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.