AZORHIZOBIUM-CAULINODANS RESPIRES WITH AT LEAST 4 TERMINAL OXIDASES

In culture, Azorhizobium caulinodans used at least four terminal oxidases, cytochrome aa(3) (cytaa(3)), cytd, cyto, and a second a-type cytochrome, which together mediated general, respiratory electron (e(-)) transport to O-2. To genetically dissect physiological roles for these various terminal oxidases, corresponding Azorhizobium apocytochrome genes were cloned, and three cytaa(3) mutants, a cytd mutant, and a cytaa(3), cytd double mutant were constructed by reverse genetics. These cytochrome 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, fixed 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 cytaa3 mutants induced a new, membrane-bound, CO-binding cytc(550), which also might serve as a cytc oxidase (a fifth terminal oxidase). The cloned Azorhizobium cytaa(3) genes were strongly expressed during exponential growth but were deactivated prior to onset of stationary phase. Azorhizobium cytd mutants showed 40% lower N-2 fixation rates in culture 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 coproporphyrin, 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 fixation required cytc oxidase activities. Assuming that the terminal oxidases function as do their homologs in other bacteria, Azorhizobium respiration 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 terminal oxidase repertoire might allow Azorhizobium spp. to flourish in wide-ranging O-2 environments.