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1 Swedish Institute for Infectious Disease Control, Department of Bacteriology, S-171 82 Solna, Sweden
2 Karolinska Institute, Microbiology and Tumor Biology Center, S-171 77 Stockholm, Sweden
Correspondence
Thomas Åkerlund
Thomas.Akerlund{at}smi.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Two supplementary tables showing primers used for quantitative real-time PCR and identified proteins in C. difficile VPI 10463, and a supplementary figure showing a proteome map of C. difficile VPI 10463, are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Toxin synthesis is normally upregulated during entry into the stationary growth phase in tryptone/peptone-yeast media (Dupuy & Sonenshein, 1998; Karlsson et al., 1999), and is associated with the induction of many other C. difficile proteins that are repressed together with the toxins by the presence of glucose, cysteine and proline (Karlsson et al., 2000). In addition, arginine, glutamine, biotin and carbonate influence toxin production, further supporting a link between carbon/amino acid metabolism and toxin production (Ikeda et al., 1998; Karasawa et al., 1997; Karlsson et al., 1999; Maegawa et al., 2002; Yamakawa et al., 1996, 1998).
The sigma factor TcdR encoded by the C. difficile pathogenicity locus (PaLoc) activates the transcription of the toxin genes tcdA and tcdB, but also tcdR itself from one of its two promoters (Dupuy & Sonenshein, 1998; Karlsson et al., 2003; Mani & Dupuy, 2001; Mani et al., 2002). This positive autoregulation indicates that other elements downregulate tcdR transcription or TcdR activity to keep C. difficile toxin production low during unrestricted growth (i.e. when nutrients such as glucose or cysteine are in excess). Indeed, another PaLoc product, TcdC, has been shown to suppress toxin A expression in a Clostridium perfringens model system by interaction with TcdR (Matamouros et al., 2007). Moreover, all PaLoc genes are induced in a codY knockout mutant (Dineen et al., 2007), and purified CodY binds to the promoter region of tcdR; this binding is enhanced by the presence of GTP and branched-chain amino acids (BCAAs). CodY is a repressor that regulates many genes in Bacillus subtilis and other Gram-positive bacteria, acting as a sensor for energy (GTP) and/or BCAAs (Guedon et al., 2001; Molle et al., 2003; Petranovic et al., 2004; Ratnayake-Lecamwasam et al., 2001; Serror & Sonenshein, 1996b; Shivers & Sonenshein, 2004). However, the control of toxin expression in C. difficile appears to be more complex, since glucose downregulates toxin production in the codY mutant also (Dineen et al., 2007). The mechanism of this apparent CodY-independent but yet nutrient-sensing pathway to control toxin production is not known. Moreover, glucose has the opposite effect on toxin production in defined media (Karlsson et al., 1999). Here, we sought to identify additional genes and intracellular processes associated with toxin production in C. difficile. The results expand the knowledge of C. difficile metabolism and toxin production (Karlsson et al., 2000, 2003), and are discussed in relation to global regulators in other Gram-positive bacteria.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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to 19 ml C. difficile culture (OD 
0.8). For 2D PAGE analysis, 2.5 ml culture aliquots were collected at OD600 0.2, 0.5, 0.8 and 1.2, and after 24 h in PY0, PY10 and PYG, each in triplicate (i.e. 45 samples). One millilitre was used to determine the toxin yield (intra- and extracellular toxin), and the remaining 1.5 ml was used for protein extraction (see below). Bacteria were pelleted by centrifugation at 16 000 g for 3 min, washed in PBS and stored at –70 °C. For quantitative real-time PCR (qRT-PCR) analysis, 0.5 ml culture aliquots collected at ODs between 0.2 and 1.3 were mixed with 1 ml RNAprotect Bacteria Reagent (Qiagen) and centrifuged for 10 min; the supernatant was removed and pellets were stored at –70 °C until RNA extraction (below). For details regarding the preparation of growth media, dilutions, culture conditions and toxin determination see Karlsson et al. (1999, 2000).
