Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region

Abstract

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.

Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins

“…The growth conditions, media, and antibiotic concentrations used to cultivate E. coli and R capsulatus strains were described previously (22,26,27). To remove traces of molybdenum, the media were treated with activated carbon as described by Schneider et al (35).…”

“…2) were used to insert DNA fragments carrying appropriate antibiotic resistance genes. The resulting hybrid plasmids (Table 1) were mobilized from E. coli S17-1 into R. capsulatus by filter matings (22), and the homogenotization of the corresponding insertion or insertion-deletion mutations was selected by the antibiotic resistance mediated by the interposon. Loss (22).…”

Purification of a sixth ferredoxin from Rhodobacter capsulatus

“…Strain BlOS, a streptomycin-resistant mutant of the wildtype strain B10 (Klipp et al, 1988), was alternatively employed as a source of FdVI. Cultures of strain BlOS were grown with excess ammonium salts (10 mM NH,').…”

“…It can grow autotrophically or heterotrophically either in the light or in darkness and can choose between five different growth modes (Madigan and Gest, 1979). It is also capable of fixing molecular nitrogen through a metabolic process which has been extensively studied by biochemical (Hallenbeck et al, 1982a; and genetic approaches (Willison et al, 1985;Klipp et al, 1988). Nitrogen fixation is catalyzed by nitrogenase and requires ATP and a low-potential reductant.…”

Detection of the in vivo incorporation of a metal cluster into a protein

“…Therefore, it was proposed that ethane formation can be used as a diagnostic criterion for the existence of a V nitrogenase . The relative yield of ethane varied depending on the reaction conditions and amounted to 2- Klipp et al, 1988Schneider et al, 1991 this work this work this work this work this work this work Simon et al, 1983 3 % under standard assay conditions (neutral pH, 30°C, component 2 (K2)lcomponent 1 (Kl) = 5-10: 1). In the case of the V nitrogenase from A. chroococcum, relative ethane production could be increased by raising the ratio K2/K1 and by increasing the assay temperature.…”