INTRODUCTION

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Reactive oxygen species are formed in aerobic organisms as by-products of normal metabolism, as a consequence of partial reduction of molecular oxygen to superoxide anion (O₂^·), hydrogen peroxide (H₂O₂), and hydroxyl radical ( ^· OH). To protect cells from damage by these reactive species to DNA, proteins, and lipids, aerobic organisms have evolved detoxification and repair systems (69). Superoxide dismutase (SOD; EC 1.15.1.1) is considered a key enzyme in oxygen defense systems by catalyzing the dismutation of O₂^· into oxygen and H₂O₂ (22), the latter being broken in turn to water by catalase or peroxidase.

SODs are classified in different groups depending on the type of metal cofactors: copper-and zinc-containing (Cu,Zn-SOD) (46), iron-containing (Fe-SOD) (83), manganese-containing (Mn-SOD) (39), and nickel-containing (Ni-SOD) (84) enzymes. Another type of small metalloprotein with superoxide dismutase activity, unrelated to classical SODs, has also been recently identified in the sulfate-reducing bacterium Desulfovibrio (65). Most bacteria possess an Mn-SOD or an Fe-SOD in their cytoplasm, while Cu,Zn-SODs have been identified in the periplasm or periphery of pathogenic and endosymbiotic bacteria (4, 5, 80). The Mn- and Fe-SODs have highly similar sequences and structures, usually have a strict metal selectivity, and are evolutionarily unrelated to other SODs. On the other hand, some SODs have been found to be active with either manganese or iron incorporated into the same apoprotein (47, 63, 82). These enzymes were named cambialistic. Some bacteria, like Escherichia coli and Pseudomonas aeruginosa, have both an Fe-SOD and an Mn-SOD, which differ not only in their primary sequences but also in their regulation (29, 72) and, at least for E. coli, in their intracellular localization, the Fe-SOD being located closer to the inner membrane than the Mn-SOD enzyme (32).

With the exception of E. coli, which has been investigated in detail (72), little is known about bacterial SOD expression and regulation, especially in gram-positive bacteria, although many genes have been cloned and sequenced. Most work concerns pathogenic species, mainly because of the relationship between antioxidant defenses and pathogenicity (1, 4, 85). Less is known about their nonpathogenic relatives. Although the amino acid-producing, telluric, and nonpathogenic Corynebacteria species have been well studied for decades, most of the work has focused on biochemical pathways or metabolic fluxes, and nothing is known about their antioxidant defenses. The so-called "amino acid-producing corynebacteria" group contains the closely related Corynebacterium glutamicum, Corynebacterium melassecola, Brevibacterium flavum, and Brevibacterium lactofermentum species (42), sometimes gathered together under the sole C. glutamicum name, and belongs to the actinomycetes suborder along with the Mycobacterium and Nocardia genera (66). Considering the high oxygen requirement of the aerobic C. melassecola species (15), we found it of great interest to study their antioxidant defenses. We therefore investigated and characterized the sodA gene and the corresponding protein of C. melassecola and studied the role of this gene in the response to superoxide and in survival after starvation.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. The bacterial strains and vectors used in this study are listed in Table 1. C. melassecola strains were grown aerobically (250 rpm) at 34°C, either in brain heart infusion (BHI; Difco) rich medium or in BMCG chemically defined medium containing 2% glucose, as described by Guillouet and Engasser (26). Growth was followed by measuring the optical density at 570 nm (OD₅₇₀) in a DU 7400 Beckman spectrophotometer. Kanamycin (25 µg/ml) and chloramphenicol (6 µg/ml) were added to the medium when needed. The E. coli DH5 strain was used for molecular biology procedures. E. coli QC1799 sodA sodB and its parental GC4468 strain were kindly provided by Danièle Touati (Institut Jacques Monod, Université Paris VII, Paris, France). E coli strains were grown aerobically at 37°C in Luria-Bertani (LB) medium (Difco) or in M63 glucose minimal medium as described by Carlioz and Touati (13). Growth was monitored at 600 nm (OD₆₀₀). Ampicillin (100 µg/ml), kanamycin (25 µg/ml), and chloramphenicol (30 µg/ml) were added to the medium when needed. All chemicals were purchased from Sigma.

