The broad objective of this investigation is to increase our understanding of a pathogenic mechanism of Pseudomonas aeruginosa in chronic respiratory infections of cystic fibrosis patients. within the airways of these patients, P. aeruginosa must have mechanisms to survive considerable oxidative stress in the forms of 1) phagocyte-mediated oxygen radicals, 2) high P02 within this milieu, and 3) pyocyanin, a toxic redox compound produced by P. aeruginosa. To combat this oxidative stress, P. aeruginosa produces superoxide dismutases (SODs, which disproportionate superoxide forming hydrogen peroxide), catalases (which destroy hydrogen peroxide), and alginate (a scavenger of extra-cellular oxygen radicals). We have recently shown that the alginate regulatory complex is strongly stimulated by oxidative stress. Little is known about these oxidative stresses and its contribution to the regulation of the alginate biosynthetic pathway, we will conduct a thorough search for mutants which no longer respond to stress induced by a redox-cycling agent that positively affects alginate gene transcription. Following characterization of the mutants obtained, we will clone a regulatory redox (rdx) gene which affects the transcription of algD, a gene encoding a key enzyme in the alginate biosynthetic pathway. Characterization of the rdx gene will include physical mapping, DNA sequence analysis, and regulation using transcription fusions. Defined mutants defective in the redox stress regulator will be constructed by gene replacement and will be characterized. To investigate the role of SOD and its regulation in the global response to oxidant stress in P. aeruginosa, we will construct sodB mutants by gene replacement to determine the role of Fe-SOD in oxidant stress and alginate production. We will also characterize the sodA gene of P. aeruginosa and generate an sodAsodB double mutant to determine its sensitivity to oxidative stress and effect on alginate production. To determine the role of catalase, and its oxidant-mediated regulation in P. aeruginosa, we will also clone a catalase (kat) gene by using genetic complementation strategies. As above, kat mutants and kat- transcriptional fusions will be constructed to evaluate the role of catalase in response to various conditions of oxidative stress.
