Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA.
J Microbiol Methods. 2012 Oct;91(1):121-7. doi: 10.1016/j.mimet.2012.07.018. Epub 2012 Aug 7.
A circular plasmid, pMyBK1, was detected in Mycoplasma yeatsii strain GIH(T). Analysis of the sequence of the 3432-bp replicon identified two predicted open reading frames (ORFs), one with sequence similarity to multiple plasmid mobilization proteins and one that matches only to hypothetical ORFs encoded by integrated chromosomal elements in the sequenced genomes of two Mycoplasma species. Shuttle vectors were constructed in Escherichia coli which could be introduced into M. yeatsii at high efficiency (10(4)-10(5) per μg DNA) by electroporation. Independent deletion analysis of the two ORFs disclosed that whereas mob was dispensable, orf2 was necessary for plasmid replication or maintenance. The absence of plasmid-encoded database matches for ORF2 indicates that pMyBK1 represents a novel plasmid family. One shuttle vector was used to demonstrate heterologous expression of the Mycoplasma fermentans malp gene and was stable during multiple passages. The host-plasmid system described has potential application for genetic manipulation in a genus for which few replicative vectors are available.
在支原体 GIH(T)中检测到一个环形质粒 pMyBK1。对 3432bp 复制子序列的分析鉴定出两个预测的开放阅读框（ORF），一个与多个质粒移动蛋白具有序列相似性，另一个仅与两种支原体测序基因组中整合的染色体元件编码的假定 ORF 相匹配。在大肠杆菌中构建了穿梭载体，通过电穿孔可高效（每微克 DNA 10(4)-10(5)个）导入支原体。对两个 ORF 的独立缺失分析表明，mob 是可有可无的，而 orf2 对于质粒复制或维持是必需的。ORF2 缺乏质粒编码的数据库匹配表明，pMyBK1 代表了一个新的质粒家族。一个穿梭载体被用于证明支原体发酵乳杆菌 malp 基因的异源表达，并且在多次传代过程中稳定。所描述的宿主-质粒系统在该属中具有遗传操作的潜力，因为该属中可用的复制载体很少。
