de Vries Johann, Wackernagel Wilfried
Genetik, Fachbereich Biologie, Universität Oldenburg, POB 2503, D-26111 Oldenburg, Germany.
Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2094-9. doi: 10.1073/pnas.042263399.
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 10(9)-fold lower than that of homologous DNA. However, integration of foreign DNA increased at least 10(5)-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.
细胞外DNA的主动摄取及其基因组整合被称为自然转化，是原核生物中一种主要的水平基因转移机制。在物种内转移的染色体DNA可通过同源重组有效整合，而序列同源性低或无序列同源性的外源DNA则依赖于罕见的非同源重组事件。通过使用nptII(+)基因（卡那霉素抗性）作为选择标记，我们发现，在转化过程中，外源DNA整合到革兰氏阴性不动杆菌属BD413基因组中的效率确实比同源DNA至少低10^9倍。然而，当外源DNA一侧与一段与受体基因组同源的DNA相连时，其整合效率至少提高了10^5倍。对外源DNA整合位点的分析揭示了供体和受体DNA之间存在短片段的序列同一性（3-8个碱基对），表明发生了非同源重组事件。这些发现表明，同源DNA作为重组锚，促进了作用于同一分子的非同源重组。低至183个核苷酸的同源片段可作为锚。用链中具有不同核苷酸序列标签的异源双链DNA进行转化表明，链以3'至5'方向进入细胞质，且具有任何一种极性的链都通过同源重组进行整合。该过程导致数千个外源核苷酸整合到基因组中，并且常常伴随着受体DNA大致相应长度的缺失。同源性促进的非同源重组可以解释在没有移动遗传元件帮助的情况下原核生物基因组中DNA的渗入。
