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-aminoacid 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.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 (O 2 ⅐Ϫ ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical ( ⅐ 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 O 2 ⅐Ϫ into oxygen and H 2 O 2 (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 the...

Differential Expression and Localization of Mn and Fe Superoxide Dismutases in the Heterocystous Cyanobacterium Anabaena sp. Strain PCC 7120

“…strain PCC 7942 (19), and the mutant strain helped in the understanding of the roles that FeSod plays in that organism. A mutant strain of Corynebacterium melassecola lacking sodA was also useful in elucidating the roles of MnSod under different growth conditions (29). Cell differentiation and nodulation in a mutant strain of Sinorhizobium meliloti lacking sodA were severely impaired (35).…”

Identification of a Stress-Induced Factor ofCorynebacterineaeThat Is Involved in the Regulation of the Outer Membrane Lipid Composition

“…Total RNA was extracted from an exponentially growing C. glutamicum culture at 30°C as described previously (27). Reverse transcription (RT) was performed using Transcriptor reverse transcriptase (Roche) according to the manufacturer's recommendations and DNase-and RNase-free (Roche).…”

Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum

“…In the recombinant strain, the sugR His gene was expressed as a single copy under the control of the native sugR promoter. Total RNA from glucose-grown cells was prepared as described by Merkamm & Guyonvarch (2001) and incubated in 0.1 M sodium acetate pH 3.0, 5 mM MgSO 4 and 0.3 U DNase I ml 21 (Roche Diagnostics) for 30 min at 37 uC. After heat inactivation for 5 min at 75 uC and phenol/chloroform extraction, the RNA was precipitated with LiCl as described by Sambrook et al (1989).…”

“…All the molecular biology procedures used in this study were as described by Sambrook et al (1989), Ausubel et al (1987) and Merkamm & Guyonvarch (2001). C. glutamicum and E. coli cells were transformed by electroporation as described by Bonamy et al (1990).…”