Transposon mutagenesis in Azospirillum brasilense: isolation of auxotrophic and Nif- mutants and molecular cloning of the mutagenized nifDNA

Abstract

We report the successful mutagenesis of Azospirillum brasilense 29710 Rif Sm with transposon Tn5. The narrow host-range plasmid pGS9 (p15A replicon), which possesses broad host-range N-type transfer genes, was used as the suicide vehicle to deliver Tn5 in Azospirillum. Out of 900 colonies tested, 0.8% proved to be auxotrophic. One mutant altered in indoleacetic acid (auxin) biosynthesis was isolated and, in addition, three mutants completely defective in nitrogen fixation (nif) were obtained. All the mutants tested contained a single copy of Tn5 integrated randomly in the genome. The Tn5-mutagenized EcoRI fragments were cloned from the three Nif- mutants. Physical analysis of cloned DNA showed that Tn5 was present on a different EcoRI fragment in each case, ranging in size from 15–17 kb. The nitrogenase structural genes (nifHDK) in A. brasilense 29710 Rif Sm were localized on a 6.7 kb EcoRI fragment. We found that Tn5 is not inserted in the nifHDK genes in the Nif- mutants reported here. Site-directed mutagenesis using the cloned, Tn5-containing DNA from mutant Nif27(pMS188), produced a large number of Nif- transconjugants of the A. brasilense 29710 Rif wild-type strain, showing the linkage between Tn5 insertion and the Nif- phenotype. This is the first time that transposon-mutagenized auxotrophic, Nif- and other mutants have been available for genetic analysis in Azospirillum. This should greatly facilitate the cloning and mapping of genes involved in nitrogen fixation as well as in many other phenotypic characteristics of Azospirillum.