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

Abstract

Background: 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 Clostridium perfringens, the two-component system, VirR/VirS, controls the expression of the ubiG operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the C. perfringens virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail.

Results: We first performed a tentative reconstruction of sulfur metabolism in C. perfringens and correlated these data with the growth of strain 13 in the presence of various sulfur sources. Surprisingly, C. perfringens 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 (cysK-cysE, cysP1 and cysP2) or an S-box riboswitch (metK and metT). We also showed that the ubiG 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.In addition, we found that expression of pfoA (theta-toxin), nagL (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 ldh gene encoding the lactate dehydrogenase was induced during cysteine limitation.

Conclusion: 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.

Figure 1

Reconstruction of sulfur metabolism in C. perfringens. We used the genomic data, growth assays and expression profiling to propose a tentative reconstruction of sulfur metabolism in C. perfringens. The cpe numbers for C. perfringens genes (strain 13) correspond to those of ClostriDB http://xbase.bham.ac.uk/clostridb/. The genes were renamed according to B. subtilis orthologues. The steps present in B. subtilis but absent in C. perfringens (sulfate assimilation and methionine biosynthesis by transsulfuration) are indicated by grey crossed arrows. A dotted arrow indicated the possible existence of a pathway. "?" indicates a step or a pathway for which a gene is lacking or remains to be identified. Serine O-acetyltransferase, cysE; OAS-thiol-lyase, cysK; anaerobic sulfite reductase, asrABC; glutamate-cysteine ligase/glutathione synthetase, gshAB ; SAM synthase, metK; adenosyl-homocysteine nucleosidase, mtnN; S-ribosyl-homocysteine lyase, luxS; cystathionine β-synthase, mccA; cystathionine γ-lyase, mccB. The following genes are absent from the genome of C. perfringens: metI (cystathionine β-synthase); metC (cystathionine β-lyase); metE (methionine synthase). AI-2, autoinducer 2; OAS, O-acetyl-serine; SAM, S-adenosyl-methionine; SAH, S-adenosyl-homocysteine; SRH, S-ribosyl-homocysteine. Ext means external.

Figure 2

Growth of C. perfringens strain 13 in the presence of various sulfur sources. Growth curves of strain 13 grown in a sulfur-free minimal medium in the presence of 1 mM sulfite (■), 1 mM glutathione (□), 0.5 mM cystine (●), 1 mM homocysteine (○), 1 mM methionine (▲) or in the absence of any sulfur source (△). We observed a similar growth for homocysteine and cystathionine, thiosulfate and cystine or sulfide and sulfite.

Figure 3

Intracellular concentration of sulfur compounds (A) and amino acids (B) in strain 13 grown in the presence of cystine or homocysteine. Grey or white boxes indicate the metabolite concentrations extracted from strain 13 grown in the presence of 0.5 mM cystine or 1 mM homocysteine, respectively. The mean value of three independent experiments is presented. # indicates that the metabolite is not detectable.

Figure 4

Genes involved in sulfur metabolism controlled by premature termination of transcription via a T-box or an S-box system. 5' untranslated region containing a T-box or an S-box motif are indicated by black or grey boxes, respectively. Loops indicate putative transcriptional terminators. Striped boxes indicate the genes encoding transporters. The genes involved in cysteine biosynthesis are indicated by dotted boxes while the SAM synthase gene, metK, is indicated by a checkerboard box. The expression ratios (homocysteine/cystine) obtained in transcriptome analysis are indicated under the genes while the expression ratios (homocysteine/cystine) obtained by qRT-PCR are indicated in parentheses. An alignment of the S-box motif of metT and metK has been previously published [9].

Figure 5

Alignment of the 4 cysteine specific T-boxes present in the C. perfringens genome. 4 genes with a T-box motif (AATTAGAGTGGAACC allowing one mismatch) were regulated in response to cysteine availability in transcriptome. We multialign a 200 bp region covering the T-box motif located upstream of these 4 genes. The conserved motifs characteristic of T-boxes (AGTA-box, F-box, AG-box, GNUG- box) are indicated. The cysteine specifier codon is boxed. Base-paired positions in the specifier hairpin (dotted arrow) are indicated by gray background. The bases involved in the formation of the antiterminator structure are underlined.

Figure 6

Northern blot analysis of the T-box controlled cysP2 transcription. Total RNA was extracted from strain 13 grown in minimal medium in the presence of cystine 1 mM (C) or homocysteine 1 mM (HC). Specific RNAs were detected using a probe hybridizing with the T-box region (A) or with the cysP2 (cpe0967) gene (B). The 16 S rRNA was used as a loading control.

Figure 7

Modulation of MccB synthesis in the presence of homocysteine or cystine in various mutants. The homocysteine γ-lyase activity of MccB was detected on zymogram. A total of 100 μg of crude extracts were charged on a native polyacrylamide gel (12%). The release of sulfide from homocysteine due to homocysteine γ-lyase activity was detected by the precipitation of insoluble PbS. The proteins were extracted from strain 13 (l and 2), TS140 (vrr::erm) (3 and 4), TS186 (virX ::erm) (5 and 6) and TS133 (virR::tet) (7 and 8) after growth in the presence of 0.5 mM cystine (1, 3, 5 and 7) or 1 mM homocysteine (2, 4, 6 and 8).