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. In brief, frozen cells were thawed, suspended in 1 ml ice-cold MilliQ water and disrupted by sonication (2x 30 s, 100 W, Labsonic 1510, B. Braun), whereafter cellular debris was removed by centrifugation (16 000 g, 10 min, 4 °C). After mixing protein samples with 2D PAGE buffer, 20 µg protein was applied on the 2D focusing gel strip. For preparative samples, cell pellets from 20–40 ml culture were sonicated for 10x 60 s, and proteins were precipitated (10 %, w/v, TCA, 0 °C, 45 min), pelleted (21 000 g, 20 min, 4 °C), washed three times in 90 % ice-cold acetone, dissolved in buffer containing 0.3 % (w/v) SDS and 200 mM DTT, vortexed for 60 min, heated for 5 min at 90 °C and finally resuspended 1 : 4 (v/v) in 2D PAGE buffer. Non-dissolved material was pelleted (38 000 g, 20 min, 20 °C) before application of 0.4 mg protein (soluble fraction) on the 2D focusing gel strips. Analytical gels were stained with silver and dried, whereas preparative gels were stained with Bio-Safe Coomassie (Bio-Rad). At least two spots of each protein taken from independent gels were used for identification.
Analysis of differentially expressed proteins.
Gels were scanned (Scanjet 3c/T, Hewlett Packard) and imported in raw format into PDQuest 7.01 (Bio-Rad). All images were cropped, filtered prior to spot detection and assembled to three match sets each representing all 15 culture samples (five time points per culture). Each set was matched and normalized with the valid spot intensity method and assembled to a high-level master. This master was used to analyse protein expression differences between growth phases and growth media representing high and low toxin-production conditions. To calculate the molecular mass and pI of each spot, a few C. difficile protein spots were used as internal markers (Supplementary Table S2).
Protein digestion and MALDI-TOF MS.
Protein digestion was performed using the Montage In-Gel Digestion kit and a vacuum manifold unit (Millipore). Briefly, Coomassie-stained spots were excised and de-stained in ammonium bicarbonate buffer with an increasing percentage of acetonitrile before a final dehydration with 100 % acetonitrile. Proteins were digested with trypsin overnight at 30 or 37 °C for 3 h. The peptides were extracted from the gel and captured on the C18 resin of the ZipPlate 96-well microtitre plate (Millipore). Peptides were eluted by centrifugation in 3.5 µl 0.1 % trifluoroacetic acid and 50 % acetonitrile. Finally, 1–2 µl of the eluted peptides was loaded on an Anchor-Chip plate (Bruker Daltronics) and covered by 1 µl matrix solution (
-cyano-4-hydroxycinnamic acid). Peptide mass mapping of tryptic peptides was performed using a Bruker Daltronics Reflex IV apparatus equipped with a nitrogen laser (337.1 nm) operated in a reflective positive mode. Spectra calibration was performed by use of the internal trypsin fragments 842.5 and 2211.1 Da and a 1000–4000 Da peptide calibration standard (Bruker Daltronics). C. difficile proteins were identified by using the MASCOT peptide mass fingerprint software at http://www.matrixscience.com/.
RNA extraction and qRT-PCR.
RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using random hexamer primers (High Capacity cDNA Reverse Transcription kit, Applied Biosystems) and qRT-PCR was performed using the Power SYBR Green assay on an ABI7500 cycler employing primers designed using Primer Express 3.0 (Supplementary Table S1). Melting standard curves using twofold dilutions of template were performed as a control for PCR efficiency and mispriming. PCR was done in triplicates of duplicate cultures (PY0) or a single culture (PY10). Results were evaluated using rpoA as the endogenous control (O'Connor et al., 2006). There was no difference in expression of target mRNAs between the two PY0 cultures, and no inconsistency between results obtained at the protein level [enzyme immunoassay (EIA) of toxins A+B, proteome data of FolD] (Figs 2 and 3, Supplementary Table S2).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and the presence of 0.9 % glucose in the inoculum strongly reduced the toxin yields: 30 U ml–1 in PY0G versus 2370 U ml–1 in PY0 at 24 h (not shown; see also PYG versus PY0 in Fig. 2). In contrast, glucose had a small impact on reducing toxin production when added to PY0 cultures prior to entry into stationary phase (Fig. 1b), whereas the addition of cysteine promptly blocked further toxin synthesis (Fig. 1c). Cysteine also reduced the growth rate and growth yield of C. difficile, possibly due to an abrupt change of the oxidation–reduction potential. Nevertheless, the nine amino acids originally found to reduce toxin production had the same effect as cysteine but without any change in growth rate (Fig. 1d). Blocking RNA transcription showed that pre-formed toxin was stable (Fig. 1e). The ambiguous result with glucose suggested that its effect was indirect or that glucose uptake was diminished prior to entry into stationary phase.
Induction of toxin synthesis is associated with utilization of alternative energy sources
Growth of C. difficile was similar in PY0, PY10 and PYG (Fig. 2a) and, as expected, toxins A and B were induced only in PY0 (Fig. 2b). A total of 1522, 1345 and 1688 protein spots were found by 2D PAGE during growth in PY0, PY10 and PYG, respectively. About 130 spots were identified, yielding 94 proteins (Supplementary Table S2, Supplementary Fig. S1). Of these proteins, 40 were upregulated under high toxin-producing conditions, i.e. in PY0, and many of the corresponding genes mapped closely to each other on the C. difficile chromosome, forming putative operons (Supplementary Table S2). One such operon (CD2339–CD3242) encodes enzymes for ATP generation via succinate/butyrate metabolism, e.g. 4-hydroxybutyrate CoA transferase (Fig. 2c). CD0717–CD0727 contained genes involved in folate and CO metabolism, e.g. carbon monoxide dehydrogenase/acetyl-CoA synthase (Fig. 2d). A majority of the genes in the latter cluster showed high similarity to ORFs in Alkaliphilus metalliredigenes and, to a lesser extent, to ones found in Carboxythermus hydrogenoformans and Moorella thermoacetica (not shown). Tetrahydrofolate is involved in numerous reactions, e.g. synthesis of purines and methionine, and together with carbon monoxide dehydrogenase/acetyl-CoA synthase also in the synthesis of acetyl-CoA from CO (Ljungdahl, 1986; Wood et al., 1986). Two more clusters with an apparent link to ATP generation and butyrate formation were CD0112–CD0118 and CD1054–CD1059 (Fig. 2e, Supplementary Table S2). Other enzymes upregulated in PY0 were involved in cysteine biosynthesis (Fig. 2f, g) and fermentation of amino acids, e.g. proline (Fig. 2h), glycine (CD2354), leucine (CD0394–CD0399; Kim et al., 2005) and aromatic amino acids (CD2828). The D-proline and glycine reductases are typical components of the ‘Stickland reaction’, by which C. difficile and other clostridia use amino acids as fuel by pair-wise fermentation (Gottschalk, 1986; Jackson et al., 2006; Stadtman & Elliot, 1957). Interestingly, we also identified a putative dual-specificity prolyl/cysteinyl-tRNA synthetase that was upregulated in PY0 (Fig. 2i). Its gene was encoded among glutamyl-, prolyl- and cysteinyl-tRNA synthetases, and its upregulation may reflect a shortage of cysteine, proline or both.