C. melassecola-specific microbial procedures. Sensitivity to paraquat and hydrogen peroxide (H₂O₂) was monitored by growth inhibition on plates. Bacteria were grown in liquid BHI rich medium to late log phase, and 100-µl aliquots from a 1,000-fold dilution were spread on BHI agar plates. A 5-mm sterile filter paper disk was placed in the middle of each plate and impregnated with 10 µl of 10-mg/ml paraquat or 10% H₂O₂. After 2 days of incubation at 34°C, sensitivity was determined by measuring the diameter of the growth inhibition zone surrounding the disk. For the determination of bacterial viability, C. melassecola cultures were serially diluted at room temperature in NaCl (0.9 g/liter), and aliquots were spread on BHI plates. After 2 days of incubation at 34°C, the colonies were counted, and viability was calculated as CFU per OD₅₇₀ unit (CFU/OD).

General molecular biology techniques. Standard molecular biology procedures were used according to Sambrook et al. (62). Enzymes were obtained from Promega and Boehringer Mannheim. Plasmid DNA was prepared with the Wizard kit from Promega, with 1 h of pretreatment at 37°C for C. melassecola in Wizard resuspension solution containing lysozyme (20 mg/ml). DNA fragments were isolated from agarose gels with the Jetsorb kit (Genomed). Preparation of C. melassecola genomic DNA and Southern blotting were performed as described elsewhere (59). DNA probes were labeled with [-³²P]dCTP (Amersham) using the T7 Quick Prime random priming kit (Pharmacia).

Cloning of the sodA gene. (i) Amplification of an internal fragment by PCR. For PCR experiments, primers S1 [5'-GGCCACACCAACCACTCCGT(C/G)TT-3'] and S3 [5'-AGGTAGAA(A/T)GCGTGCTCCCACAT-3'] (see Fig. 3) were chosen on the basis of highly conserved regions in bacterial Fe- and Mn-SODs (30) and according to the codon preference in Corynebacterium-related species (45). PCR was carried out with 2.5 U of thermostable DNA polymerase (AmpliTaqGold; Perkin-Elmer) in a reaction mixture containing 30 ng of genomic C. melassecola DNA, 0.2 mM each deoxynucleotide triphosphates (Promega), 0.5 µM each of both primers, 2 mM MgCl₂, and 1× AmpliTaqBuffer in a final volume of 50 µl. For the amplification reaction, after 10 min at 94°C, 35 identical cycles (1 min at 94°C, 1 min at 50°C, and 1 min at 72°C) were followed by a final elongation step of 5 min at 72°C. The amplified DNA fragment of the expected size was cloned into the pGEM-T vector (Promega) to give plasmid pMM7. Partial sequencing of the insertion showed high homology with known Fe- and Mn-SODs.

(ii) Cloning of the entire sodA gene. The amplified fragment isolated from pMM7 was radiolabeled and used as a probe for colony hybridization (62). One clone (pMM8) was selected from a previously described C. melassecola ATCC 17965 genomic library (59). The pMM8 plasmid was shown to carry a 10-kb inserted DNA fragment. From Southern experiments, this DNA fragment clearly originated from the C. melassecola chromosomal DNA, without detectable structural alteration. The DNA sequence was determined for 2.2 kb on both stands by primer walking in the region covering the sodA gene, using an ABI model 373 DNA sequencer (Applied Biosystem).

Disruption of the sodA chromosomal gene. An internal sodA fragment (see Fig. 3) was amplified by PCR using primers S6 (5'-GCAGGATCCAACGCAGCACTCGAGGCACTA-3'), including a BamHI site (underlined), and S7 (5'-ATCAAGCTTCAGAACTGGGGTGATGTCGAT-3'), including an HindIII site (underlined). The 371-bp PCR fragment was digested with BamHI and HindIII and inserted into the E. coli-C. melassecola shuttle vector pCGL243 (59) to create pMM12. This vector was used to construct the integron, essentially as described by Reyes et al. (59). The pMM12 plasmid was introduced into C. melassecola by electroporation (9). Plasmid pMM12 was extracted from this host and digested by NotI. The 1.8-kb fragment containing the aphIII kanamycin resistance gene and the internal sodA fragment was isolated and self-ligated to create the viMM12 integron. Transformants from C. melassecola wild-type strain with the integron were the result of a single crossing-over, as verified by Southern blotting. The disrupted strain was named CGL10016, and two independant integrants were used for physiological analysis.

Construction of an expression vector for C. melassecola sodA. The trc promoter was isolated from pKK388-1 (11) as a 0.36-kb BamHI-NcoI fragment, and sodA was isolated from pMM8 as a 0.8-kb NcoI-SmaI fragment, including the entire open reading frame (ORF) and the putative terminator (see Fig. 3). The two fragments were ligated together and inserted into a BamHI-SmaI-digested pCGL243 shuttle vector to give plasmid pMM23.

