Cloning of the sodA gene from Corynebacterium melassecola and role of superoxide dismutase in cellular viability

Abstract

The sodA gene encoding the Corynebacterium melassecola manganese-cofactored superoxide dismutase (SOD) has been cloned in Escherichia coli and sequenced. The gene is transcribed monocistronically; the predicted polypeptide is 200 amino acids long and associates in a homotetrameric, manganese-dependent form, able to complement an SOD-deficient E. coli mutant. A second open reading frame, coding for a putative 217-amino-acid protein with high homology to peptide methionine sulfoxide reductases from various origins, has been identified immediately upstream of sodA in the opposite transcription orientation. The sodA gene was inactivated by insertion of an integrative vector carrying a kanamycin resistance gene. The growth rate of the SOD-deficient integrant was only slightly affected in BHI rich medium as well as in BMCG chemically defined medium, but was strongly affected by the presence of the redox-cycling agent paraquat. The SOD deficiency had, on the other hand, a deleterious effect on viability as soon as the culture entered the stationary phase of growth in BHI medium. Surprisingly, SOD deficiency was able to rescue the dramatic loss of viability observed for the wild-type strain in BMCG synthetic medium when glucose was not the limiting growth factor.

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.

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.

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).

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).

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%.

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%.