Interspecies homology of nitrogenase genes ( recombinant DNA / restriction endonuclease mapping / DNA - DNA hybridization ) GARY B . RUVKUN

Cloned nitrogen fixation (nifl) genes from Klebsiella pneumoniae hybridize to DNA from 19 out of 19 widely divergent nitrogen-fixing bacterial strains but do not hybridize to DNA from 10 different non-nitrogen-fixing species. K pneumoniae nifDNA fragments that hybridize to DNA from other species contain part of the three structural genes that code for nitrogenase polypeptides. We have utilized this homology to clone an EcoRI restriction endonuclease fragment from Rhizobium meliloti that hybridizes to the K. pneumoniae nif structural genes. Some of the species whose DNA hybridizes with K. pneumoniae nif DNA have been postulated to have diverged from K pneumoniae 3 X 109 years ago. Nitrogenase genes are the only known example of such highly conserved prokaryotic translated genes. Nitrogenase genes are either extraordinarily conserved in evolution or have been exchanged between different nitrogen-fixing species relatively recently in evolutionary time. The ability to fix atmospheric nitrogen is widely distributed among divergent prokaryotic taxonomic groups, including Azotobacteraceae, Enterobacteriaceae, Rhodospirillaceae, Bacillaceae, Rhizobiaceae, Actinomycetaceae, and Cyanobacteria (1). Although the physiological conditions under which prokaryotes fix nitrogen vary considerably, the enzymatic apparatus involved in nitrogen fixation is remarkably similar. Each nitrogen-fixing species that has been studied produces an enzyme complex called nitrogenase composed of two characteristic components: a molybdenumiron protein (MoFe protein), which reduces substrate, and an iron protein (Fe protein), which binds MgATP and transfers electrons to the MoFe protein (2). Neither purified component in the absence of the other reduces nitrogen, but it is possible to reconstitute an active nitrogenase complex in vitro from purified MoFe and Fe proteins (2). In some cases, purified MoFe protein from one bacterial species reconstitutes an enzymatically active hybrid nitrogenase with Fe protein from another species (3). In addition, amino acid compositions of nitrogenases from several species are very similar (4). These observations suggest that the structure of nitrogenase has been conserved between evolutionarily distant organisms. We hypothesized, therefore, that the DNA sequences coding for nitrogenase proteins may also have been conserved and that DNA that codes for nitrogenase from one nitrogen-fixing species would hybridize specifically to DNA from other nitrogen-fixing species. Cannon et al. (5, 6) have previously constructed amplifiable plasmids carrying 14 out of 15 Klebsiella pneumoniae nitrogen fixation (nif) genes, including those that code for the MoFe and Fe proteins of nitrogenase. In the experiments described here, purified DNA fragments containing K. pneumoniae nif genes were labeled with 32P and hybridized to restriction endonuclease-digested DNAs from various nitrogen-fixing and nonnitrogen-fixing bacteria, using the gel transfer technique of Southern (7). We found that K. pneumoniae DNA containing nitrogenase genes hybridized to DNA from 19 out of 19 nitrogen-fixing bacterial strains but did not hybridize to DNA from 10 non-nitrogen-fixing species. MATERIALS AND METHODS Southern Hybridizations. Total DNA was isolated from each species by using a simplified Marmur technique (8). Approximately 5 ,tg of each DNA was digested with 20 units of EcoRI purified and used as described (9), and the DNA was electrophoresed in a 13 X 24 X 0.5 cm 0.8% agarose horizontal gel (Dankar plastics) at 2 V/cm for 12-16 hr. The DNA in the gel was transferred to nitrocellulose sheets (Schleicher and Schuell type BA85) by the method of Southern (7), using modifications from Botchan et al. (10). Supercoiled plasmids were prepared by the cleared lysate technique (11). Purified restriction fragments were obtained by gel electrophoresis and eluted by inserting a gel slice containing the desired fragment into a dialysis bag and placing the bag in electrophoresis buffer, followed by electrophoresis at 5 V/cm for 16-24 hr. Intact plasmid DNA and purified restriction fragments were labeled by the nick translation (12, 13) method to a specific activity of 107 to 108 cpm/,ug of DNA with thymidine 5'-[32P]triphosphate (350 Ci/mmol, Amersham; 1 Ci = 3.7 X 1010 becquerels) and DNA polymerase I (Boehringer Mannheim). Hybridizations were performed essentially as described by Botchan et al. (10) in a Seal-a-Meal boilable bag (Sears and Roebuck) in a 650C water bath, with approximately 106 dpm of labeled DNA probe per 24-slot 13 X 24 cm filter. Kodak XR-1 x-ray film was exposed to the dried nitrocellulose filter with an intensifying screen (Du Pont Cronex Lightning Plus) at -700C for 48-96 hr. Melting Temperature Experiments. Approximately 50 ig of Rhizobium DNA was digested with EcoRI restriction endonuclease, mixed with 1 Mig of K. pneumoniae DNA digested with EcoRI or Sal I, and subjected to electrophoresis in a single 12-cm-long slot on a 13 X 24 X 0.5 cm 1% agarose horizontal gel. The DNA was transferred to nitrocellulose and hybridized to 32P-labeled K. pneumoniae nifDNA as described above. The filter was cut into identical strips, each of which was washed at 650C in various concentrations of standard saline/citrate (NaCI/Cit: 0.15 M NaCI/0.015 M sodium citrate, pH 7). After washing and drying, the strips were lined up and autoradiographed. Cloning of the Rhizobium meliloti DNA Fragment Homologous to K. pneumoniae nif. Approximately 10 ,ug of R. meliloti strain M2011 DNA was digested with EcoRI and ligated to 5 Mug of EcoRI-linearized plasmid pBR322 (14) DNA by using phage T4 ligase as described (6). A sample of the liAbbreviations: NaCI/Cit, standard saline/citrate (0.15 M NaCI/0.015 M sodium citrate, pH 7); kb, kilobase pairs; Nif, relating to nitrogen fixation. 191 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 192 Biochemistry: Ruvkun and Ausubel gated DNA was used to transform CaCl2-treated Escherichia coli strain HB101 cells (15). DNAs from a total of 4000 transformed colonies were hybridized in duplicate to 32P-labeled K. pneumoniae nif DNA by using the colony hybridization technique of Grunstein and Hogness (16) as modified by D. Hanahan (personal communication).