative bacteria (10,11). Apart from the sequence-unrelated SufR found in cyanobacteria (31,32), no IscR homolog was described in Grampositive bacteria, with the possible exception of some species of the Clostridium genus for which functional data are still lacking (30). In T. potens, the TherJR_1914 gene encodes a protein of the Rrf2 family of transcriptional regulators, sharing only 37% identity with E. coli IscR but with full conservation of the cysteine residues known to coordinate the [2Fe-2S] cluster (Cys 92, 98, 104 , E. coli numbering) (Fig.…”

Section: Resultsmentioning

confidence: 99%

The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium

Santos

Alonso-García

Macedo-Ribeiro

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Iron-sulfur clusters function as cofactors of a wide range of proteins, with diverse molecular roles in both prokaryotic and eukaryotic cells. Dedicated machineries assemble the clusters and deliver them to the final acceptor molecules in a tightly regulated process. In the prototypical Gram-negative bacterium Escherichia coli, the two existing iron-sulfur cluster assembly systems, iron-sulfur cluster (ISC) and sulfur assimilation (SUF) pathways, are closely interconnected. The ISC pathway regulator, IscR, is a transcription factor of the helix-turn-helix type that can coordinate a [2Fe-2S] cluster. Redox conditions and iron or sulfur availability modulate the ligation status of the labile IscR cluster, which in turn determines a switch in DNA sequence specificity of the regulator: cluster-containing IscR can bind to a family of gene promoters (type-1) whereas the clusterless form recognizes only a second group of sequences (type-2). However, iron-sulfur cluster biogenesis in Gram-positive bacteria is not so well characterized, and most organisms of this group display only one of the iron-sulfur cluster assembly systems. A notable exception is the unique Gram-positive dissimilatory metal reducing bacterium Thermincola potens, where genes from both systems could be identified, albeit with a diverging organization from that of Gram-negative bacteria. We demonstrated that one of these genes encodes a functional IscR homolog and is likely involved in the regulation of iron-sulfur cluster biogenesis in T. potens. Structural and biochemical characterization of T. potens and E. coli IscR revealed a strikingly similar architecture and unveiled an unforeseen conservation of the unique mechanism of sequence discrimination characteristic of this distinctive group of transcription regulators.Rrf2-like regulator | transcription regulation | helix-turn-helix motif | DNA recognition | specificity modulation I ron-sulfur (Fe/S) proteins play crucial roles for the functioning of both prokaryotic and eukaryotic cells, being required for biological functions ranging from electron transport to redox and nonredox catalysis, and from DNA synthesis and repair to sensing in regulatory processes (1). The main role of the Fe/S cluster assembly machineries is to mobilize iron and sulfur atoms from their storage sources, assemble the two components into an Fe/S cluster, and then transfer the newly formed cluster to the final protein acceptors (2). In Escherichia coli, there are two of these Fe/S cluster ''factories,'' the ISC (iron-sulfur cluster) and SUF (sulfur assimilation) systems, whose corresponding genes are organized in two operons, iscSUA-hscBA-fdx and sufABCDSE, respectively (2, 3). Deletion mutants of the ISC system display a variety of growth defects due to loss of Fe/S cluster-containing enzyme activity and disruption of sulfur metabolism whereas failure of both the ISC and SUF systems leads to synthetic lethality (4, 5).In E. coli, the ISC machinery is considered the housekeeping system responsible for the maturation of a lar...

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

Cited by 21 publications

References 63 publications

Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile

The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium

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