Preparation of crude extracts. Bacterial cells were harvested and resuspended in 50 mM sodium phosphate buffer (pH 7.8) to 50 OD₅₇₀ units for C. melassecola. Cells were disrupted by vortexing three times 1 ml of suspension with 1 g of 0.5-mm glass beads for 2 min in a 2-ml Eppendorf tube. The suspension was centrifuged at 10,000 × g for 15 min, and the resulting supernatant was used as the crude extract for enzymatic determinations. The protein concentration was measured by the method of Lowry et al. (44) using bovine immunoglobulin G as a standard.

Determination of SOD, catalase, aconitase, and isocitrate dehydrogenase activities. SOD specific activity was determined by the method of Ukeda et al. (74) based on the inhibition of a tetrazolium salt reduction by superoxide generated from a xanthine-xanthine oxidase reaction. Purified E. coli Mn-SOD (Sigma) was used as a standard, and 1 U is defined as the amount of enzyme that inhibits the rate of reduction of cytochrome c by 50% in the standard xanthine oxidase/cytochrome c assay. SOD activity in nondenaturing 10% acrylamide gels was visualized by tetrazolium negative staining according to Schmidt et al. (64). Catalase activity was determined according to Goldberg and Hochman (24) by measuring the disappearance of H₂O₂ at 240 nm, with an initial concentration of 20 mM. Aspergillus niger catalase (Sigma) was used as a standard, and 1 U is defined as the amount of enzyme catalyzing the dismutation of 1 µmol of H₂O₂ per min. Aconitase and isocitrate dehydrogenase activities were determined as described by Hanson and Cox (28).

Purification of SOD from C. melassecola. One liter of bacterial culture in BHI medium was harvested at an OD₅₇₀ of 8.5, washed twice in 0.5 volume of 25 mM sodium phosphate (pH 7.5) buffer, and resuspended in 40 ml of the same buffer containing 10 mM MgCl₂, RNase A (1 µg/ml), and DNase I (1 µg/ml). Cells were disrupted in a French press (SLM Aminco) at 16,000 lb/in². The extract was centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was ultracentrifuged at 130,000 × g for 2 h at 4°C, and the resulting supernatant was used as the cytosoluble fraction. This supernatant was fractionated with ammonium sulfate. Proteins precipitating in the range of 90 to 100% ammonium sulfate saturation, the fraction containing most of the SOD activity, were collected by centrifugation, dissolved in 50 mM Tris-Cl (pH 8.0) buffer, dialyzed against the same buffer, and applied to a Bioscale Q2 (Bio-Rad) ion-exchange column equilibrated with the same buffer. Proteins were eluted with a linear gradient of 0 to 0.5 M NaCl in the same buffer. The most active fractions were pooled, dialyzed against the Tris-Cl (pH 8.0) buffer, and run on a Sephacryl S300 (Pharmacia) column equilibrated with the same buffer. The fractions containing the eluted SOD were pooled and concentrated with a Centriprep-30 (Amicon). After this step, 5 µg of protein was analyzed for purity. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining according to Laemmli (40), only a single band could be observed (see Fig. 2).

Nucleotide sequence accession number. The assigned accession number for the nucleotide and deduced amino acid sequences of sodA and msrA is AF236111 in the GenBank nucleotide sequence database.

