At least four genes are known to affect formation of the cytochrome bd-type terminal oxidase of Escherichia coli. In addition to the genes (cydA and cydB) encoding the two constituent subunits of this complex, a further two genes (cydC and cydD) map near 19 min on the E. coli chromosome. We report here the cloning of both genes on a 5.3 kb ClaI-HindIII restriction fragment, which, when used to transform either a cydC or cydD mutant, restored the ability of these mutants to grow on a selective medium containing azide and zinc ions and also restored the spectral signals associated with the cytochrome components of the oxidase complex. A subcloned 1.8 kb DdeI fragment similarly restored growth and cytochrome content of a cydD mutant, but not a cydC mutant. The complete nucleotide sequence of the ClaI-HindIII fragment reveals three open reading frames, one being trxB (19.3 min on the E. coli chromosome map, encoding thioredoxin reductase), confirming the mapping position of cydD previously established by P1-mediated transduction. Two ORFs identified by complementation experiments as cydD and cydC encode proteins with predicted molecular masses, respectively, of 65,103 and 62,946 Da. The hydropathy profile of each protein reveals an N-terminal hydrophobic domain and a C-terminal hydrophilic domain containing a putative nucleotide-binding site. The gene products probably constitute an ABC (ATP-binding cassette) family membrane transporter, the function of which is necessary for the formation of the cytochrome bd quinol oxidase. The CydDC system appears to be the first prokaryotic example of a heterodimeric ABC transport system in which each polypeptide contains both hydrophobic and ATP-binding domains.

We report the isolation and characterization of a mutant of Escherichia coli unable to grow aerobically on nonfermentable substrates, except for very slow growth on glycerol. The mutant contains cytochrome oxidases o and d, and grows anaerobically with alternative electron acceptors. Oxygen consumption rates of cell-free extracts were low relative to activities in an isogenic control strain, but were restored in vitro by adding ubiquinone-1 to cell-free extracts. Transformation with a cloned 2.8 kb ClaI-EcoRV fragment of chromosomal DNA restored the ability of this mutant (AN2571) to grow on succinate and also restored cellular quinone levels in this strain. The plasmid also complemented a previously isolated ubiG mutant (AN151) for aerobic growth on succinate. The nucleotide sequence revealed a 0.7kb portion of gyvA. Unidirectional nested deletions from this fragment and complementation analysis identified an open reading frame encoding a protein with a predicted molecular mass of 26-5 kDa. This gene (u6iG) encodes the enzyme 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase, which catalyses the terminal step in the biosynthesis of ubiquinone. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by three palindromic unit sequences. Comparison of the inferred amino acid sequence of UbiG with the sequence of other S-adenosylmethionine (AdoMet)-dependent methyltransferases reveals a highly conserved AdoMet-binding region. The cloned 2.8 kb fragment also contains a sequence encoding the C-terminus of a protein with 42-44 % identity to fungal acetyl-CoA synthetases.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa 3 pathway were identified by the inability to oxidize N,N-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa 3 and d were present, the latter at a higherthan-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.

A mutant of Escherichia coli has been isolated that is unable to grow aerobically on non-fermentable substrates, but able to grow anaerobically on glycerol with alternative electron acceptors such as fumarate. Nitrate as electron acceptor supports anaerobic growth on glycerol, but not on succinate or lactate. Oxygen consumption rates by cellfree extracts with succinate, lactate or glycerol 3-phosphate as substrates were low relative to activities in an isogenic control strain but were restored in vitro by adding ubiquinone-1. Transformation of the mutant with a cloned 2.6 kb CZaI-PvuII fragment of chromosomal DNA restored cellular quinone levels and growth on succinate. The plasmid also complemented a previously isolated ubiA mutant for aerobic growth on non-fermentable substrates. The nucleotide sequence of the cloned fragment revealed a fragment of plsB (91.7 min on the E. coli chromosome map) and three open reading frames (ORFs), one of which (ORF3) encodes a protein with a predicted molecular mass of 32511 Da. The hydrophobicity profile of the OW3 protein is characteristic of a membrane protein with five hydrophobic regions and is very similar to that of the Saccharomyces cerevisiae COQ2 gene product (p-hydroxybenzoate : polyprenyltransferase, required for the second step of ubiquinone biosynthesis) and to the product of the E. cdi cyoE gene. Complementation of ubi mutants with various deletion derivatives of the cloned DNA fragment confirms that ORF3 is ubiA. ORF3 is closely linked to ubiC (ORF2), which encodes chorismate lyase.

Azotobacter vinelandii cydAB mutants lacking cytochrome bd lost viability in stationary phase, irrespective of temperature, but microaerobiosis or iron addition to stationary phase cultures prevented viability loss. Growth on solid medium was inhibited by a diffusible factor from neighbouring cells, and by iron chelators, In(III) or Ga(III); microaerobic growth overcame inhibition by the extracellular factor. Siderophore production and total Fe(III)-chelating activity were not markedly affected in Cyd(-) mutants, and remained responsive to iron repression. Cyd(-) mutants were hypersensitive to Cu(II), Zn(II), and compounds exerting oxidative stress. Failure to synthesise haemoproteins does not explain the complex phenotype since mutants retained significant catalase activity. We hypothesise that Cyd(-) mutants are defective in maintaining the near-anoxic cytoplasm required for reductive iron metabolism and nitrogenase activity.