Isolation of behavioral mutants of Azospirillum brasilense by using Tn5 lacZ

Tn5 lacZ mutants were generated with Azospirillum brasilense 7030 by mating that strain with Escherichia coli strains carrying suicide plasmid pCIB100 or pCIB110. Kanamycin-resistant Azospirillum colonies were obtained with a maximum frequency of 10(-6) per recipient cell. The potential of Tn5 lacZ for random transposon mutagenesis coupled to transcription analysis in A. brasilense 7030 was demonstrated. Sixty percent of all Kmr A. brasilense 7030 mutants expressed beta-galactosidase activity. Mutants affected in motility (Fla-) and general chemotaxis (Che-) were identified. Chromosomal insertions of Tn5 lacZ are involved, except for two Che- mutants. The latter che loci reside on a 90-megadalton plasmid. Expression of an acidic protein (Mr, 110,000) was abolished in these mutants.