	RESULTS

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Heterologous expression of the cloned gene. The C. melassecola sodA gene was cloned, through a PCR-based method, as a pMM8-borne DNA fragment as described in Materials and Methods. Since many C. melassecola genes have been proven to be expressed in E. coli (56), the cloned DNA fragment was tested for its ability to complement the QC1799 sodA sodB mutant of E. coli. This mutant cannot grow aerobically in minimal medium without addition of a subset of amino acids and exhibits enhanced sensitivity to the superoxide generator paraquat (13). The QC1799 E. coli mutant was transformed with plasmid pMM8. The resulting transformed strain showed a growth pattern in minimal medium similar to that of the parental strain. To ensure that E. coli complementation depended only on C. melassecola sodA gene expression, and for further expression studies in C. melassecola, the sodA coding region, identified after DNA sequencing (see below), was placed under the control of the E. coli hybrid trc promoter, which was previously shown to promote efficient expression of genes in both E. coli (11) and C. glutamicum (17). The resulting pMM23 plasmid was used to transform QC1799, and the recombinant strain showed a growth pattern in minimal medium (Fig. 1a) and in LB medium containing 50 µM paraquat (Fig. 1b) similar to that of the parental SOD-proficient strain and grew even faster than the nontransformed E. coli SOD-deficient strain in LB without paraquat (Fig. 1b).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Complementation of SOD defect in E. coli by the C. melassecola sodA gene. (a) Growth in M63 minimal medium was measured by OD600 for the QC1799 sodA sodB mutant transformed by the control vector pCGL482 (), for QC1799 transformed by pMM23 (), for the nontransformed parental strain GC4468 (), and for GC4468 transformed by pMM23 (). Transformed strains were grown in the presence of chloramphenicol (30 µg/ml). (b) Growth of GC4468 (, ), nontransformed QC1799 (, ), and QC1799 transformed by pMM23 () was monitored by OD600 in LB medium in the presence (, , ) or absence (, ) of 30 µM paraquat.

Purification and biochemical characterization of SOD. To obtain information on the characteristics of the C. melassecola SOD, it was purified as described in Materials and Methods from the wild-type strain. A representative scheme for purification is given in Table 2. The isolated protein was estimated to be pure from SDS-PAGE analysis (Fig. 2). The SOD specific activity of the purified protein was estimated to 3,900 U/mg of protein. The amino acid sequences of the N terminus and two internal peptides isolated from the purified protein were determined (see below). These sequences unambiguously confirmed that the purified protein corresponded to an SOD. The molecular weight of native SOD was estimated to approximately 80,000 from gel filtration chromatography. Its monomeric molecular weight, determined by SDS-PAGE, was 24,000 (Fig. 2). Atomic absorption spectrometry for identification of the metal cofactor in the purified protein was carried out. Manganese was clearly identified, and the calculated stoichiometry was 0.43 atom/subunit. This unbalanced stoichiometry could be explained by either release of manganese from the enzyme during purification or manganese limitation in the growth medium. No signal corresponding to the presence of iron was detected.

View larger version (60K): [in this window] [in a new window]   FIG. 2.   Purification of SOD from C. melassecola. SDS-PAGE of cytosoluble extract from C. melassecola (lane 1), extract after ammonium sulfate fractionation (lane 2), and SOD-containing fractions after Bioscale Q2 chromatography (lane 3), and Sephacryl S300 chromatography (lane 4). Lane M, molecular weight standards as described in Materials and Methods.

Nucleotide sequence analysis of the cloned DNA fragment. The nucleotide sequence of a 2,200-bp fragment from pMM8 was determined by a primer walking strategy from both strands by dideoxy chain termination. Computer analysis revealed two complete and one incomplete ORF, as shown in Fig. 3, extending from ATG (bp 1139) to TAA (bp 1741) for ORF1, from ATG (bp 892) to TAA (bp 239) for ORF2, and from ATG (bp 188) to bp 1 for the incomplete ORF3. ORF2 and ORF3 are in the reverse orientation with respect to ORF1. Database searches with the deduced polypeptides of these three ORFs revealed that the amino acid sequence deduced from ORF1 shows significant homology to known Fe- and Mn-SODs (see below), that deduced from ORF2 shows homology to peptide methionine sulfoxide reductases (PMSR; EC 1.8.4.5) from various organisms (see below), and that deduced from the incomplete ORF3 shows homology to N-acyl-L-aminoacid hydrolases from the Campylobacter jejuni hippuricase family (27). These results indicated that ORF1 may correspond to the sodA gene of C. melassecola and ORF2 may correspond to the msrA gene. ORF3 has not been named.

View larger version (77K): [in this window] [in a new window]   FIG. 3.   Nucleotide sequence of C. melassecola sodA region. ORF1 corresponds to the SOD and ORF2 to the PMSR encoding sequences; ORF3 is incomplete. The nucleotide sequence corresponds to the sodA coding and to the msrA-ORF3 noncoding strand. The deduced amino acid sequences are given below the nucleotide sequence. Amino acids that were determined by amino acid sequencing of the purified C. melassecola SOD are indicated in bold. Putative ribosome-binding sites (RBS) are boxed, and possible promoter regions are underlined. The two inverted sequences (IR) in the promoter region and the putative strem-loop structures downstream of the ORFs are shown with arrows. The NcoI and SmaI restriction sites used for pMM23 construction and the locations of PCR primers are underlined (5'-3' orientation for S6 and S1, 3'-5' orientation for S7 and S3).

