Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination

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Abstract
The active uptake of extracellular DNA and its genomic integration is termed natural transformation and constitutes a major horizontal gene-transfer mechanism in prokaryotes. Chromosomal DNA transferred within a species can be integrated effectively by homologous recombination, whereas foreign DNA with low or no sequence homology would rely on illegitimate recombination events, which are rare. By using the nptII+ gene (kanamycin resistance) as selectable marker, we found that the integration of foreign DNA into the genome of the Gram-negative Acinetobacter sp. BD413 during transformation indeed was at least 109-fold lower than that of homologous DNA. However, integration of foreign DNA increased at least 105-fold when it was linked on one side to a piece of DNA homologous to the recipient genome. Analysis of foreign DNA integration sites revealed short stretches of sequence identity (3–8 bp) between donor and recipient DNA, indicating illegitimate recombination events. These findings suggest that homologous DNA served as a recombinational anchor facilitating illegitimate recombination acting on the same molecule. Homologous stretches down to 183 nucleotides served as anchors. Transformation with heteroduplex DNA having different nucleotide sequence tags in the strands indicated that strands entered the cytoplasm 3′ to 5′ and that strands with either polarity were integrated by homologous recombination. The process led to the genomic integration of thousands of foreign nucleotides and often was accompanied by deletion of a roughly corresponding length of recipient DNA. Homology-facilitated illegitimate recombination would explain the introgression of DNA in prokaryotic genomes without the help of mobile genetic elements.

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