Characterization of DNA Segments Adjacent to the nifHDK Genes of Azospirillum Brasilense Sp7 by Tn5 Site-Directed Mutagenesis
Three recombinants containing DNA inserts of 13 kb (λAb5), 15 kb (λAb2c) and 16 kb (λAb4) were isolated from a gene library of A. brasilense Sp7. These DNA inserts are overlapping DNA segments covering approximately 40 kb of the A. brasilense genome in the region of the nifHDK genes. The λAb2c recombinant containing the nifHDK genes localized in a 6.5 kb EcoRI DNA fragment was characterized. The EcoRI DNA fragments of this recombinant (6.5, 4.15, 2.4, 1.2 and 0.8 kb) were subcloned into the plasmid pACYC184 and after Tn5 mutagenesis, recloned into the plasmid pRK290 and transferred to A. brasilense. Five Nif− mutants were obtained, one with Tn5 inserted in the nifHDK genes and four mapping approximately 4 to 5 kb downstream from the nifHDK genes.
Key wordsnif genes site-directed mutagenesis gene library EMBL4 vector
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