Identification and sequence of a nifJ‐like gene in Rhodospirillum rubrum: partial characterization of a mutant unaffected in nitrogen fixation

Abstract

A nifJ-like gene was identified in the photosynthetic purple non-sulphur bacterium Rhodospirillum rubrum. A DNA segment hybridizing to Klebsiella pneumoniae nifJ was isolated, the gene was inactivated, and a mutant strain, SNJ-1, was constructed by allele replacement. The mutation was confirmed by DNA sequencing. Northern blotting and by the lack of pyruvate oxidoreductase activity. This is the first report of a nifJ-like gene in photosynthetic bacteria. Unexpectedly, SNJ-1 was capable of nitrogen fixation, and growth was similar to the wild-type strain under all conditions investigated. Therefore, this is also the first demonstration that a nifJ homologue, when present, is not essential for nitrogen fixation in a diazotroph. The nifJ-like gene was sequenced and found to have considerable similarity to published nifJ gene sequences from other organisms. By primer extension, the initiation site for transcription was located, and a typical sigma 54 promoter sequence was identified.

Electron transport to nitrogenase in Rhodospirillum rubrum: the role of NAD(P)H as electron donor and the effect of fluoroacetate on nitrogenase activity

“…The results previously obtained in studies of the nifJ mutant of R. rubrum indicate that unlike in K. pneumoniae the nifJ product is not primarily involved in electron transfer to nitrogenase in R. rubrum [7]. We suggest that generation of reductant to nitrogenase is dependent on the TCA cycle, which as well as pyruvate dehydrogenase has been previously demonstrated in R. rubrum [15–18].…”

“…We have sequenced the nifJ gene from R. rubrum and a mutant devoid of the pyruvate oxidoreductase has been generated. This mutant showed no significant differences in growth under any conditions studied nor in nitrogenase activity compared with the wild‐type [7]. Taken together it is unlikely that the pyruvate oxidoreductase/ferredoxin (flavodoxin) couple constitutes the major pathway of electron transfer to nitrogenase in R. rubrum .…”

The fixABCX Genes in Rhodospirillum rubrum Encode a Putative Membrane Complex Participating in Electron Transfer to Nitrogenase

“…A nifJ-like gene encoding a pyruvate-ferredoxin oxidoreductase has been identified (6,32). This pyruvate-ferredoxin oxidoreductase has been shown to support nitrogenase activity in vitro, linking the oxidation of pyruvate to the reduction of nitrogenase by using partially purified ferredoxin fractions (6).…”

“…This pyruvate-ferredoxin oxidoreductase has been shown to support nitrogenase activity in vitro, linking the oxidation of pyruvate to the reduction of nitrogenase by using partially purified ferredoxin fractions (6). Studies of a knockout mutant strain revealed no phenotype with respect to nitrogen fixation, indicating that the nifJ-like gene product is not likely to be part of the major pathway for electron transfer to nitrogenase in R. rubrum (32). Similar results have been reported for the nifJ gene from R. capsulatus (54).…”

Two pathways of electron transport to nitrogenase inRhodospirillum rubrum: the major pathway is dependent on thefixgene products

“…In Klebsiella pneumoniae, electrons are transferred via two nitrogen fixation specific proteins, pyruvate:flavodoxin oxidoreductase and a flavodoxin, linking the oxidation of pyruvate to nitrogen reduction (Shah et al, 1983). Similar systems have been shown to be present, although not required for nitrogen fixation in a number of other diazotrophic bacteria (Bauer et al, 1993;Yakunin & Hallenbeck, 1998) including Rhodospirillum rubrum (Lindblad et al, 1996).…”