Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 rue du docteur Roux, 75724 Paris Cedex 15, France
Correspondence
Gilles Reysset
greysset{at}pasteur.fr
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ABSTRACT |
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INTRODUCTION |
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The genes involved in the oxidative stress response of anaerobic Gram-positive bacteria have not all been identified. However, rubrerythrin (rbr) and superoxide dismutase (sod) genes have been identified in C. perfringens strain NCIMB8875 (Lehmann et al., 1996; Geissmann et al., 1999
), but it was shown that neither sod nor rbr transcription was influenced by oxidative stress, and these results did not lead to any conclusions about the role of the two genes in the defence against oxygen in C. perfringens. In parallel, studies of sulfate-reducing Desulfovibrio species have recently given new insight in the area of stress resistance in anaerobic bacteria (Pianzzola et al., 1996
; Lumppio et al., 1997
; Voordouw & Voordouw, 1998
; Coulter et al., 1999
; Jenney et al., 1999
; Lumppio et al., 2001
; Emerson et al., 2002
). Homologues of some of the oxidative stress response genes found in Desulfovibrio vulgaris have thus been sought in C. perfringens. Analysis of the genome sequence of strain 13 shows that C. perfringens possesses genes potentially encoding (i) a classical Mn-SOD (sod), (ii) an alkyl hydroperoxide reductase (ahpC), (iii) two rubredoxins (rub) suspected to belong to two superoxide reductase systems and (iv) three non-haem iron rubrerythrins (rbr), with hypothetical H2O2 reductase activity.
We used various strategies to evaluate the role of these genes in the oxidative stress response of C. perfringens. First, we carried out functional complementation experiments in Escherichia coli strains impaired in either SOD, alkyl hydroperoxide reductase or catalase activity to evaluate the catalytic properties of these proteins. Second, we compared the level of transcription of these genes in stressed and unstressed cells of C. perfringens. Finally, we studied the physiological properties of regulatory mutants constructed by gene inactivation, to confirm whether inactivated genes were indeed involved in the oxidative stress response.
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METHODS |
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Kinetics of C. perfringens cell survival under aerobic conditions.
Bacteria were grown to mid-exponential phase in MMP medium supplemented with 0·01 % yeast extract at 37 °C in an anaerobic chamber. The bacterial cultures supplemented without and with 2·5 µM plumbagin (Pl) were then transferred to aerobic conditions at 37 °C with shaking in a rotary incubator (250 r.p.m.), in flasks with a capacity 510 times greater than the volume of the culture. At various times, aliquots were returned to the anaerobic chamber and viability was measured. The rate of cell survival was calculated as the number of viable cells after exposure to air divided by the total number of viable cells before treatment.
Inhibition of E. coli growth with paraquat (PQ), H2O2 and ter-butylhydroperoxide (t-BT) on solid medium.
E. coli cells were grown overnight in M63casa liquid medium and then spread onto M63casa solid medium, with or without Ap (50 µg ml1). Then, 5 µl of 25, 50 or 25 mM PQ, H2O2 or t-BT solutions, respectively, was spotted onto small circles (diam. 6 mm) of blotting paper that had been placed on the agar plate. The plates were then incubated for 24 h at 37 °C and the diameter of inhibition was measured. Means of four to eight inhibition zones were determined for each strain.
Peroxidase activity gels.
Peroxidase activity was detected in native polyacrylamide gels as described by Heym & Cole (1992). A crude extract of E. coli cells was prepared as follows. The strains were cultivated in LB medium until stationary phase, harvested by centrifugation and washed twice in 50 mM PB buffer, pH 7·0. The cells were then resuspended in 1/25 of the initial volume of PB buffer and disrupted by sonication (3x3 min with 5 min cooling intervals). Cell debris was removed by centrifugation (20 min at 15 000 g) and the supernatant was stored frozen in aliquots. Proteins were quantified using the method of Bradford (1976)
.
Molecular and genetic techniques.