Two rubrerythrin/rubredoxin Fe(Cys)4-like proteins were upregulated in PY10 (CD1474 and CD1524; Supplementary Table S2). The corresponding chromosomal genes were nearly identical and clustered together with genes encoding other iron–sulphur proteins or ABC transporters in each of two operons. In addition, the F0F1 ATP synthase β subunit (CD3468) and several chaperones (CD0193, CD0194 and CD2461) were induced in PY10. Enzymes such as glyceraldehyde-3-phosphate dehydrogenase (CD3174) and fructose bisphosphate aldolase (CD0403) were more abundant in PYG, indicating an upregulation of the glycolytic pathway (Supplementary Table S2).
Coordinated transcription of tcdA, folD, tcdR and sigH
To confirm and refine the expression kinetics of genes of particular interest, mRNA levels of tcdA, folD (CD0720 in the CO/folate operon), the toxin operon control genes tcdR and tcdC, and the global regulators codY, sigH, sigL and sigB were followed during induction of the C. difficile toxins. The growth rates of the PY0 and PY10 cultures were nearly identical (Fig. 3a). Transcription of tcdA was abruptly induced at OD 0.7 in PY0 but not in PY10 (Fig. 3b), and folD expression parallelled that of tcdA (Fig. 3c). At this time also, the mRNA levels of tcdR and sigH increased (by 3.5-fold), while minor variations in expression were observed for tcdC, codY, sigL and sigB (Fig. 3d–i). The precise timing of transcription of tcdA, tcdR, folD and sigH in PY0 suggests a common regulator for these genes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), but the sequenced clostridial species do not contain ORFs with high similarities to B. subtilis GlnR or TnrA. In contrast, C. difficile (and other clostridia) encodes a CodY homologue in its genome (55 % similarity to CodY in B. subtilis). The global repressor CodY is deactivated by low intracellular concentrations of GTP and/or BCAAs (i.e. isoleucine, leucine and valine), leading to induction of genes involved in histidine utilization, synthesis of isoleucine/valine, motility and competence (see Introduction). C. difficile CodY was recently found to bind to the promoter of the toxin-specific sigma factor tcdR and block expression of all PaLoc genes in C. difficile strain 630 (Dineen et al., 2007), but several observations imply a more complex control network: (i) glucose downregulates toxin production in the codY mutant also (Dineen et al., 2007), implying that the toxin genes must be controlled by yet another nutrient-sensing regulator. This regulation is most likely not due to glucose-mediated catabolite repression of the toxin operon (Karlsson et al., 1999). (ii) The amino acids cysteine and proline, which efficiently downregulate toxin production (Karlsson et al., 2000), are not known to be direct effectors of CodY. One of the known effectors, isoleucine, has even been found to induce toxin production in C. difficile strain VPI 10463 (Ikeda et al., 1998), a result that may be explained by the high rate of cysteine depletion under these conditions. (iii) We observed a coordinated transcription of tcdA, folD, tcdR and sigH. In B. subtilis, sigH is controlled by the transition state repressor AbrB (see below). (iv) Similar to tcdR, the negative regulator of toxin expression tcdC had a putative CodY binding site at its promoter and was induced together with the other PaLoc genes in the C. difficile codY mutant (Dineen et al., 2007). Although the level of tcdC induction was apparently lower than for the other PaLoc genes, it was not consistent with the opposite transcription pattern of tcdC found in C. difficile strain VPI 10463 (Hundsberger et al., 1997). We observed only small changes of tcdC mRNA level in strain VPI 10463 during induction of tcdA and tcdR (Fig. 3). However, the expression patterns of tcdR and tcdC were more divergent in PY0 compared to that in PY10, indicating that these genes were regulated differently in the two media.
The complexity of toxin regulation in C. difficile is underscored by the observation that arginine limitation induces toxin production in a few isolates (Karasawa et al., 1997), and that the excessive toxin production during biotin limitation is abolished by adding glutamate/glutamine (Yamakawa et al., 1998). In B. subtilis, glutamate and glutamine are key amino acids involved in both the synthesis of other amino acids and the TCA cycle intermediate 2-oxoglutarate, providing a link between nitrogen and carbon metabolism (Sonenshein, 2007). However, an amino acid analysis of culture supernatants revealed that neither arginine nor glutamate/glutamine was significantly consumed by strain VPI 10463, regardless of the medium tested, whereas the BCAAs were preferentially used in PY0 and PY10 but conserved in PYG cultures (S. Karlsson and others, unpublished results).
