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

Abstract

The 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.

Figure 1.

C. difficile cre_CD consensus of the CcpA binding motif. The motif sequence logo was created based on the alignment of all the proposed direct targets of CcpA listed in Table 2, using the weblogo website (http://weblogo.berkeley.edu). The height of the letters is proportional to their frequency.

Figure 2.

Binding of CcpA to different targets with a putative cre_CD sequence identified by ChIP-on-Chip experiments. EMSAs of DNA fragments containing the cre_CD binding sites of the CD1893, spoVS (CD1935), nanE (CD2241), CD3218, CD3664, virS (CD0576), CD1768, spo0A (CD1214) and prdA (CD3244) genes. The ³²P-labelled DNA fragments were incubated with increasing concentrations of purified CcpA–His₆ as described in the ‘Materials and Methods’ section: (A) 0, 32, 65, 97 and 129 nM of CcpA–His₆; (B) 0, 97, 129, 194 and 259 nM of CcpA–His₆; (C) 0, 129, 259, 388 and 518 nM of CcpA–His₆. (D) Negative controls corresponding to the DNA fragments containing promoter regions of CD1893 and CD3218 without putative cre_CD sites were incubated with 0, 65, 97 and 129 nM of CcpA–His₆, and 0, 97, 129, 194 and 259 nM of CcpA–His₆, respectively. The autoradiograms of the gels are shown. F = free DNA; C = protein–DNA complex.

Figure 3.

Impact of cre_CD deletion on the binding of CcpA to the hadA, acnB, feoA, CD1536, gatA and cstA target genes. EMSAs of DNA fragments containing the cre_CD binding site (cre_CD) or the same DNA fragment where the cre_CD site has been deleted (Δcre_CD) of the hadA (CD0395), feoA (CD1477), gatA (CD2327), acnB (CD0833), CD1536 and cstA (CD2600) genes. The ³²P-labelled DNA fragments were incubated with increasing concentrations of purified CcpA–His₆ as described in the ‘Materials and Methods’ sections: (A) 0, 13, 32 and 65 nM of CcpA–His₆; (B) 0, 65, 97 and 129 nM of CcpA–His₆. (C) Internal ³²P-labelled DNA fragments of feoA and gatA genes without putative cre_CD sites were incubated with 0, 65, 97 and 129 nM of CcpA–His₆ (negative controls). The autoradiograms of the gels are shown. F: free DNA; C: protein–DNA complex.

Figure 4.

Binding of CcpA to the cre_CD sites of tcdC, grdX, tcdR and spoIIAA target genes. EMSAs of DNA fragments containing the cre_CD sites of the tcdC (CD0664), grdX (CD2357), tcdR (CD0659) and spoIIAA (CD0770) genes. The ³²P-labelled DNA fragments were incubated with increasing concentrations of purified CcpA–His₆ as described in the ‘Materials and Methods’ section: (A) 0, 32, 65, 97 and 129 nM of CcpA–His₆ for tcdC and grdX; (B) 0, 129, 259, 388 and 518 nM of CcpA–His₆ for tcdR-cre_CD1,tcdR-cre_CD2 and spoIIAA and 0, 65, 97, 129, 259, 388 and 518 nM of CcpA–His₆ for tcdR-cre_CD1-CD2. The autoradiograms of the gels are shown.

Figure 5.

Role of glucose and CcpA in the regulation of carbohydrate utilization, central carbon metabolism and fermentation. Genes encoding enzymes or transporters, which are regulated by CcpA and/or glucose in transcriptome. Green: gene downregulated by glucose; red: gene upregulated by glucose; black: gene not regulated by glucose; blue CcpA: regulation by glucose through CcpA; blue and underlined CcpA: other type of control by CcpA; perpendicular: gene downregulated by CcpA; arrow: gene upregulated by CcpA. glgCDA (CD0882–CD0884), glycogen biosynthesis; fbp (CD1191), fructose-1,6-bisphosphatase; fba (CD0403), fructose-1,6-bisphosphate aldolase; tpi (CD3172), triosephosphate isomerase; gapA (CD3174) and gapB (CD1767), glyceraldehyde-3-phosphate dehydrogenase; pgk (CD3173), phosphoglycerate kinase; gpmI (CD3171), 2,3-bisphosphoglycerate-mutase; eno (CD3170), enolase; pykF (CD3394), pyruvate kinase; fumB (CD1004), fumarate hydratase subunit B; CD1005, putative nicotinamide adenine dinucleotide-dependent malic enzyme; rpe (CD2319), putative ribulose-phosphate 3-epimerase; rpiB1 (CD2320), ribose-5-phosphate isomerase B1; rpiB2 (CD3480), ribose-5-phosphate isomerase B2; rbsK (CD0299), ribokinase; tkt’ (CD2321), transketolase, central and C-terminal; tkt (CD2322), transketolase, N-terminal; CD0764–CD0768, PTS sorbitol IIC, IIB, IIA; CD3089–CD3088, PTS cellobiose IIBC; CD2327–CD2325, PTS galactitol IIA, IIB, IIC; CD0208–CD0206, PTS fructose-like IIA, IIB, IIC; CD2486–CD2488, PTS fructose-like IIC, IIB, IIA; CD3015–CD3013, PTS mannose-specific IIA, IIB, IIC; ptsG (CD2667, CD2666), PTS glucose-specific IIA, IICB; pmi (CD2491), mannose-6-phosphate isomerase; rbsBAC (CD0300–CD0302), ribose ABC transporter; adhE (CD2966) and CD3006, aldehyde-alcohol dehydrogenase; thlA (CD1059), acetyl-CoA acetyltransferase; hbd (CD1058), 3-hydroxybutyryl-CoA dehydrogenase; crt2 (CD1057), 3-hydroxybutyryl-CoA dehydratase; cat1 (CD2343), succinyl-CoA: coenzyme A transferase; sucD (CD2342), succinate-semialdehyde dehydrogenase; 4hbd (CD2338), 4-hydroxybutyrate dehydrogenase; cat2 (CD2339), 4-hydroxybutyrate CoA transferase; abfD (CD2341), vinylacetyl-coa-δ-isomerase; bcd2 (CD1054), butyryl-CoA dehydrogenase; etfBA (CD1055–CD1056), electron transfer flavoproteins; ptb (CD0715, CD0112), phosphate butyryltransferase; buk (CD0113), butyrate kinase.