Analysis of the sodA gene. The predicted sodA gene product consists of 200 amino acids (Fig. 3), with a molecular weight of 22,104, which is in agreement with the size of the subunit of the purified C. melassecola SOD protein (M_r 24,000), as estimated from SDS-PAGE. The G+C content of 57% in the coding region corresponds to a clear preference for C or G at the wobble position, with a codon bias index of 0.79 as calculated according to Malumbres et al. (45). This is consistent with the relative abundance (about 1% of cytosoluble proteins) calculated by the ratio between specific SOD activity in cytosoluble extract (45 U/mg of protein) and for the purified enzyme (3,900 U/mg). In order to confirm the predicted translation initiation site, the N-terminal amino acid sequence of the purified SOD from C. melassecola was determined. This sequence was identical to the predicted one with the exception of the initial formyl-methionine, which was missing in the purified enzyme, as described for SODs from various origins (35, 70) and in some C. glutamicum proteins (31). The sequences of two internal peptides obtained by trypsin digestion of the purified enzyme were also identical to the deduced sequence. A ribosome-binding site (5'-GAAAGGA-3'), complementary to the 3' end of the Brevibacterium lactofermentum 16S rRNA (3'-AAUCUUUCCUCC-5') (3), was identified 10 bp upstream from the ATG initiation codon (Fig. 3). Two putative promoter regions with nucleotide sequences close to the consensus sequence for C. glutamicum promoters (56) are located upstream from the putative ribosome-binding site. An almost perfect palindromic sequence covers the 35 region from the distal putative promoter. Such a structure could be a binding site for a regulatory factor. Two inverted repeats with putative stable RNA stem-loop structures (G, 61.6 and 87.6 kJ mol¹), located 18 and 88 bp downstream of the stop codon, may represent transcription termination regions. Northern hybridization experiments were performed in order to analyze the size of the sodA transcript. Hybridization of C. melassecola RNA to an internal sodA probe resulted in a single signal at approximately 700 nucleotides (Fig. 4). This size is consistent with the translation initiation and termination regions described above and indicates that the sodA gene is monocistronic.

View larger version (62K): [in this window] [in a new window]   FIG. 4.   Northern analysis of the C. melassecola sodA gene. RNA was extracted from wild-type C. melassecola (lanes 1 and 3) and from the SOD-deficient C. melassecola strain CGL10016 (lanes 2 and 4). RNAs were monitored either with a radioactively labeled sodA internal probe (lanes 3 and 4) or by methylene blue staining (lanes 1 and 2) as described in Materials and Methods. The positions of RNA standards (Promega) are given (lane M) (in nucleotides).

Analysis of the deduced SOD amino acid sequence from C. melassecola. Comparison of the deduced SOD amino acid sequence with protein sequences from databases using the National Center for Biotechnology Information Blast program (2) revealed high similarity to Fe- and Mn-SODs of prokaryotic and eukaryotic origin. The highest scores were obtained with deduced partial sequences of SODs from the pathogenic Corynebacterium diphtheriae and Corynebacterium pseudodiphtheriticum species (81% identity) (86) and from the closely related Mycobacterium species (61% identity with Mycobacterium leprae Mn-SOD). Homology was higher with mitochondrial Mn-SODs (54% identity with human Mn-SOD) than with other bacterial species like Bacillus stearothermophilus (51% identity) and E. coli Mn-SOD (42%) and Fe-SOD (40%). The 12 strictly conserved residues in Fe/Mn-SODs (33), including the four metal-binding residues His₂₇, His₇₆, Asp₁₆₀, and His₁₆₄ (C. melassecola SOD numbering), could be identified. Among the residues involved in metal selectivity, Met₂₄, Leu₂₆, Gly₇₁, Gly₇₂, His₇₃, Phe₇₉, Gln₁₄₅, and Gln₁₆₉ are predicted to be discriminant for an Mn-dependent enzyme (37, 55). Manganese as a cofactor was clearly identified in the purified C. melassecola enzyme by atomic adsorption spectrometry, as described previously.

