Abstract
In Acinetobacter sp. ADP1, growth on benzoate requires regulation of the cat genes by two transcriptional activators. Here, mutants were obtained from a strain lacking both activators by selecting for growth on benzoate medium. The mutants, which arose within 3 weeks at a frequency of approximately 10-8, carried amplified chromosomal regions (amplicons) encompassing the cat genes. Multiple occurrences of low-level expression of catA and the catBCIJFD operon provided sufficient transcription for growth. The amplicons of four independently isolated mutants varied in size from approximately 30-100 kbp of the normally 3.8 Mbp chromosome. Mutants had approximately 10-20 copies of an amplicon in adjacent head-to-tail orientations. At the amplicon's chromosomal endpoint, an atypical junction juxtaposed normally distant DNA regions from opposite sides of the cat genes. The sequences of these junctions revealed the precise recombination sites underlying amplification. Additionally, amplicon stability was evaluated in the absence of selective pressure. The natural competence of Acinetobacter for transformation by linear DNA has allowed the development of a powerful new model system for investigating chromosomal rearrangements and for engineering DNA amplifications for wide-ranging applications. The frequent spontaneous amplification of these large chromosomal segments demonstrated the importance of supra-operonic gene clustering in the evolution of catabolic pathways.