Unless otherwise stated, all DNA manipulations were performed by standard techniques (Sambrook et al., 1989). DNA preparation and C. perfringens electroporation were done as described by Briolat & Reysset (2002)
.
Cloning of genes or genetic regions of C. perfringens.
The genes encoding the alkyl hydroperoxide reductase (ahpC), the SOD (sod), the rubrerythrins (rbr1, rbr2 and rbr3), the rubredoxins (rub1 and rub2) and the genetic regions GRI (from CPE0776 to CPE0778) and GRII (from CPE0779 to CPE0781) (see Fig. 1) were amplified by PCR using specific sets of primers (Table 2
). After purification, the PCR products were cloned into the pGEMTeasy vector (Promega) and introduced into E. coli DH5
by electroporation. The multiple cloning sequence of the pGEMTeasy vector is flanked by promoters recognized by the T7 and SP6 RNA polymerases, respectively. However, as the two polymerases are both absent from the E. coli strains used in this study, the C. perfringens genes are likely to be expressed from their own promoter.
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Preparation and quantification of RNA.
C. perfringens (50 ml) was harvested 0, 20, 40 and 60 min after the onset of each stress condition and total RNA was extracted using the trizol reagent (Gibco-BRL) (Dupuy & Sonenshein, 1998). RNA concentration was determined by the orcinol reaction (Schneider, 1957
). Various concentrations (5, 2·5, 1 and 0·5 µg) of the denatured DNA-free RNA preparations from each time point were blotted onto two nylon filter membranes. The filters were subjected to Northern hybridization with an internal 106 d.p.m. [
-32P]dCTP (Amersham) radiolabelled probe of the gene of interest and 106 d.p.m. of a specific C. perfringens 16S RNA probe. Filters were hybridized overnight at 55 °C in rapid-hyb buffer (Amersham) and then washed once for 15 min in 1x SSC (0·15 M NaCl plus 0·015 M sodium citrate)/0·1 % SDS, and three times for 10 min each in 0·2x SSC/0·1 % SDS at 55 °C. Radioactivity was quantified using the Storm Phospho-Imager (Molecular Dynamics). Standardization was done by dividing the value obtained for a given probe by the corresponding 16S rRNA value. The results are expressed as the means of the various RNA concentrations used.
Computer methods.
The whole-genome sequence of C. perfringens was visualized and analysed using Artemis software (Rutherford et al., 2000). C. perfringens gene sequences were compared with sequences present in public databases using FASTA, BLASTN and BLASTX (Altschul et al., 1994
). Protein and regulatory or consensus sequences were determined using the MOTIF programs of the Staden package (Staden et al., 2003
).
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RESULTS |
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(i) The sod gene potentially encoding a SOD showing extensive similarity to amino acid sequences typically conserved in Mn-SOD from both Gram-positive and Gram-negative bacteria. An orthologous SOD gene has previously been cloned from C. perfringens strain NCIMB8875 by functional complementation of an E. coli strain lacking superoxide activity (Geissmann et al., 1999).
(ii) The rbr1, rbr2 and rbr3 genes, potentially encoding three rubrerythrin proteins. These proteins, which are 39 to 48 % identical, are closely related to the well characterized rubrerythrin of Desulfovibrio vulgaris (GenBank accession no. AAB39991-1) and to other rubrerythrins from strictly anaerobic bacteria and archaea. The rubrerythrin and related nigerythrin protein from the anaerobic sulphate-reducing bacterium D. vulgaris are suspected to possess NADH peroxidase activity at least in an in vitro assay system (Coulter et al., 1999).