Several proteins induced in PY0 were encoded in operons containing RpoN-dependent transcriptional activators, e.g. CD0394–CD0397 and CD2380, suggesting that sigL-dependent genes are induced under high toxin-producing conditions. Sigma factors of the RpoN family are known to regulate chemotaxis, electron transport, other alternative sigma factors, virulence and nitrogen metabolism (Buck et al., 2000; Merrick, 1993). For example, transcription of isoleucine/leucine utilization genes and butyrate kinase is dependent on SigL (Debarbouille et al., 1999), and glucose downregulates sigL transcription in a roadblock manner via catabolite repression/CcpA in B. subtilis (Choi & Saier, 2005). A regulation of such genes by catabolite repression in C. difficile is in accord with the reduced uptake of BCAAs in PYG medium (S. Karlsson and others, unpublished results; see above). However, expression of apparent SigL-dependent genes usually preceded toxin induction, and the C. difficile sigL and tcdR transcript levels were not consistently correlated (see Fig. 3). We found a more apparent co-expression for tcdR and sigH. SigH (Spo0H) is among the first proteins expressed during B. subtilis sporulation and is transcriptionally regulated by the transition state repressor AbrB (Weir et al., 1991). CodY and AbrB regulons may also overlap, since both these repressors have been found to bind the dipeptide permease (dpp) promoter sequence in B. subtilis (Serror & Sonenshein, 1996a). It is interesting to note that toxin synthesis in Bacillus anthracis is regulated by AbrB and SigH, and, similar to C. difficile, induced by elevated carbonate concentrations in the medium (Hadjifrangiskou et al., 2007; Karlsson et al., 1999). In conclusion, the results presented here further support the hypothesis that glucose acts indirectly by preventing exhaustion of certain amino acids that in turn signal induction of C. difficile toxin synthesis (Karlsson et al., 1999), and that cysteine has a profound impact on both metabolism and toxin production in C. difficile. Our data also showed a transcriptional coordination among C. difficile tcdR/tcdA, folD and sigH. Although regulation of folD and sigH by CodY cannot be ruled out, current data indicate that additional global regulators, e.g. the transition state regulator AbrB (Mani et al., 2002), are involved in coordinating C. difficile toxin expression, alternative metabolism and early sporulation events.
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ACKNOWLEDGEMENTS |
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Edited by: S. J. Foster
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REFERENCES |
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54 (N) transcription factor. J Bacteriol 182, 4129–4136.Choi, S. K. & Saier, M. H., Jr (2005). Regulation of sigL expression by the catabolite control protein CcpA involves a roadblock mechanism in Bacillus subtilis: potential connection between carbon and nitrogen metabolism. J Bacteriol 187, 6856–6861.
Debarbouille, M., Gardan, R., Arnaud, M. & Rapoport, G. (1999). Role of bkdR, a transcriptional activator of the sigL-dependent isoleucine and valine degradation pathway in Bacillus subtilis. J Bacteriol 181, 2059–2066.
Dineen, S. S., Villapakkam, A. C., Nordman, J. T. & Sonenshein, A. L. (2007). Repression of Clostridium difficile toxin gene expression by CodY. Mol Microbiol 66, 206–219.[CrossRef][Medline]
Dupuy, B. & Sonenshein, A. L. (1998). Regulated transcription of Clostridium difficile toxin genes. Mol Microbiol 27, 107–120.[CrossRef][Medline]
Fisher, S. H. (1999). Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!. Mol Microbiol 32, 223–232.[CrossRef][Medline]
Gottschalk, G. (1986). Bacterial Metabolism, 2nd edn. New York: Springer Verlag.
