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Analysis of IS1236-mediated gene amplification events in Acinetobacter baylyi ADP1.

ABSTRACT: Recombination between insertion sequence copies can cause genetic deletion, inversion, or duplication. However, it is difficult to assess the fraction of all genomic rearrangements that involve insertion sequences. In previous gene duplication and amplification studies of Acinetobacter baylyi ADP1, an insertion sequence was evident in approximately 2% of the characterized duplication sites. Gene amplification occurs frequently in all organisms and has a significant impact on evolution, adaptation, drug resistance, cancer, and various disorders. To understand the molecular details of this important process, a previously developed system was used to analyze gene amplification in selected mutants. The current study focused on amplification events in two chromosomal regions that are near one of six copies of the only transposable element in ADP1, IS1236 (an IS3 family member). Twenty-one independent mutants were analyzed, and in contrast to previous studies of a different chromosomal region, IS1236 was involved in 86% of these events. IS1236-mediated amplification could occur through homologous recombination between insertion sequences on both sides of a duplicated region. However, this mechanism presupposes that transposition generates an appropriately positioned additional copy of IS1236. To evaluate this possibility, PCR and Southern hybridization were used to determine the chromosomal configurations of amplification mutants involving IS1236. Surprisingly, the genomic patterns were inconsistent with the hypothesis that intramolecular homologous recombination occurred between insertion sequences following an initial transposition event. These results raise a novel possibility that the gene amplification events near the IS1236 elements arise from illegitimate recombination involving transposase-mediated DNA cleavage.

SUBMITTER: Cuff LE

PROVIDER: S-EPMC3416208 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Analysis of IS1236-mediated gene amplification events in Acinetobacter baylyi ADP1.

Cuff Laura E LE Elliott Kathryn T KT Seaton Sarah C SC Ishaq Maliha K MK Laniohan Nicole S NS Karls Anna C AC Neidle Ellen L EL

Journal of bacteriology 20120615 16

Recombination between insertion sequence copies can cause genetic deletion, inversion, or duplication. However, it is difficult to assess the fraction of all genomic rearrangements that involve insertion sequences. In previous gene duplication and amplification studies of Acinetobacter baylyi ADP1, an insertion sequence was evident in approximately 2% of the characterized duplication sites. Gene amplification occurs frequently in all organisms and has a significant impact on evolution, adaptatio ...[more]

PMID: 22707704

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Project description:BackgroundAcinetobacter baylyi ADP1 is an ideal bacterial strain for high-throughput genetic analysis as the bacterium is naturally transformable. Thus, ADP1 can be used to investigate DNA mismatch repair, a mechanism for repairing mismatched bases. We used the mutS deletion mutant (XH439) and mutL deletion mutant (XH440), and constructed a mutS mutL double deletion mutant (XH441) to investigate the role of the mismatch repair system in A. baylyi.ResultsWe determined the survival rates after UV irradiation and measured the mutation frequencies, rates and spectra of wild-type ADP1 and mutSL mutant via rifampin resistance assay (RifR assay) and experimental evolution. In addition, transformation efficiencies of genomic DNA in ADP1 and its three mutants were determined. Lastly, the relative growth rates of the wild type strain, three constructed deletion mutants, as well as the rifampin resistant mutants obtained from RifR assays, were measured. All three mutants had higher survival rates after UV irradiation than wild type, especially the double deletion mutant. Three mutants showed higher mutation frequencies than ADP1 and favored transition mutations in RifR assay. All three mutants showed increased mutation rates in the experimental evolution. However, only XH439 and XH441 had higher mutation rates than the wild type strain in RifR assay. XH441 showed higher transformation efficiency than XH438 when donor DNA harbored transition mutations. All three mutants showed higher growth rates than wild-type, and these four strains displayed higher growth rates than almost all their rpoB mutants. The growth rate results showed different amino acid mutations in rpoB resulted in different extents of reduction in the fitness of rifampin resistant mutants. However, the fitness cost brought by the same mutation did not vary with strain background.ConclusionsWe demonstrated that inactivation of both mutS and mutL increased the mutation rates and frequencies in A. baylyi, which would contribute to the evolution and acquirement of rifampicin resistance. The mutS deletion is also implicated in increased mutation rates and frequencies, suggesting that MutL may be activated even in the absence of mutS. The correlation between fitness cost and rifampin resistance mutations in A. baylyi is firstly established.

Dataset Information

Analysis of IS1236-mediated gene amplification events in Acinetobacter baylyi ADP1.

Publications

Analysis of IS1236-mediated gene amplification events in Acinetobacter baylyi ADP1.

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