Analysis of the msrA and adjacent ORF3 genes and products. The 654-bp-long msrA ORF is located only 246 bp upstream from the sodA ORF, in the opposite transcriptional orientation (Fig. 3). The predicted msrA gene product consists of 217 amino acids, with a calculated molecular weight of 23,892, in agreement with the size of known bacterial PMSRs. The predicted msrA gene product showed similarities along its entire length to PMSRs from mammals (50% identity with bovine PMSR [52]) and from bacteria (48% with E. coli [58] and 36% with Streptococcus pneumoniae [79]). A codon bias index of 0.48 was calculated for msrA as described by Malumbres et al. (45), which may indicate a rather low expression level for this gene. A possible ribosome-binding site with the sequence 5'-GAAAGGA-3' is located 12 bp upstream from the initiation codon, and two possible promoter regions with reasonable homologies to consensus sequences are located further upstream. Two short inverted repeats, one covering the ribosome-binding site and the other located at the distal 35, corresponding also to a potential 35 for the sodA gene (see above), could be regions involved in transcriptional regulation.

1

Disruption of the chromosomal sodA gene. To study the physiological effects of sodA deficiency, the chromosomal copy of this gene was disrupted in C. melassecola. For this purpose, the integrative vector viMM12, carrying an internal fragment of the sodA coding region and the aphIII gene, which confers resistance to kanamycin, was constructed as described in Materials and Methods. Integration of viMM12 by a single crossing-over event in the sodA chromosomal locus led to sodA gene disruption, as seen by Southern blotting on several transformants (data not shown). Lack of sodA transcription and expression in the disrupted CGL10016 strain was confirmed as follows. In Northern blotting experiments with CGL10016 RNAs, no sodA transcript was seen (Fig. 4). Activity staining on native polyacrylamide gels did not reveal the presence of any active SOD (not shown). Furthermore, SOD activity in crude extracts from the disrupted strain was 0.24 ± 0.18, versus 17.4 ± 1 U/mg of protein in crude extracts from the wild-type strain. The residual SOD activity in cell extracts from the disrupted strain probably resulted from the presence of about 0.5% genotypic revertants at the stage of cell harvesting (see below).

Effect of sodA disruption on growth. Various growth characteristics of the SOD-proficient wild-type C. melassecola strain ATCC 17965 and the corresponding SOD-deficient strain CGL10016 were compared.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Effect of paraquat on growth and SOD and catalase activities in C. melassecola. (a) Growth of ATCC 17965 (, ) and SOD-deficient CGL10016 (, ) was monitored in BHI or BMCG medium and in BHI with 100 µM paraquat (ATCC 17965, ; CGL10016, ), by measuring OD570. CGL10016 was cultivated in the presence of kanamycin (25 µg/ml). (b) SOD (, ) and catalase (, ) activities in ATCC 17965 crude extracts were measured as described in Materials and Methods in the presence (, ) and absence (, ) of 100 µM paraquat in BHI medium. Activity values are the means of at least three experiments that differed by less than 20%.

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Effect of sodA disruption on oxidative stress response. Responses of the mutant, the pMM23-transformed mutant, and wild-type strains to oxidative stresses generated by paraquat and H₂O₂ were investigated by growth inhibition tests on plates (Table 3) as described in Materials and Methods. Growth of the wild-type strain was not inhibited in the presence of 0.1 mg of paraquat, but the disrupted strain showed a bright inhibition zone. This growth inhibition of the SOD-deficient strain was completely abolished after transformation by pMM23. On the other hand, all strains exhibited the same sensitivity to H₂O₂.

Effect of sodA disruption on viability during stationary phase of growth. To test the effect of SOD deficiency on cell viability after extensive stationary-phase cultivation, cultures of wild-type ATCC 17965 and SOD-deficient CGL10016 C. melassecola strains in BHI rich medium and BMCG chemically defined medium were maintained under aeration at 34°C for 6 days. All reached the stationary growth phase in less than 1 day, the OD₅₇₀ then remaining stable at approximatly 20 U (Fig. 6). Viability was estimated at various cultivation times by the CFU/OD ratio. Microscopic observations at each cultivation time did not reveal cellular aggregation.

View larger version (37K): [in this window] [in a new window]   FIG. 6.   Viability and SOD and catalase activities of C. melassecola during the stationary phase of growth. (a) Growth (, ), viability (, ), and proportion of phenotypic revertants () for SOD-deficient CGL10016 (, , ), and parental ATCC 17965 (, ) strains cultivated in BHI, in the presence or absence of kanamycin (25 µg/ml) for CGL10016. Growth was measured by OD570, and cellular viability was determined by plating on BHI and calculated as CFU/OD. The experiment was done at least three times without significant differences. (b) Growth (, , ) and viability (, , +) determined as described above for ATCC 17965 (, ) and CGL10016 (, ) in BMCG (2% glucose) and for ATCC 17965 in BMCG containing only 1% glucose (, +). Kanamycin (25 µg/ml) was added for CGL10016. (c and d) SOD () and catalase (, ) activities measured as described in Materials and Methods, from crude extracts of ATCC 17965 (, ) and CGL10016 () strains cultivated in BHI (c) or BMCG with 2% glucose (d) in the presence of kanamycin (25 µg/ml) for CGL10016. Values are the means of at least two experiments that differed by less than 20%.

