Isolation and physical mapping of nitrogen fixation genes from the cyanobacterium Anabaena 7120.

DNA restriction fragments containing nitrogen fixation (nifi genes of the cyanobacterium Anabaena 7120 were isolated as inserts in bacteriophage X, using plasmids containing Klebsiella pneumoniae nif genes as heterologous hybridization probes.Four contiguous E m RI fragments, comprising a 39-kilobase pair segment of the Anabaena chromosome, were physically mapped and those regions homologous to IL pneumoniae nif genes were identified by Southern hybridization and by electron microscope examination of heteroduplex DNA molecules.Regions of Anabaena DNA were found homologous to the following K. pneumoniae nif genes: nif H, the structural gene for nitrogenase reductase; nif D and nif K, the structural genes for the two subunits of nitrogenase; and either nif V or nif S, genes whose products are required for the maturation of nitrogenase.The four Anabaena nif genes so identified are transcribed in the same direction, as is the case in K. pneumoniae.However, the genes are rearranged with respect to the map of K. pneumoniae.In K. pneumoniae, nif H, nif D, and nif K are adjacent and cotranscribed.In Anabaena, nif H and nif D are adjacent but nif K is separated from nif D by 11 kilobase pairs of DNA.In K.pneumoniae, the nif V-nif S genes are to the left of the nif HDK cluster, while in Anabaena, the homologous region is to the right of the nif H gene. Hybridization of labeled subclones of Anabaena DNA to filter-bound RNA prepared from cells induced for nitrogenase indicated that most of the 11 kilobase pairs between nif K and nif D are not transcribed, while fragments containing the identified nif genes are transcribed abundantly.An additional fragment of Anabaena 7120 DNA was isolated, in recombinant X, which is apparently unlinked to the 39-kilobase pair region described above.This fragment contains a region homologous to K. pneumoniae nif H, but not to nif D. The restriction map of the nif H-homologous region of this Anabaena fragment is different from that of the nif H gene described above.