Nucleotide sequence and genetic analysis of the Rhodobacter capsulatus ORF6-nifUI SVW gene region: possible role of NifW in homocitrate processing.

DNA sequence analysis of a 3494-bp HindIII-BclI fragment of the Rhodobacter capsulatus nif region A revealed genes that are homologous to ORF6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiella pneumoniae. R. capsulatus nifU, which is present in two copies, encodes a novel type of NifU protein. The deduced amino acid sequences of NifUI and NifUII share homology only with the C-terminal domain of NifU from A. vinelandii and K. pneumoniae. In contrast to nifA and nifB, which are almost perfectly duplicated, the predicted amino acid sequences of the two NifU proteins showed only 39% sequence identity. Expression of the ORF6-nifUISVW operon, which is preceded by a putative sigma 54-dependent promoter, required the function of NifA and the nif-specific rpoN gene product encoded by nifR4. Analysis of defined insertion and deletion mutants demonstrated that only nifS was absolutely essential for nitrogen fixation in R. capsulatus. Strains carrying mutations in nifV were capable of very slow diazotrophic growth, whereas ORF6, nifUI and nifW mutants as well as a nifUI/nifUII double mutant exhibited a Nif+ phenotype. Interestingly, R. capsulatus nifV mutants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative nitrogenase system. Homocitrate added to the growth medium repressed ethane formation and cured the NifV phenotype in R. capsulatus. Higher concentrations of homocitrate were necessary to complement the NifV phenotype of a polar nifV mutant (NifV-NifW-), indicating a possible role of NifW either in homocitrate transport or in the incorporation of this compound into the iron-molybdenum cofactor of nitrogenase.