Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria.
Pseudomonas aeruginosa is a strict aerobe which is likely exposed to oxygen reduction products including superoxide and hydrogen peroxide during the metabolism of molecular oxygen. To counterbalance the potentially hazardous effects of elevated endogenous levels of superoxide, most aerobic organisms possess one or more superoxide dismutases or compounds capable of scavenging superoxide. We have previously shown that P. aeruginosa possesses both an iron- and a manganese-cofactored superoxide dismutase (D. J. Hassett, L. Charniga, K. A. Bean, D. E. Ohman, and M. S. Cohen, Infect. Immun. 60:328-336, 1992). In this study, the genes encoding manganese (sodA)- and iron (sodB)- cofactored superoxide dismutase were cloned by using a cosmid library of P. aeruginosa FRD which complemented an Escherichia coli (JI132) strain devoid of superoxide dismutase activity. The sodA and sodB genes of P. aeruginosa, when cloned into a high-copy-number vector (pKS-), partially restored the aerobic growth rate defect, characteristic of the Sod- strain, to that of the wild type (AB1157) when grown in Luria broth. The nucleotide sequences of sodA and sodB have open reading frames of 612 and 579 bp that encode dimeric proteins of 22.9 and 21.2 kDa, respectively. These data were also supported by the results of in vitro expression studies. The deduced amino acid sequence of the P. aeruginosa manganese and iron superoxide dismutase revealed approximately 50 and 67% similarity with manganese and iron superoxide dismutases from E. coli, respectively. There was also remarkable similarity with iron and manganese superoxide dismutases from other phyla. The mRNA start site of sodB was mapped to 174 bp upstream of the ATG codon. A likely promoter with similarity to the -10 and -35 consensus sequence of E. coli was observed upstream of the ATG start codon of sodB. Regions sequenced 519 bp upstream of the sodA electrophoresis, sodA gene revealed no such promoter, suggesting an alternative mode of control for sodA. By transverse field electrophoresis, sodA and sodB were mapped to the 71- to 75-min region on the P. aeruginosa PAO1 chromosome. Strikingly, mucoid alginate-producing bacteria generated greater levels of manganese superoxide dismutase than nonmucoid revertants, suggesting that mucoid P. aeruginosa is responding to oxidative stress and/or changes in the redox status of the cell.
Hassett
D J
DJ
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45257-0524.
Woodruff
W A
WA
Wozniak
D J
DJ
Vasil
M L
ML
Cohen
M S
MS
Ohman
D E
DE
D000464:Alginates / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000235:genetics*
D000464:Alginates; D013482:Superoxide Dismutase