Guedon, E., Serror, P., Ehrlich, S. D., Renault, P. & Delorme, C. (2001). Pleiotropic transcriptional repressor CodY senses the intracellular pool of branched-chain amino acids in Lactococcus lactis. Mol Microbiol 40, 1227–1239.[CrossRef][Medline]
Hadjifrangiskou, M., Chen, Y. & Koehler, T. M. (2007). The alternative sigma factor H is required for toxin gene expression by Bacillus anthracis. J Bacteriol 189, 1874–1883.
Hundsberger, T., Braun, V., Weidmann, M., Leukel, P., Sauerborn, M. & von Eichel-Streiber, C. (1997). Transcription analysis of the genes tcdA–E of the pathogenicity locus of Clostridium difficile. Eur J Biochem 244, 735–742.[Medline]
Ikeda, D., Karasawa, T., Yamakawa, K., Tanaka, R., Namiki, M. & Nakamura, S. (1998). Effect of isoleucine on toxin production by Clostridium difficile in a defined medium. Zentralbl Bakteriol 287, 375–386.[Medline]
Jackson, S., Calos, M., Myers, A. & Self, W. T. (2006). Analysis of proline reduction in the nosocomial pathogen Clostridium difficile. J Bacteriol 188, 8487–8495.
Karasawa, T., Maegawa, T., Nojiri, T., Yamakawa, K. & Nakamura, S. (1997). Effect of arginine on toxin production by Clostridium difficile in defined medium. Microbiol Immunol 41, 581–585.[Medline]
Karlsson, S., Burman, L. G. & Åkerlund, T. (1999). Suppression of toxin production in Clostridium difficile VPI 10463 by amino acids. Microbiology 145, 1683–1693.
Karlsson, S., Lindberg, A., Norin, E., Burman, L. G. & Åkerlund, T. (2000). Toxins, butyric acid, and other short-chain fatty acids are coordinately expressed and down-regulated by cysteine in Clostridium difficile. Infect Immun 68, 5881–5888.
Karlsson, S., Dupuy, B., Mukherjee, K., Norin, E., Burman, L. G. & Åkerlund, T. (2003). Expression of Clostridium difficile toxins A and B and their sigma factor TcdD is controlled by temperature. Infect Immun 71, 1784–1793.
Kim, J., Darley, D. & Buckel, W. (2005). 2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile. FEBS J 272, 550–561.[CrossRef][Medline]
Ljungdahl, L. G. (1986). The autotrophic pathway of acetate synthesis in acetogenic bacteria. Annu Rev Microbiol 40, 415–450.[Medline]
Maegawa, T., Karasawa, T., Otha, T., Wang, X., Kato, H., Hayashi, H. & Nakamura, S. (2002). Linkage between toxin production and purine biosynthesis in Clostridium difficile. J Med Microbiol 51, 34–41.
Mani, N. & Dupuy, B. (2001). Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci U S A 98, 5844–5849.
Mani, N., Lyras, D., Barroso, L., Howarth, P., Wilkins, T., Rood, J. I., Sonenshein, A. L. & Dupuy, B. (2002). Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression. J Bacteriol 184, 5971–5978.
Matamouros, S., England, P. & Dupuy, B. (2007). Clostridium difficile toxin expression is inhibited by the novel regulator TcdC. Mol Microbiol 64, 1274–1288.[CrossRef][Medline]
Merrick, M. J. (1993). In a class of its own – the RNA polymerase sigma factor sigma 54 (sigma N). Mol Microbiol 10, 903–909.[Medline]
Molle, V., Nakaura, Y., Shivers, R. P., Yamaguchi, H., Losick, R., Fujita, Y. & Sonenshein, A. L. (2003). Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin immunoprecipitation and genome-wide transcript analysis. J Bacteriol 185, 1911–1922.