(iii) The rub1 and rub2 genes, both of which encode highly conserved rubredoxin proteins. Such non-haem iron proteins have been found in numerous species of anaerobic eubacteria and archaea. These two rub genes, which encode polypeptides of 53 and 54 aa, respectively, are located in the same chromosomal region of C. perfringens strain 13 and share a similar genetic organization. Both genes are associated with a gene encoding a flavoprotein and a gene that encodes a product displaying similarity with transcriptional regulators. The rub1 gene is located 664 bp upstream of CPE0778, which encodes a probable flavohaemoprotein (haemoglobin-like protein) sharing similarity to other proteins possessing a probable NADH : rubredoxin-oxidoreductase (Rbo) activity. Likewise, rub2 forms an operon with the CPE0781 gene, encoding a hypothetical flavoprotein. The product shares 29 % identity and 47 % similarity with the rubredoxin-oxygen-oxidoreductase (Roo) from Desulfovibrio gigas, which is believed to catalyse the reduction of dioxygen to water (Gomes et al., 1997; Kurtz & Coulter, 2002
). Two putative regulatory genes (CPE0776 and CPE0779) are located upstream of two rubredoxin-encoding genes. The two corresponding proteins, share 76 % identity at the amino acid level and belong to the cAMP-binding protein (CRP)/FNR superfamily of transcriptional factors which includes the CRP, FNR and FLP (FNR-like protein) regulatory proteins (Green et al., 2001
). The genetic region including CPE0776, rub1 and CPE0778 is designated GRI and that including CPE0779, rub2 and CPE0781 is designated GRII (Fig. 1
).
The sequence data also show that the rub2-CPE0781 operon is separated by 208 bp from the ahpC gene of C. perfringens. Associated with the specific electron transporter (AhpF), AhpC is suspected of being the main scavenger of both endogenous alkyl peroxide and H2O2 in E. coli (Seaver & Imlay, 2001). No clear orthologue of the ahpF gene was found in the entire C. perfringens genome. However, a BLAST search identified three genes (CPE0783, CPE2440, CPE2354) sharing about 30 % identity with ahpF, but annotated as probable thioredoxin reductase proteins. Such similarity with a family of thioredoxin reductase proteins has already been pinpointed for the AhpF polypeptide of Salmonella typhimurium (Tartaglia et al., 1990
). Notice that one of these genes (CPE0783) is located just downstream (247 bp) of the ahpC gene of C. perfringens (Fig. 1
).
Functional complementation of a sodA
sodB E. coli strain
To evaluate the superoxide scavenging activity of the sod, rub and rbr genes of C. perfringens, the recombinant plasmids carrying these genes were introduced into E. coli strain QC1799 deficient in both sodA and sodB genes, encoding manganese- and iron-SODs, respectively. This strain was very sensitive to superoxide-generating compounds (e.g. PQ, Pl) and unable to grow in minimal medium in the absence of amino acids. This last property was due to superoxide damage of ironsulphur enzymes in the biosynthetic pathway of branched-chain amino acids (Touati, 2000). The diameter of growth inhibition of the transformants with 25 mM PQ revealed that all the C. perfringens genes tested in strain QC1799 provided partial (rbr2, rbr3, GRI and sod) or complete (rbr1 and GrII) protection against this agent (Fig. 2
). These results were in good agreement with those obtained in liquid minimal medium (Fig. 3
). Plasmids carrying the rbr1 gene and the GRII region of C. perfringens fully restored the aerobic growth of strain QC1799, whereas the expression of sod, rbr2 and rbr3 genes or the entire GRI region were less efficient.
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Functional complementation of a catalase-deficient E. coli strain
The recombinant plasmids described above were also introduced into E. coli QC2476 devoid of catalase activity. Whatever the constructions used, the inhibition concentrations of H2O2 were identical to that of the E. coli mutant strain in either aerobic or anaerobic conditions (data not shown). However, a peroxidase activity was detected in a protein crude extract of strain QC2476(pGrbr1) (Fig. 4). This suggests that only one of the three rubrerythrin genes (CPE0135) possesses an in vitro peroxidase activity and that this activity is not sufficient to protect in vivo a catalase-negative strain of E. coli from the toxic action of H2O2.
