Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

In order to improve the gene transfer/mutagenesis system for Azospirillian brasilense, gene-cartridge mutagenesis was used to replace the nif D gene w ith the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integratio n into the chromosome. Nevertheless, all 12 colonies were deficient in acet ylene reduction, indicating an Nif(-) phenotype. Four Nif(-) mutants were a nalyzed by Southern blot, using six different probes spanning the nif and K m(r) genes and the plasmid vector. Apparently, several recombination events occur-red in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagen esis combined with electrotransformation.