War/d Journ& of Microbio/ogy and Biotechno/ogy, 9, 387-389 Short Communication Identification of structural nitrogen-fixation (I-M) genes in B~i/hs p/ymyxa and Bacillus macerans S.S. Oliveira, L. Seldin and M.C.F. Bastes* Some naturally occurring Nif + and Nif - strains of Bacihs po&rnyxa and Bacillus macerans were tested for DNA homology to the Klebsiella pneumoniae nif genes by Southern hybridization. Genomic DNA from all Nif’ strains carried homologous sequences only to structural nif genes. The homology detected was limited to nifH and nim. The hybridization pattern observed suggested that both genes are contiguous on the chromosome of the Bacillus strains. No homology was found between the genomic DNA from Nif - strains and the K. pneumoniae nif genes. The genetics of nitrogen fixation has been studied in great detail in K/ebsida pmmoniae, where a cluster of twenty nif genes is arranged in seven major operons (Triplett ef al. 1%~). The structural proteins of nitrogenase are coded by three genes, tiifi, rtiJ3 and Hi/K, which are arranged in a single operon. The structural nif gene sequences are conserved between diverse nitrogen-fixing organisms, including methanogenic archaebacteria (Triplett et ul. 1989). Up to now, all Bucihs strains described as nitrogen-fixers belong to the species B. pojyrny~a, B, muceruns, B. circdms and B. azofojxans. In a previous study, homology to nigh and r#D was found in 22 strains of B. uz~t~fixu~ and the possibility of reiteration of nigh was suggested (Seldin ef ul. 1989). However, no genetic and molecular characterization of nif genes of other nitrogen-fixing Bacihs has been reported. In this work, the presence of nif genes in naturally occurring Nif + and Nif - strains of B. po/ymy~u and B. maceraws was investigated. The patterns of hybridization to r$ probes observed among strains were also compared in order to detect the occurrence of restriction polymorphisms and to find a strain, for cloning purposes, in which a single restriction fragment contained all the nitrogenase structural genes. S.S. Oliveira, L. Seldin and M.C.F. Bastes are with the lnstituto de Microbiologia, Departamento de Microbiologia Geral, Universidade Fed- eral do Rio de Janeiro, CCS, Bloco I, Cidade Universitaria, 21.944-970, Rio de Janeiro, Brazil. ‘Corresponding author. @I 1993 Rapid Communications of Oxford Ltd Materials and Methods Bucterid Shins und Cdfure Condifions Most of the Bucihs polymyxu and Bucih macerans strains used in this study were isolated from Brazilian soils and described previously (Seldin et al. 1983). In addition, we used the following reference strains: B. polyrnyxu: L, DSM356, NRRL-B371 and LMD24.16; B. rnaceruns: LMD24.3 and NCTC6355. The LMD and NCTC strains were received from the Laboratory for Microbio- logy, Delft, Netherlands. The strain designated L was provided by Prof M.W. Loutit, University of Otago, New Zealand. The DSM and NRRL strains were provided by Dr P. Jurtshuk, Jr, University of Houston, USA. The liquid medium used (unless otherwise indicated) was GB (Seldin et a/. 1983). Acetylene Reduction Tests Acetylene reduction was tested by measuring the ethylene production of cultures in IS-ml vials as described previously (Seldin et a/, 1983). Isolation and In Vitro Mmipulafion of DNA The total genomic DNA was isolated from Bucihs strains as described by Seldin et al. (1983). Plasmid DNA purification, restriction endonuclease digestions, agarose gel electrophoresis, Southern blots and hybridizations were performed essentially as described by Sambrook ef al. (1989). Results and Discussion To identify the presence of DNA sequences homologous to the K pneumoniue nij genes in the genome of strains of B.