DNA sequence analysis of a 12236 bp fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames. With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins. Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation. Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation). Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X2-C-X2-C-X3-C-P) typical for [4Fe--4S] proteins. Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase. In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase. The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole. Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins.

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A DNA region showing homology to Klebsiella pneumoniae nifA and nifB is duplicated in Rhodobacter capsulatus. The two copies of this region are called nifA/nifB copy I and nifA/nifB copy II. Deletion mutagenesis demonstrated that either of the two copies is sufficient for growth in nitrogen-free medium. In contrast, a double deletion mutant turned out to be deficient in nitrogen fixation. The complete nucleotide sequence of a 4838 bp fragment containing nifA/nifB copy I was determined. Two open reading frames coding for a 59,653 (NifA) and a 49,453 (NifB) dalton protein could be detected. Comparison of the amino acid sequences revealed that the R. capsulatus nifA and nifB gene products are more closely related to the NifA and NifB proteins of Rhizobium meliloti and Rhizobium leguminosarum than to those of K. pneumoniae. A rho-independent termination signal and a typical nif promoter region containing a putative NifA binding site and a consensus nif promoter are located within the region between the R. capsulatus nifA and nifB genes. The nifB sequence is followed by an open reading frame (ORF1) coding for a 27721 dalton protein in nifA/nifB copy I. DNA sequence analysis of nifA/nifB copy II showed that both copies differ in the DNA region downstream of nifB and in the noncoding sequence in front of nifA. All other regions compared, i.e. the 5' part of nifA, the intergenic region and the 3' part of nifB, are identical in both copies.

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon TnS mutagenesis. The TnS insertion sites of 30 Nif mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of TnS insertions (21 mutants) map within nif region A, characterized by two CloT fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::TnS insertions, and the three remaining mutations are located on a 32-kb Clal fragment of nif region C. Hybridization experiments using all 17 KlebsieUa pneumonie nifgenes individually as probes revealed homology to nifE, nifS, nifA, and nijB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nifregion A, was identified.The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata [15]) fixes atmospheric dinitrogen (N2) under anaerobic or microaerobic conditions (36). The structural genes (nifHDK) for nitrogenase of R. capsulatus hybridized to Klebsiella pneumoniae nifHDK DNA (28). This conservation helped to clone the nifHDK genes from R. capsulatus (3). Mutations in the corresponding region of the R. capsulatus chromosome result in a Nif-phenotype (31). In addition, several copies of sequences hybridizing to njfHDK were found (31). There is also evidence that one of these sequences contains a gene coding for 16S rRNA, but no copy of the nif structural genes (30). A putative nitrogenase reductase gene has also been localized in the nucleotide sequences from the photosynthetic gene cluster (12).Other R. capsulatus nif genes have been identified by the isolation of Nif mutants (2,34,35,38). Genetic mapping of these nif genes by the gene transfer agent revealed several linkage groups (34), and DNA fragments containing nifgenes were isolated by complementation analysis (2). Willison et al. (37) established a circular linkage map of the R. capsulatus chromosome showing nonclustering of genes involved in nitrogen fixation. One of these nifgene regions was shown to carry a nifA-like regulatory gene contiguous with the niJHDK genes (1). This regulatory gene seems to be identical to the nifR4 gene identified by Kranz and Haselkorn (19). The nifR4 gene and other regulatory genes were characterized using lac gene fusions (19). A detailed review of these results is given by Haselkorn (11).In contrast to the mutants published earlier, the R. capsulatus Nif mutants described in this paper were generated by random TnS mutagenesis. This method allowed easy cloning and exact mapping of a large number of insertional mutations. In addition, we identified and localized R. capsulatus nif genes homologous to nifgenes from K. pneumoniae by hybridization experiments using Klebsiella probes specific for individual genes.

In most bacteria, nitrogen metabolism is tightly regulated and P II proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK ) encoding P II -like proteins. The glnB gene forms part of a glnB-glnA operon and the glnK gene is located immediately upstream of amtB, encoding a (methyl-) ammonium transporter. Expression of glnK is activated by NtrC under nitrogen-limiting conditions. The synthesis and activity of the molybdenum and iron nitrogenases of R. capsulatus are regulated by ammonium on at least three levels, including the transcriptional activation of nifA1, nifA2 and anfA by NtrC, the regulation of NifA and AnfA activity by two different NtrC-independent mechanisms, and the post-translational control of the activity of both nitrogenases by reversible ADP-ribosylation of NifH and AnfH as well as by ADP-ribosylation independent switch-off. Mutational analysis revealed that both P II -like proteins are involved in the ammonium regulation of the two nitrogenase systems. A mutation in glnB results in the constitutive expression of nifA and anfA. In addition, the post-translational ammonium inhibition of NifA activity is completely abolished in a glnB-glnK double mutant. However, AnfA activity was still suppressed by ammonium in the glnB-glnK double mutant. Furthermore, the P II -like proteins are involved in ammonium control of nitrogenase activity via ADP-ribosylation and the switch-off response. Remarkably, in the glnB-glnK double mutant, all three levels of the ammonium regulation of the molybdenum (but not of the alternative) nitrogenase are completely circumvented, resulting in the synthesis of active molybdenum nitrogenase even in the presence of high concentrations of ammonium.