Molecular cloning and expression analysis of the Rhodobacter capsulatus sodB gene, encoding an iron superoxide dismutase.
Genetic complementation of a sodA sodB Escherichia coli mutant strain was used to clone Rhodobacter capsulatus genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The R. capsulatus sodB gene was expressed in E. coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The sodB gene was expressed at low levels in R. capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a sodB mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodB gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R. capsulatus in aerobic cultures.
Programa Multidisciplinario de Biología Experimental (PROMUBIE), National University of Rosario and CONICET, RA2000 Rosario, Argentina cortez@arnet.com.ar
N
N
Cortez
N
N
Carrillo
C
C
Pasternak
A
A
Balzer
G
G
Klug
D000332:Aerobiosis / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004351:Drug Resistance; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D020043:Genes, Essential; D005816:Genetic Complementation Test; D007501:Iron; D008667:Metalloproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000235:genetics*; D013481:Superoxides / Q000494:pharmacology
D001426:Bacterial Proteins; D008667:Metalloproteins; D016877:Oxidants; D013481:Superoxides; D007501:Iron; C087504:SodB protein, Bacteria; D013482:Superoxide Dismutase