Characterization and mutagenesis of the leucine biosynthetic genes of Azotobacter vinelandii : an analysis of the rarity of amino acid auxotrophs

Abstract

An 8.7 kb region of the Azotobacter vinelandii chromosome has been analyzed by genetic complementation and nucleotide sequencing. The sequence reveals that leuC and leuD comprise an operon- and leuB is adjacent to leuD. leuA was not detected. Experiments involving lac fusion constructs have confirmed the existence of separate promoters for leuC-leuD and for leuB. Primer extension studies have localized the transcription initiation sites of the leuC-leuD operon and also of the leuB operon. Five more open reading frames showing homology with the Escherichia coli genes yoh1, ibpB, cynR, asd and usg1 have also been found. Auxotrophic mutations are rare in A. vinelandii. We have been able to generate, for the first time, stable mutations in leuB, leuC and leuD by insertion of various gene blocks in vitro and integration by double crossover in vivo. Homogenotization of the mutation into all of the multiple chromosomes of A. vinelandii has been achieved. Evidence has been obtained suggesting the presence of a permease in A. vinelandii capable of leucine transport. Possible reasons for the dearth of auxotrophic mutations in A. vinelandii are discussed.