Figure 6.

Fermentation end products of the strain JIR8094 and the ccpA mutant grown in the presence or absence of glucose. Gas–liquid chromatography analysis of the metabolic end products from JIR8094 and ΔccpA mutant strains after 48 h of growth in TY or TYG medium was performed. The mean and standard error of three experiments are shown. Asterisk corresponds to significative statistical difference analysed by the student t-test (P < 0.05).

Figure 7.

Role of glucose and CcpA in the control of peptide and amino acid metabolisms. Genes encoding enzymes or transporters, which are regulated by CcpA and/or glucose in transcriptome. Green: gene downregulated by glucose; red: gene upregulated by glucose; black: gene not regulated by glucose; blue CcpA: regulation by glucose through CcpA; blue and underlined CcpA: other type of control by CcpA; hash: localization unknown; perpendicular: gene downregulated by CcpA; arrow: gene upregulated by CcpA. lysC (CD2054), aspartokinase; asd (CD3224), aspartate-semialdehyde dehydrogenase; dapA1 (CD3000), dihydrodipicolinate synthase 1; dapA2 (CD3223), dihydrodipicolinate synthase 2; dapA3 (CD3225), dihydrodipicolinate synthase 3; dapB1 (CD3226), dihydrodipicolinate reductase; dapH (CD3227), 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate; lysA (CD2053), diaminopimelate decarboxylase; thrC (CD2118), threonine synthase, thrB (CD2119), homoserine kinase; tdcB (CD2514), threonine dehydratase II; serA (CD0995), putative D-3-phosphoglycerate dehydrogenase; sdaB (CD3222), L-serine dehydratase; cysE (CD1595), serine acetyltransferase; cysK (CD1594), O-acetylserine sulphydrylase; iscS2 (CD1279), cysteine desulphurase; oppBCADF (CD0853–CD0857), oligopeptide ABC transporter; CD2177–CD2174, cystine ABC transporter; prdF (CD3237), proline racemase; prdE (CD3239), proline reductase; prdA (CD3244), D-proline reductase proprotein; prdB (CD3241), proline reductase; trxB3 (CD2356), thioredoxin reductase 3; trxA2 (CD2355), thioredoxin 2; grdX (CD2357), glycine reductase complex component; grdE (CD2354), glycine reductase complex component B subunits α and β; grdA (CD2352), glycine reductase complex selenoprotein A; grdB (CD2351), glycine reductase complex component B γ subunit; grdC (CD2349), glycine reductase complex component C subunit β; grdD (CD2348), glycine reductase complex component C subunit α; ldhA (CD0394), 2-hydroxyisocaproate dehydrogenase; hadA (CD0395), 2-hydroxyisocaproate CoA transferase; hadI (CD0396), activator of dehydratase; acdB (CD0399), acyl-CoA dehydrogenase; gcvTPA (CD1657), bi-functional glycine dehydrogenase/aminomethyl transferase protein; gcvPB (CD1658), Glycine decarboxylase; CD1228, putative protease; CD3183, putative peptidase; CD2485, putative Xaa-Pro aminopeptidase; CD2347, putative Xaa-Pro dipeptidase; gcp (CD0152), putative O-sialoglycoprotein endopeptidase; CD2129, putative membrane-associated peptidase; pepI (CD3041), proline iminopeptidase.

Figure 8.

Effect of glucose addition or CcpA inactivation on the sporulation efficiency. Comparison of sporulation efficiency of C. difficile JIR8094 and ccpA mutant strains grown in SM medium supplemented or not supplemented with 0.5% of glucose at 24 h (A), 48 h (B) and 96 h (C). Total cell counts were obtained by enumeration of CFUs derived from serial dilutions of the initial culture plated on BHI agar supplemented with 0.1% of taurocholate. The same procedure was applied for spore counts with an additional step of heat treatment at 60°C for 30 min, to select for heat resistant spores. Percentage of sporulation is the ratio between the spore titer and total cell titer at each time point. The mean and standard error of three experiments are shown.