Deoxyribonucleic Acid Homology Among Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and Other Nitrogen-Fixing Bacillus Strains

Abstract

Deoxyribonucleic acid homology experiments were performed among representative strains of Bacillus polymyxa, Bacillus macerans, and Bacillus azotofixans and other nitrogen-fixing Bacillus strains identified as B. polymyxa-like. All B . azotofixans strains showed less than 20% homology when B . polymyxa or B . macerans was used as a probe. The range of homology among B. azotofixans strains (strain P3L-5T [T = type strain] was used as the probe) varied from 54 to loo%, indicating that these strains comprise a relatively homogeneous species.Strain Hino appears to be a variant of B . azotofixans because it exhibited only 28 to 44% homology with this species. Strains P3L-2, CR5D-16, and CR5D-23 showed less than 20% homology with B. polymyxa, B . macerans, and B . azotofixans, indicating that these strains do not belong to these species. Additional data will be necessary to fully clarify the taxonomy of these nitrogen-fixing Bacillus strains. In this work, we performed deoxyribonucleic acid (DNA) homology experiments with representative strains of B. polymyxa and B. macerans and the strains reported to be B . azotojixans and B . polymyxa-like (10, 18) in an attempt to clarify the taxonomy of the nitrogen-fixing bacilli. MATERIALS AND METHODSBacterial strains. All of the strains used in this work are listed in Table 1.Propagation of cultures. Portions (2 ml) from 24-h cultures grown in liquid medium [glucose, 10 g; MgS04 -7H20, 0.2 g; NaCI, 0.1 g; yeast extract, 1 g; (NH&HP04, 1 g; K2HP04, 4 g; KH2P04, 2.8 g; distilled water, 1,000 ml; pH 6.51 were inoculated into 200 ml of the same medium and incubated at 32°C for 18 h.Isolation of chromosomal DNA. Cells from 200-ml cultures were concentrated in 5 ml of lysis buffer [25% sucrose, 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.001 M ethylenediaminetetraacetate, pH 8.01 and treated with 1 mg of lysozyme per ml for 30 min at 37°C. B . polymyxa strains were treated for 2.5 h at the same temperature. Sodium dodecyl sulfate was added to a final concentration of 1%, and the preparations were incubated for 30 min at 65°C. In order to purify the DNA, 1 mg of pronase (catalog no. P5147; Sigma Chemical Co.) per ml was added, and the preparations were incubated at 50°C for 2 h. The DNA was then precipitated with 2 volumes of cold ethanol, spooled with a glass stirring rod, dissolved in 0 . 1~ SSC (IX SSC is 0.15 M NaCI plus 0.015 M sodium citrate) (131, and shaken with an * Corresponding author. equal volume of phenol neutralized with TES buffer (50 mM NaCI. 30 mM Tris, 5 mM ethylenediaminetetraacetate) at pH 7.5 for 15 min. The phenol treatment was repeated several times until very little interface was observed. The aqueous phase containing the DNA was spooled after ethanol precipitation and suspended in 0 . 1~ SSC. Then, the preparations were treated with pancreatic ribonuclease (50 pglml) and ribonuclease TI (1 U/ml) for 30 min at 37°C. After two treatments with phenol the DNA was precipitated, spooled, and dissolved in 1 ml of DNA buffer (4 mM NaCI, 10 mM Tris, 0.1 mM ethyl...

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