Yang Joyce C, Lessard Philip A, Sengupta Neil, Windsor Steven D, O'brien Xian M, Bramucci Michael, Tomb Jean-Francois, Nagarajan Vasantha, Sinskey Anthony J
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Plasmid. 2007 Jan;57(1):55-70. doi: 10.1016/j.plasmid.2006.08.002. Epub 2006 Sep 25.
Pulsed-field gel electrophoresis (PFGE) revealed three previously uncharacterized megaplasmids in the genome of Rhodococcus erythropolis AN12. These megaplasmids, pREA400, pREA250, and pREA100, are approximately 400, 250, and 100kb, respectively, based on their migration in pulsed-field gels. Genetic screening of an AN12 transposon insertion library showed that two megaplasmids, pREA400, and pREA250, are conjugative. Mobilization frequencies of these AN12 megaplasmids to recipient R. erythropolis SQ1 were determined to be approximately 7x10(-4) and 5x10(-4) events per recipient cell, respectively. It is known for other bacterial systems that a relaxase encoded by the traA gene is required to initiate DNA transfer during plasmid conjugation. Sequences adjacent to the transposon insertion in megaplasmid pREA400 revealed a putative traA-like open reading frame. A targeted gene disruption method was developed to generate a traA mutation in AN12, which allowed us to address the role of the traA gene product for Rhodococcus megaplasmid conjugation. We found that the AN12 traA mutant is no longer capable of transferring the pREA400 megaplasmid to SQ1. Furthermore, we confirmed that the conjugation defect was specifically due to the disruption of the traA gene, as pREA400 megaplasmid conjugation defect is restored with a complementing copy of the traA gene.
脉冲场凝胶电泳（PFGE）显示，在红平红球菌AN12的基因组中存在三个以前未被表征的大质粒。根据它们在脉冲场凝胶中的迁移情况，这些大质粒pREA400、pREA250和pREA100的大小分别约为400kb、250kb和100kb。对AN12转座子插入文库的遗传筛选表明，两个大质粒pREA400和pREA250是可接合的。这些AN12大质粒向受体红平红球菌SQ1的转移频率分别确定为每个受体细胞约7×10⁻⁴和5×10⁻⁴次转移事件。对于其他细菌系统而言，已知在质粒接合过程中启动DNA转移需要由traA基因编码的松弛酶。大质粒pREA400中转座子插入位点附近的序列揭示了一个推定的类traA开放阅读框。开发了一种靶向基因破坏方法，以在AN12中产生traA突变，这使我们能够研究traA基因产物在红球菌大质粒接合中的作用。我们发现，AN12的traA突变体不再能够将pREA400大质粒转移到SQ1。此外，我们证实接合缺陷是由于traA基因的破坏所致，因为pREA400大质粒的接合缺陷可通过traA基因的互补拷贝得以恢复。
