Roles of the bradyrhizobium japonicum terminal oxidase complexes in microaerobic H2-dependent growth
Spectral, inhibitor, and O2-consumption studies on membranes from free-living and bacteroid forms of Bradyrhizobium japonicum have revealed the existence of a number of terminal oxidases, and four terminal oxidase gene clusters within the heme-copper cytochrome family have been cloned. Here the complexes encoded by coxMNOP and coxWXYZ, genes with homology to CuA-containing cytochrome c oxidases and b-type ubiquinol oxidases respectively, are studied by analysis of mutants in each of the two oxidases and a double mutant in both of the terminal oxidase genes. Membranes from microaerobically incubated strain JHK12 (which contains an insertion in coxWXYZ) were deficient in levels of CO-reactive heme b, and both strains JHK12 and Bj3430 (the latter lacks coxMNOP) were deficient in CN--reactive cytochrome b. Membranes of the double mutant (strain JHKS4) retained less than 7% of the cytochrome b3 and 25% of the total CN--reactive cytochrome b of the wild type. Cyanide inhibition curves of oxygen uptake by wild-type membranes were triphasic, and only the phases inhibited by the highest (at about 50 &mgr;M CN-, attributed to cytochrome aa3) and the lowest (at approximately 0.1 &mgr;M) CN- were identifiable in the membranes from the two individual oxidase mutants. Membrane respiratory activity of the double mutant was resistant to CN- over a broad inhibitor concentration in the micromolar range. Consistent with our findings that these oxidases are expressed when cells are incubated in a low O2 environment, the double mutant was severely deficient in H2-dependent chemolithotrophic growth. The latter growth condition requires prolonged incubation in an atmosphere of H2, CO2, and a low (1% or less) partial pressure of oxygen. The double mutant was also deficient in whole cell O2 dependent H2 oxidation, with H2 uptake rates 31% of the wild type. Copyright 1998 Elsevier Science B.V.
Surpin
MA
Department of Biology, The Johns Hopkins University, Baltimore, MD 21218-2685, USA.
Maier
RJ