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Transcriptional analysis of the sod, rub, rbr and ahpC genes of C. perfringens
To evaluate the roles of the sod, rub, rbr and ahpC genes in the oxidative stress response, we monitored the transcriptional level of these genes in stressed C. perfringens cells. Induction (-fold difference) was determined by comparing stressed samples to the t=0 control sample. The cells were grown to mid-exponential phase in MMP medium under anaerobic conditions and then maintained under anaerobic conditions after the addition of subinhibitory H2O2 (50 µM) or t-BT (25 µM) to the medium, or transferred into the normal atmosphere with vigorous shaking with or without Pl (2·5 µM), H2O2 (50 µM) and t-BT (25 µM). Samples were harvested after 0, 20, 40 and 60 min. RNA was then extracted and hybridized either with radiolabelled internal probes corresponding to the various genes or with a 16S rRNA probe used as an internal standard. Some of the data are illustrated in Fig. 5. The main results may be summarized as follows.
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(b) The sod gene was induced three- to fourfold under aerobic conditions when the intracellular superoxide-producing drug Pl was present. The induction was detected 20 min after addition and remained maximal for at least 60 min. This suggested that the sod gene also takes part in the superoxide stress response. When H2O2 or t-BT was added to the medium in both aerobic and anaerobic conditions, the sod gene was not significantly induced. It is nevertheless possible that the basal level of transcription was sufficient to produce superoxide activity in the presence of oxygen, H2O2 or t-BT, as already suggested by Geissmann et al. (1999).
(c) The mRNA induction level of the three rubrerythrin-encoding genes (rbr1, rbr2 and rbr3) was only significant (three- to fourfold) when the cells were shaken in air in the presence of t-BT.
(d) The ahpC gene of C. perfringens was highly induced (up to 50-fold) whatever the aerobic stress conditions used. Under anaerobic conditions, the induction was less important, about eightfold in cells challenged with H2O2 and between 10- and 20-fold in cells challenged with t-BT.
Physiological properties of mutants impaired in the potentially regulatory genes of GRI and GRII of C. perfringens
To confirm the physiological role of the genes studied in the oxidative stress response of C. perfringens, we attempted to create insertional mutants of the genes of interest. Unfortunately, despite numerous experiments, we failed to inactivate the sod gene. However, we successfully disrupted the regulatory genes of the GRI and GRII regions (CPE0776 and CPE0779, respectively). The survival of the CPE0776 : : catP and CPE0779 : : catP mutants was monitored under aerobic conditions with and without Pl. The survival curves were identical to those obtained with the wild-type strain, but the sizes of the colonies were considerably smaller compared to those of strain 13R (data not shown). The results suggested that even if the activity of Rub1 and CPE0778 is not essential for the survival of C. perfringens under aerobic conditions, and that lowering the activity of the CPE0781-rub2 operon was not sufficient to impair survival at least during the time-course (6 h) of the experiment, they might be both required when the cells are maintained for a longer period of time under aerobic conditions.
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DISCUSSION |
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Geissmann et al. (1999) have shown that the sod mRNA level of C. perfringens strain NCIMB8875 increases during exponential growth and peaks upon entry into stationary phase, independently of whether C. perfringens is grown under anaerobic or partially aerobic conditions. In the more drastic stress conditions used in this study, exposure to oxygen induced the sod gene by a maximum of threefold. Due to the rather high level of SOD still present under anaerobic conditions, this response is undoubtedly sufficient to protect cells against superoxide generated under aerobic conditions. The fact that we failed to generate a sod mutant of C. perfringens strain 13R suggests that the sod gene is essential for survival. However, superoxide-deficient mutants of aerobic organisms [e.g. E. coli (Carlioz & Touati, 1986
), Candida albicans (Hwang et al., 2002
), Streptococcus agalactiae (Poyart et al., 2001
)], or of microaerophilic bacteria exemplified by Helicobacter pylori (Seyler et al., 2001
), have been readily obtained. These mutants are generally more sensitive to oxygen or to oxidative stress than the wild-type and display an increased susceptibility to bacterial killing by macrophages. No such mutants have been obtained in strictly anaerobic Gram-positive bacteria, but sod mutants have been reported in Porphyromonas gingivalis (Nakayama, 1994
; Lynch & Kuramitsu, 1999
).
