title: The cycHJKL genes of Rhizobium meliloti involved in cytochrome c biogenesis are required for "respiratory" nitrate reduction ex planta and for nitrogen fixation during symbiosis.
abstract: We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix-) and "respiratory" nitrate reduction (Rnr-). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr- and Fix- phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Cc11 and Cc12 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multisubunit cytochrome c haem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.
authors.affiliation: Institute of Genetics, Hungarian Academy of Sciences, Szeged.
authors.forename: A
authors.initials: A
authors.lastname: Kereszt
authors.forename: K
authors.initials: K
authors.lastname: Slaska-Kiss
authors.forename: P
authors.initials: P
authors.lastname: Putnoky
authors.forename: Z
authors.initials: Z
authors.lastname: Banfalvi
authors.forename: A
authors.initials: A
authors.lastname: Kondorosi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D000455:Medicago sativa / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016254:Mutagenesis, Insertional; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics; D009586:Nitrogen Fixation / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment; D016962:Sinorhizobium meliloti / Q000201:enzymology / Q000235:genetics*; D013053:Spectrophotometry / Q000379:methods; D013559:Symbiosis
chemical_list: D001426:Bacterial Proteins; C093104:CycJ protein, Bacteria; C093105:CycK protein, Bacteria; D003574:Cytochrome c Group; D004269:DNA, Bacterial; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D009565:Nitrate Reductases; D050901:Nitrate Reductase