Genomic plasticity and chromosomal copy number of Acinetobacter baylyi ADP1
Cuff, Laura Elizabeth
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Acinetobacter baylyi ADP1, a ubiquitous soil bacterium, is an excellent model organism for systematic studies of gene duplication and amplification. This dissertation describes the role of a mobile insertion sequence element, IS1236, in chromosomal duplication and amplification events. Genetic rearrangements were further explored by characterizing the potential for recombination between copies of its single circular chromosome. Genomic copy number was evaluated at various growth stages and rates. Gene amplification events are difficult to study due to their transient nature; however, by exploiting the pathway for benzoate consumption in A. baylyi, chromosomal amplification can be selected and maintained. A. baylyi strains lacking two transcriptional activators acquire the ability to grow on benzoate via amplification of a chromosomal region encompassing the catA gene and the catBCIJFD operon. Growth on benzoate selects amplification mutants, allowing detailed study of the recombination events generating the initial duplication. To expand this experimental system, the cat-gene DNA was relocated for the selection of amplification mutants in different chromosomal regions. Analysis of these amplification mutants revealed that proximity to one of six chromosomal copies of IS1236 increases the likelihood that the insertion sequence is involved in the initial duplication event. Surprisingly, the mechanism underlying duplication formation differs from the well-characterized method of transposition and intra-molecular recombination between duplicated IS copies. To determine whether duplication events might involve transposition and inter-molecular recombination between IS copies, the ploidy level of A. baylyi was studied. Quantitative PCR analysis of DNA content in cells was carried out on bacterial cultures at various growth stages and rates. This analysis showed A. baylyi contains a single copy of the chromosome per cell in both exponential and early-stationary phase cultures. Additionally, a single copy of the chromosome per cell was found in cultures with varying doubling times. These results make it unlikely that frequent recombination occurs between IS elements on different chromosomal copies within the same cell. The studies described herein clarify the ploidy level in A. baylyi and suggest a novel method of duplication involves DNA cleavage by the transposase encoded by IS1236.