The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium japonicum.
We present an extended genetic analysis of the previously identified cycH locus in Bradyrhizobium japonicum. Three new open reading frames found in an operon-like structure immediately adjacent to the 3' end of cycH were termed cycJ, cycK and cycL. A deletion mutant (delta cycHJKL) and biochemical analysis of its phenotype showed that the genes of the cluster are essential for the biogenesis of cellular c-type cytochromes. Mutations in discrete regions of each of the genes were also constructed and shown to affect anaerobic respiration with nitrate and the ability to elicit an effective symbiosis with soybean, both phenotypes being a consequence of defects in cytochrome c formation. The CycK and CycL proteins share up to 53% identity in amino acid sequence with the Rhodobacter capsulatus Cc11 and Cc12 proteins, respectively, which have been shown previously to be essential for cytochrome c biogenesis, whereas cycJ codes for a novel protein of 169 amino acids with an M(r) of 17857. Localisation studies revealed that CycJ is located in the periplasmic space; it is probably anchored to the cytoplasmic membrane via an N-terminal hydrophobic domain. Based on several considerations discussed here, we suggest that the proteins encoded by the cycHJKL-cluster may be part of a cytochrome c-haem lyase complex whose active site faces the periplasm.
Ritz
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Mikrobiologisches Institut, Eidgenössiche Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
Thöny-Meyer
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Hennecke
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D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008190:Lyases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics; D011487:Protein Conformation; D015183:Restriction Mapping; D012230:Rhizobiaceae / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013559:Symbiosis
D001426:Bacterial Proteins; C093104:CycJ protein, Bacteria; C093105:CycK protein, Bacteria; D003574:Cytochrome c Group; D004269:DNA, Bacterial; D008565:Membrane Proteins; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D008190:Lyases; C031581:cytochrome C synthetase