Molecular characterization and quantitative analysis of superoxide dismutases in virulent and avirulent strains of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A. salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB. The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O₂^--catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative sodA were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of A. salmonicida subsp. salmonicida strain A449 when cultured in vitro and in vivo.