Douglas Rice

Cloning of a nitrogen fixation (nif) gene cluster of Azospirillum brasilense.

Homology was detected between the structural genes for the nitrogenase complex of K. pneumoniae (nifHDK genes) and the total DNA of several Azospirillum strains. Bacteriophage lambda gt 7-ara6 was used to construct a gene bank of A. brasilense strain 7000 DNA and a recombinant phage carrying a 6.7 kb Eco RI fragment, termed AbRI, was selected by hybridization with the K. pneumoniae nif probe. Using heteroduplex analysis the extent of the homology of the AbRI fragment and the K. pneumoniae nif genes was found to be approximately 5 kb. Proteins encoded by the AbRI fragment were examined after infection of E. coli minicells.

Organization and transcription of genes important in Anabaena heterocyst differentiation

The structural genes for nitrogenase and nitrogenase reductase have been cloned from Anabaena and physically mapped. The map differs from that of Klebsiella in several ways, including the insertion of 11 kbp between nifK and nifD in Anabaena. One nif RNA transcript has been studied in detail and shown to originate from a site in the Anabaena chromosome which lacks good correspondence with a typical prokaryotic strong promoter, suggesting the possibility of a need for positive activation. The nifH message is unstable or repressed or both under aerobic conditions. This feature is sufficient to account for the need for heterocyst differentiation in order for Anabaena to fix nitrogen aerobically. Structural genes for glutamine synthetase and the large subunit of RuBP carboxylase were also cloned, mapped and used to study transcription. In each case, the level of messenger RNA following nitrogenase induction is consistent with regulation of these genes at the level of transcription.

Organization and Transcription of Nitrogen Fixation Genes in the Cyanobacterium Anabaena

The mechanisms for protection of nitrogen fixation enzymes from inactivation by oxygen appear to be as richly varied in the cyanobacteria as they are in the bacteria. An extra challenge to the cyanobacteria is provided by one property they all share: photosynthetic evolution of oxygen in the light. For many species of cyanobacteria, both unicellular and filamentous, this feature means that nitrogen fixation is principally a laboratory phenomenon, made possible by experimental inhibition of photosystem II and continuous removal of oxygen. Under these circumstances nif gene expression is formally regulated like that of Klebsiella; repressed by either oxygen or combined nitrogen (ammonia, nitrate, urea or amino acids). For other species, capable of differentiating heterocysts at regular intervals along filaments, aerobic nitrogen fixation is accomplished by restricting such activity to the anaerobic internal milieu of the heterocyst1. In such species, both differentiation and nif gene expression are repressed by combined nitrogen sources. Finally, some unicellular and filamentous cyanobacterial species have recently been shown to fix nitrogen under aerobic conditions without the benefit of morphologically evident structures, such as heterocyst walls, to protect against oxygen2,3. These species are particularly puzzling because, unlike Azotobacter which can fix nitrogen aerobically by consuming oxygen through vigorous respiration, the cyanobacteria evolve oxygen photosynthetically.