	DISCUSSION

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We identified the sodA gene encoding SOD in C. melassecola ATCC 17965. As estimated from activity-staining experiments, Northern experiments, SOD and aconitase activity measurements, and response of the wild-type and corresponding sodA-disrupted mutant to paraquat, the cloned gene seems to be the unique SOD-encoding gene in this organism. From biochemical experiments on the purified enzyme, SOD from C. melassecola was shown to be a homotetramer. From the deduced amino acid sequence, the enzyme belongs to the Mn-dependent SOD family, as experimentally confirmed by atomic adsorption spectrometry. Since SOD from Mycobacterium tuberculosis contains iron even though its sequence is closer to that of Mn-SODs than Fe-SODs (85), and since the Mycobacterium smegmatis cambialistic enzyme contains both metals (82), it would be interesting to determine if only manganese is present in the SOD of C. melassecola after growth in different media or if an iron-substituted enzyme would retain activity.

Putative promoter sequences for the vegetative sigma 70 factor were proposed upstream of the initiation codon, with respect to the promoter consensus sequences from C. glutamicum (56). The initiation site for transcription should be determined to clearly identify the actual promoter. The gene was shown to be monocistronic by Northern experiments, and the transcript size is fully compatible with the putative initiation and termination signals of transcription. We were unable to demonstrate regulation of C. melassecola sodA gene expression under various growth conditions. No standard binding site for the transcription factors SoxS (41) and Fur (20), involved in the regulation of sod genes in other species (29, 72), were found. The SOD specific activity was not significantly different in the wild-type strain grown in the presence or in the absence of paraquat, even if we assumed from experimental data that paraquat-mediated redox cycling readily occurred in this strain. The absence of a change in activity profile upon paraquat addition could be explained either by considering an immediate removal of superoxide radicals by the existing SOD enzyme or by the failure of superoxide radicals to induce C. melassecola sodA expression. Nevertheless, the expression of sodA could be somehow regulated, as in standard laboratory growth conditions in BHI medium the SOD specific activity was shown to vary. Activity increased during the logarithmic phase of growth and reached a plateau as soon as the culture entered stationary phase, with a maximum at transition phase. It would therefore be interesting to follow the expression of sodA in different conditions, in the presence of other stress inducers, for instance, H₂O₂, or when the strain is less likely to produce superoxide radicals, as should be the case under low aeration. It would thus be important for further study of sodA expression to put a reporter gene under the control of the upstream sequences of sodA.

We identified a second gene, msrA, immediately upstream from sodA, coding for a putative PMSR (52, 58, 79), whose catalytic activity is the reduction of oxidized methionine residues, a modification leading to functional inactivation of numerous proteins (75). A role for PMSR in protecting against oxidative damage has been described in bacteria (51) and yeasts (50). Thus, the location of the msrA gene upstream of sodA in C. melassecola could be more than just a coincidence. The genes are in opposite transcription orientations, and the two initiation codons are separated by only 246 bp. This kind of divergent organization, with two genes involved in the same physiological process and with coordinate regulation, has often been characterized. For example, it is the case for the E. coli soxR and soxS genes, involved in superoxide response (81), and for the Fur-regulated fepA-fes genes, involved in siderophore production (21). In M. tuberculosis and M. leprae, the alkyl hydroperoxide reductase and peroxide stress regulator homolog-encoding genes ahpC and oxyR are divergently transcribed (18). Furthermore, a sod gene has been localized upstream from a peroxidase gene in Helicobacter pylori (76). Nevertheless, this is the first time that divergent sod and msrA genes have been reported. Although a sodA transcript has been clearly identified in the C. melassecola wild-type strain, no msrA transcript could be detected, even in the sodA-disrupted strain, in which the superoxide level is assumed to be increased. This may be due either to a low transcription level of this gene under our conditions or to poor technology for the extraction and/or detection of large RNA of C. melassecola in the case of an operon organization, since msrA is immediately followed by ORF3, the physiological role of which is unknown. We now need to look for conditions and methods to detect msrA gene expression in C. melassecola and to determine if the expression of the divergent sodA and msrA genes is connected. Northern identification of the msrA transcript will also highlight the possible operon organization suggested by the presence of ORF3 immediately downstream of msrA.

