PSEUDOMONAS-AERUGINOSA SODA AND SODB MUTANTS DEFECTIVE IN MANGANESE-COFACTORED AND IRON-COFACTORED SUPEROXIDE-DISMUTASE ACTIVITY DEMONSTRATE THE IMPORTANCE OF THE IRON-COFACTORED FORM IN AEROBIC METABOLISM

The consumption of molecular oxygen by Pseudomonas aeruginosa can lead to the production of reduced oxygen species, including superoxide, hy drogen peroxide, and the hydroxyl radical, As a first line of defense against potentially toxic levels of endogenous superoxide, P. aerugino sa possesses an iron- and manganese-cofactored superoxide dismutase (S OD) to limit the damage evoked by this radical. In this study, we have generated mutants which possess an interrupted sodA (encoding mangane se SOD) or sodB (encoding iron SOD) gene and a sodA sodB double mutant , Mutagenesis of sodA did not significantly alter the aerobic growth r ate in rich medium (Luria broth) or in glucose minimal medium in compa rison with that of wild-type bacteria. In addition, total SOD activity in the sodA mutant was decreased only 15% relative to that of wild-ty pe bacteria. In contrast, sodB mutants grew much more slowly than the sodA mutant or wild-type bacteria in both media, and sodB mutants poss essed only 13% of the SOD activity of,wild-type bacteria, There was al so a progressive decrease in catalase activity in each of the mutants, with the sodA sodB double mutant possessing only 40% of the activity of wild-type bacteria. The sodA sodB double mutant grew very slowly in rich medium and required similar to 48 h to attain saturated growth i n minimal medium, There was no difference in growth of either strain u nder anaerobic conditions. Accordingly, the sodB but not the sodA muta nt demonstrated marked sensitivity to paraquat, a superoxide-generatin g agent. P. aeruginosa synthesizes a blue, superoxide-generating antib iotic similar to paraquat in redox properties which is called pyocyani n, the synthesis of which is accompanied by increased iron SOD and cat alase activities (D, J, Hassett, L, Charniga, K, A, Bean, D, E. Ohman, and hi. S. Cohen, Infect. Immun, 60:328-336, 1992). Pyocyanin product ion was completely abolished in the sodB and sodA sodB mutants and was decreased similar to 57% in sodA mutants relative to that of the wild -type organism. Furthermore, the addition of sublethal concentrations of paraquat to wild-type bacteria caused a concentration-dependent dec rease in pyocyanin production, suggesting that part of the pyocyanin b iosynthetic cascade is inhibited by superoxide. These results suggest that iron SOD is more important than manganese SOD for aerobic growth, resistance to paraquat, and optimal pyocyanin biosynthesis in P. aeru ginosa.