Decreased electroporation efficiency in Borrelia burgdorferi containing linear plasmids 1p25 and 1p56:: Impact on transformation of infectious B-burgdorferi
Decreased electroporation efficiency in Borrelia burgdorferi containing linear plasmids 1p25 and 1p56:: Impact on transformation of infectious B-burgdorferi
复制标题
DOI：
10.1128/iai.70.9.4798-4804.2002
复制
发表时间：
2002-09-01
影响因子：
3.1
通讯作者：
Norris, SJ
中科院分区：
文献类型：
--
作者：
Lawrenz, MB;Kawabata, H;Norris, SJ
研究发现，伯氏疏螺旋体B31的线性质粒lp25和lp56的存在显著降低了与穿梭载体pBSV2的电孔转化速率，pBSV2是一种自主复制的质粒，具有卡那霉素抗性（P. E. Stewart， R. Thalken， J. L. Bono， P. Rosa, Mol. Microbiol. 339:714-721, 2001）。伯氏疏螺旋体B31克隆的转化效率有低、中、高三种类型，其表型与lp25和lp56的存在与否相关。在本研究条件下，同时含有lp25和lp56的克隆中未检测到转化子；少数的卡那霉素耐药菌落不含pBSV2，表明耐药是由突变引起的。lp25（-）和Ip56（+）或lp25（+）和lp56（-）的B31克隆获得了中间电穿孔率（10 ~ 200个菌落/杯DNA）。在这个群体中，最初含有lp25的克隆在pBSV2转化体中缺乏这种质粒，这一发现与lp25-变体的选择性转化一致。在缺乏lp25和lp56的克隆中，转化率很高（每杯DNA有1000个菌落）。序列分析表明，lp25和lp56含有可能编码限制和/或修饰系统的基因，这些基因可能导致含有这些质粒的菌株获得较低的转化率。先前报道的lp25与小鼠传染性之间的相关性，以及lp25对转化的障碍，可能解释了为何难以获得伯氏疏螺旋体的毒力转化体。
The presence of the linear plasmids lp25 and lp56 of Borrelia burgdorferi B31 was found to dramatically decrease the rate of transformation by electroporation with the shuttle vector pBSV2, an autonomously replicating plasmid that confers kanamycin resistance (P. E. Stewart, R. Thalken, J. L. Bono, and P. Rosa, Mol. Microbiol. 39:714-721, 2001). B. burgdorferi B31 clones had transformation efficiencies that were either low, intermediate, or high, and this phenotype correlated with the presence or absence of lp25 and lp56. Under the conditions utilized in this study, no transformants were detected in clones that contained both lp25 and lp56; the few kanamycin-resistant colonies isolated did not contain pBSV2, indicating that the resistance was due to mutation. Intermediate electroporation rates (10 to 200 colonies per mug of DNA) were obtained with B31 clones that were either lp25(-) and Ip56(+) or lp25(+) and lp56(-). Clones in this group that initially contained lp25 lacked this plasmid in pBSV2 transformants, a finding consistent with selective transformation of lp25- variants. High transformation rates (> 1,000 colonies per mug of DNA) occurred in clones that lacked both lp25 and lp56. Sequence analysis indicated that lp25 and lp56 contain genes that may encode restriction and/or modification systems that could result in the low transformation rates obtained with strains containing these plasmids. The previously reported correlation between lp25 and infectivity in mice, coupled with the barrier lp25 presents to transformation, may explain the difficulty in obtaining virulent transformants of B. burgdorferi.