A sodA null mutant was constructed by disruption of the chromosomal gene by an integrative vector carrying a kanamycin resistance gene. Unlike the wild-type strain, the disrupted strain showed an increased sensitivity to superoxide radicals generated by the redox cycling agent paraquat. This indicated that the sodA mutation could not be rescued by another activity in C. melassecola cells. The growth rate of the disrupted strain was only slightly impaired in BHI rich medium, with a generation time of 60 min, compared to 50 min for the wild-type strain. This is consistant with the behavior of other organisms (13, 36). Much more surprising was the growth of the disrupted strain in BMCG chemically defined medium. Even without supplementary amino acids, its growth rate was the same as in BHI rich medium, whereas the growth of other species of sod mutants was dramatically impaired in minimal medium without amino acids (13, 36). Oxidative stress has been proven to exist in the C. melassecola mutant strain, as revealed by the decrease in aconitase activity. The prototrophy of the C. melassecola SOD-deficient strain could then be explained by a lower sensitivity of amino acid biosynthesis pathways to superoxide radicals generated by aerobic metabolism. Another explanation could be partial compensation for SOD deficiency by either an unidentified enzymatic activity or the presence of an intracellular metabolite or metal ion able to detoxify superoxide radicals. Since the BMCG medium contains 70 µM FeSO₄, 10 times the concentration in E. coli M63 minimum medium, and since it has been reported that growth in iron-enriched medium can partially compensate for SOD deficiency in E. coli (7), iron-mediated compensation of SOD deficiency could be arguable in our case. Note that, as even the wild-type C. melassecola ATCC 17965 strain cannot grow in M63, the control experiment was not possible. Carotenoid pigments can play a role in singlet oxygen and also superoxide scavenging, (8, 12, 49). Carotenoids have been identified in some Corynebacterium species (54, 77). Even if it has not been demonstrated that the C. melassecola ATCC 17965 pink pigment belongs to this family, its role in superoxide scavenging could be arguable.

While only a small difference in growth rate on BHI medium was observed between wild-type and SOD-deficient strains, they behave in a different way in late stationnary phase with regard to their viability. The viability of the SOD-deficient strain decreased dramatically soon after entry into stationary phase, while the wild-type strain kept a high viability for at least 2 days. Viability of the disrupted strain dropped to 5% of the initial value within 2 days of culture, and almost no sodA mutant cells could be recovered after 4 days, when close to 100% of the population consisted of genotypic revertants. Viability loss in SOD-deficient C. melassecola is not surprising, since the same type of behavior has been described for other organisms (6, 14, 43, 48), but it still remains unclear why the viability of the wild-type strain decreased after 2 days of culture to reach only 2% of the initial value after 4 days.

When viability was followed in BMCG chemically defined medium, the behavior of the two strains was much more surprising. Viability of the SOD-deficient strain decreased less rapidly than in BHI medium, but continued to decrease for 6 days, while no real increase in reversion was observed, contrary to the behavior in BHI. The most important difference concerns the evolution of viability of the wild-type strain. Viability loss was so dramatic that no viable cells could be recovered after 4 days of cultivation. As the viability profile of the wild-type strain was similar to that of the disrupted strain when the initial glucose concentration was lowered from 2 to 1%, we can conclude that this dramatic loss of viability was the result of an excess of glucose, by a physiological mechanism that is still to be defined, and which does not take place, or takes place to a lesser extent, in the SOD-deficient strain.

Even if wild-type and SOD-deficient strains do not behave in the same way in BHI rich medium and in BMCG chemically defined medium concerning viability, it appeared that loss of viability during the culture cannot be attributed to a decrease in SOD or catalase antioxidant activities. Moreover, catalase activity increased in the SOD-deficient strain in BHI and SOD-proficient strain in BMCG, but only after viability started to decrease.

We have to consider that nonviable cells, as determined by our viability test, could in fact be viable but noncultivable cells, or in a so-called dormant state (19, 38). Nevertheless, this is the first time that a SOD-deficient strain has shown a selective advantage over a wild-type strain. Efforts will now be engaged to understand this at the metabolic level.