Cytochrome c terminal oxidase pathways of Azotobacter vinelandii: analysis of cytochrome c4 and c5 mutants and up-regulation of cytochrome c-dependent pathways with N2 fixation.
The Azotobacter vinelandii cytochrome c5 gene (termed cycB) was cloned and sequenced. Mutants in this c-type cytochrome as well as cytochrome c4 mutants (mutations in cycA) and double mutants in both of the c-type respiratory pathways were characterized. Spectral and heme staining experiments on membranes from the mutants were consistent with the anticipated characteristics of all the gene-directed mutants. Membranes of the individual cytochrome c4 or c5 mutants had normal respiratory rates with physiological substrates but respiration significantly lower than the wild-type rate with ascorbate-N,N,N',N',-tetramethyl-p-phenylenediamine (TMPD) as a reductant. The growth rates of the individual cytochrome c4 or c5 mutants were not markedly different from that of the wild-type strain, but the cycA cycB double-mutant strain was noticeably growth retarded at and below 7.5% O2 on both N-containing and N-free media. The double-mutant strain was unable to grow on agar plates at O2 tensions of 2.5% or less on N-free medium. As the wild-type growth was unaffected by varying the O2 tension, the results indicate that the role of the cytochrome c-dependent pathways is to provide respiration at intermediate (5 to 10%) and low (below 5%) O2 tensions. The two c-type cytochrome genes are transcriptionally up-regulated with N2 fixation; N starvation caused 2.8-fold and 7- to 10-fold increases in the promoter activities of cycA and cycB, respectively, but these activities were affected little by the O2 level supplied to the cultures.
Rey
L
L
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Maier
R J
RJ
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D003576:Electron Transport Complex IV / Q000378:metabolism*; D015870:Gene Expression; D006418:Heme / Q000378:metabolism; D007763:Lac Operon; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics*; D010100:Oxygen / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013057:Spectrum Analysis; D013772:Tetramethylphenylenediamine / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation
D003574:Cytochrome c Group; D004269:DNA, Bacterial; C034186:cytochrome C4; D006418:Heme; C046450:cytochrome C5; D003576:Electron Transport Complex IV; D009584:Nitrogen; D013772:Tetramethylphenylenediamine; D010100:Oxygen