CLONING AND CHARACTERIZATION OF THE PSEUDOMONAS-AERUGINOSA SODA AND SODB GENES ENCODING MANGANESE-COFACTORED AND IRON-COFACTORED SUPEROXIDE-DISMUTASE - DEMONSTRATION OF INCREASED MANGANESE SUPEROXIDE-DISMUTASEACTIVITY IN ALGINATE-PRODUCING BACTERIA

Pseudomonas aeruginosa is a strict aerobe which is likely exposed to o xygen reduction products including superoxide and hydrogen peroxide du ring the metabolism of molecular oxygen. To counterbalance the potenti ally hazardous effects of elevated endogenous levels of superoxide, mo st aerobic organisms possess one or more superoxide dismutases or comp ounds capable of scavenging superoxide. We have previously shown that P. aeruginosa possesses both an iron- and a manganese-cofactored super oxide 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 superoxid e dismutase were cloned by using a cosmid library of P. aeruginosa FRD which complemented an Escherichia coli (JI132) strain devoid of super oxide dismutase activity. The sodA and sodB genes of P. aeruginosa, wh en cloned into a high-copy-number vector (pKS(-)), partially restored the aerobic growth rate defect, characteristic of the Sod(-) strain, t o that of the wild type (AB1157) when grown in Luria broth. The nucleo tide sequences of sodA and sodB have open reading frames of 612 and 57 9 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 s tudies. The deduced amino acid sequence of the P. aeruginosa manganese and iron superoxide dismutase revealed approximate to 50 and 67% simi larity with manganese and iron superoxide dismutases from E. coli, res pectively. There was also remarkable similarity with iron and manganes e 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 obs erved upstream of the ATG start codon of sodB. Regions sequenced 519 b p upstream of the sodA gene revealed no such promoter, suggesting an a lternative mode of control for sodA. By transverse field electrophores is, sodA and sodB were mapped to the 71- to 75-min region on the P. ae ruginosa PAO1 chromosome. Strikingly, mucoid alginate-producing bacter ia generated greater levels of manganese superoxide dismutase than non mucoid revertants, suggesting that mucoid P. aeruginosa is responding to oxidative stress and/or changes in the redox status of the cell.