C. perfringens 13R possesses three genes encoding a non-haem iron protein belonging to the rubrerythrin family. Some members of this family possess NADH peroxidase activity in an in vitro system containing NADH and H2O2, and a bacterial NADH oxidoreductase (Coulter et al., 1999). However, the specific activity is very low and the activity detected in vitro may be different from that expressed in vivo. As some of these proteins can complement E. coli strains devoid of SOD, they may possess SOD-like activity (Lehmann et al., 1996
). However, according to Lumppio et al. (1997)
, this activity was one or two orders of magnitude lower than that reported for classical iron SOD. They thus suggested that these proteins protect cells against oxidative stress by both scavenging iron from and donating iron to oxidatively damaged [4Fe4S] proteins. The proteins may also have peroxidase activity. A mutant of P. gingivalis, an obligate anaerobe, devoid of rubrerythrin-like proteins, was more sensitive to dioxygen and H2O2 than the wild-type strain (Sztukowska et al., 2002
). Our data indicated that the three rubrerythrin genes were only significantly induced when the cultures were shaken in air in the presence of t-BT. Thus, a possible role of the rubrerythrin proteins in the redox reaction required to eliminate organic peroxides under aerobic conditions is not excluded, but an efficient hydrogen peroxidase activity of these proteins in vivo remains questionable. This latter hypothesis was also suggested by Geissmann et al. (1999)
, who showed that in C. perfringens NCIMB8875, rubrerythrin (rbr) gene transcription was not influenced by oxidative stress.
The C. perfringens genes or operons able to restore the growth of a sodA sodB E. coli mutant on minimal medium included the two rubredoxin genes associated with flavoprotein- or flavohaemoprotein-encoding genes (CPE0778 and CPE0781, respectively). The rbo genes from Desulfovibrio species have the same properties (Pianzzola et al., 1996). It has been proposed that the Rbo protein catalyses the reduction of the superoxide anion to H2O2 at the expense of cellular reducing agents such NAD(P)H (Liochev & Fridovich, 1997
; Emerson et al., 2002
; Kurtz & Coulter, 2002
). It has also been proposed that the flavohaemoprotein is a rubredoxin-oxygen oxidoreductase (Roo), the final component of a soluble electron transfer chain that produces energy by recycling NAD(P)+ from carbon reserves in an aerobic environment (Santos et al., 1993
). The fact that in C. perfringens these genes might be regulated by a CRP-like protein, which controls the response to glucose starvation, and that the size of the colonies of the mutants impaired in these regulatory genes is smaller than those of the wild-type strain when plated after oxidative stress, favour the latter hypothesis. Moreover, orthologues of putative NADH : rubredoxin-oxidoreductase and rubredoxin-oxygen-oxidoreductase genes are widespread in anaerobic micro-organisms, even though the genetic organization may be different (Das et al., 2001
; Lumppio et al., 2001
)
The ahpC gene was rapidly and fully induced when exponentially growing wild-type C. perfringens was exposed to air or to various oxidative stress under anaerobic conditions. As a general rule, anaerobic bacteria do not possess catalase activity. It is thus possible that in strict anaerobic bacteria AhpC plays a pivotal role in scavenging the various peroxide species, and accumulates the activity of the two major scavenging enzymes, alkyl hydroperoxide reductase and catalase found in aerobic bacteria (Seaver & Imlay, 2001).
Thus, taken together this specialized enzymic equipment may explain how C. perfringens and other strictly anaerobic bacteria can survive occasional oxidative stress, despite the fact that the key metabolic enzymes required for normal growth are promptly damaged by oxygen or superoxide (Imlay, 2002; Pan & Imlay, 2001
).
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ACKNOWLEDGEMENTS |
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Received 6 January 2004;
revised 4 March 2004;
accepted 19 March 2004.