AZORHIZOBIUM-CAULINODANS RESPIRES WITH AT LEAST 4 TERMINAL OXIDASES

In culture, Azorhizobium caulinodans used at least four terminal oxida ses, cytochrome aa(3) (cytaa(3)), cytd, cyto, and a second a-type cyto chrome, which together mediated general, respiratory electron (e(-)) t ransport to O-2. To genetically dissect physiological roles for these various terminal oxidases, corresponding Azorhizobium apocytochrome ge nes were cloned, and three cytaa(3) mutants, a cytd mutant, and a cyta a(3), cytd double mutant were constructed by reverse genetics. These c ytochrome oxidase mutants were tested for growth, oxidase activities, and N-2 fixation properties both in culture and in symbiosis with the host plant Sesbania rostrata. The cytaa(3) mutants grew normally, fixe d N-2 normally, and remained fully able to oxidize general respiratory e(-) donors (NADH, succinate) which utilize a cytc-dependent oxidase. By difference spectroscopy, a second, a-type cytochrome was detected in the cytaa(3) mutants. This alternative a-type cytochrome (A(max) = 610 nm) was also present in the wild type but was masked by bona fide cytaa(3) (A(max) = 605 nm). In late exponential-phase cultures, the cy taa3 mutants induced a new, membrane-bound, CO-binding cytc(550), whic h also might serve as a cytc oxidase (a fifth terminal oxidase). The c loned Azorhizobium cytaa(3) genes were strongly expressed during expon ential growth but were deactivated prior to onset of stationary phase. Azorhizobium cytd mutants showed 40% lower N-2 fixation rates in cult ure and in planta, but aerobic growth rates were wild type. The cytaa( 3), cytd double mutant showed 70% lower N-2 fixation rates in planta. Pleiotropic cytc mutants were isolated by screening for strains unable to use N,N,N',N'-tetramethyl-p-phenylenediamine as a respiratory e(-) donor. These mutants synthesized no detectable cytc, excreted copropo rphyrin, grew normally in aerobic minimal medium, grew poorly in rich medium, and fixed N-2 poorly both in culture and in planta. Therefore, while aerobic growth was sustained by quinol oxidases alone, N-2 fixa tion required cytc oxidase activities. Assuming that the terminal oxid ases function as do their homologs in other bacteria, Azorhizobium res piration simultaneously employs both quinol and cytc oxidases. Because Azorhizobium terminal oxidase mutants were able to reformulate their terminal oxidase mix and grow more or less normally in aerobic culture , these terminal oxidases are somewhat degenerate. Its extensive termi nal oxidase repertoire might allow Azorhizobium spp. to flourish in wi de-ranging O-2 environments.