Mukherjee, K., Karlsson, S., Burman, L. G. & Åkerlund, T. (2002). Proteins released during high toxin production in Clostridium difficile. Microbiology 148, 2245–2253.
O'Connor, J. R., Lyras, D., Farrow, K. A., Adams, V., Powell, D. R., Hinds, J., Cheung, J. K. & Rood, J. I. (2006). Construction and analysis of chromosomal Clostridium difficile mutants. Mol Microbiol 61, 1335–1351.[CrossRef][Medline]
Petranovic, D., Guedon, E., Sperandio, B., Delorme, C., Ehrlich, D. & Renault, P. (2004). Intracellular effectors regulating the activity of the Lactococcus lactis CodY pleiotropic transcription regulator. Mol Microbiol 53, 613–621.[CrossRef][Medline]
Poxton, I. R., McCoubrey, J. & Blair, G. (2001). The pathogenicity of Clostridium difficile. Clin Microbiol Infect 7, 421–427.[CrossRef][Medline]
Ratnayake-Lecamwasam, M., Serror, P., Wong, K. W. & Sonenshein, A. L. (2001). Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels. Genes Dev 15, 1093–1103.
Serror, P. & Sonenshein, A. L. (1996a). Interaction of CodY, a novel Bacillus subtilis DNA-binding protein, with the dpp promoter region. Mol Microbiol 20, 843–852.[Medline]
Serror, P. & Sonenshein, A. L. (1996b). CodY is required for nutritional repression of Bacillus subtilis genetic competence. J Bacteriol 178, 5910–5915.
Shivers, R. P. & Sonenshein, A. L. (2004). Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids. Mol Microbiol 53, 599–611.[CrossRef][Medline]
Sonenshein, A. L. (2007). Control of key metabolic intersections in Bacillus subtilis. Nat Rev Microbiol 5, 917–927.[CrossRef][Medline]
Stadtman, T. C. & Elliot, P. (1957). Purification and properties of D-proline reductase and a proline racemase from Clostridium sticklandii. J Biol Chem 228, 983–997.
Weir, J., Predich, M., Dubnau, E., Nair, G. & Smith, I. (1991). Regulation of spo0H, a gene coding for the Bacillus subtilis H factor. J Bacteriol 173, 521–529.
Wood, H. G., Ragsdale, S. W. & Pezacka, E. (1986). A new pathway of autotrophic growth utilizing carbon monoxide or carbon dioxide and hydrogen. Biochem Int 12, 421–440.[Medline]
Yamakawa, K., Karasawa, T., Ikoma, S. & Nakamura, S. (1996). Enhancement of Clostridium difficile toxin production in biotin-limited conditions. J Med Microbiol 44, 111–114.
Yamakawa, K., Karasawa, T., Ohta, T., Hayashi, H. & Nakamura, S. (1998). Inhibition of enhanced toxin production by Clostridium difficile in biotin-limited conditions. J Med Microbiol 47, 767–771.
Received 22 April 2008;
revised 21 July 2008;
accepted 27 July 2008.
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) and toxin levels (
) of C. difficile VPI 10463 growing in PY0 supplemented with 1 ml PY0 (a, control) or 1 ml PY0 containing the indicated compounds (b–e) at OD600 
), PY10 (
). (b) Relative toxin levels (by EIA) were normalized to 1.0 for PY0 at time zero. (c–i) Expression of CD2339/4-hydroxybutyrate CoA transferase (c), CD0727/carbon monoxide dehydrogenase/acetyl-CoA synthase subunit (d), CD1058/3-hydroxybutyryl-CoA dehydrogenase (e), CD1594/putative O-acetylserine thiolase (f), CD5414/cysteine synthase A (g), CD3244/proline reductase subunit proprotein (h) and CD0050/putative dual-specificity prolyl/cysteinyl-tRNA synthetase (i). All expression data (panels c–i) are means of three experiments and shown as relative values (1.0=PY0 at time zero, OD 0.2). Bars, SEM.