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 Rhodob acter capsulatus nif region A revealed genes that are homologous to OR F6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiel la pneumoniae. R. capsulatus nifU, which is present in two copies, enc odes a novel type of NifU protein. The deduced amino acid sequences of NifU(I) and NifU(II) share homology only with the C-terminal domain o f NifU from A. vinelandii and K. pneumoniae. In contrast to nifA and n ifB, which are almost perfectly duplicated, the predicted amino acid s equences of the two NifU proteins showed only 39% sequence identity. E xpression of the ORF6-nifU(I)SVW operon, which is preceded by a putati ve sigma54-dependent promoter, required the function of NifA and the n if-specific rpoN gene product encoded by nifR4. Analysis of defined in sertion and deletion mutants demonstrated that only nifS was absolutel y essential for nitrogen fixation in R. capsulatus. Strains carrying m utations in nifV were capable of very slow diazotrophic growth, wherea s ORF6, nifU(I) and nifW mutants as well as a nifU(I)/nifU(II) double mutant exhibited a Nif+ phenotype. Interestingly, R. capsulatus nifV m utants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative n itrogenase system. Homocitrate added to the growth medium repressed et hane formation and cured the NifV phenotype in R. capsulatus. Higher c oncentrations of homocitrate were necessary to complement the NifV phe notype of a polar nifV mutant (NifV-NifW-), indicating a possible role of NifW either in homocitrate transport or in the incorporation of th is compound into the iron-molybdenum cofactor of nitrogenase.