Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis
Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis
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DOI：
10.1128/aem.00988-07
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发表时间：
2007-08-01
影响因子：
4.4
通讯作者：
Munderloh, Ulrike G.
中科院分区：
文献类型：
--
作者：
Baldridge, Gerald D.;Burkhardt, Nicole Y.;Munderloh, Ulrike G.
直到最近在猫立克次体中发现 pRF 之前，人们认为立克次体属（立克次体目：立克次体科）的专性细胞内细菌不具有质粒。我们描述了 pRM，一种来自 monacensis 立克次体的质粒，通过脉冲场凝胶电泳和 Southern 印迹分析对来自两个独立的 monacensis 立克次体种群的 DNA 进行检测，这两个独立的 monacensis 种群通过转座子介导将偶联的绿色荧光蛋白和氯霉素乙酰转移酶标记基因插入 pRM 中进行转化。二维电泳显示pRM以圆形和线性异构体存在于立克次体细胞中。通过限制性内切酶消化的转化体 DNA 片段的氯霉素标记拯救和使用衍生自重叠限制性片段序列的引物的 PCR，从转化体群体中克隆出 23,486 个核苷酸（31.8% G/C）的 pRM 质粒。对质粒进行测序。根据 GenBank 数据库的 BLAST 搜索，pRM 包含 23 个预测基因或假基因，与较大的 pRF 质粒非常相似。在已测序的立克次体基因组中，这 23 个基因中有 2 个是 pRM 和 pRF 所独有的，而 pRM 和 pRF 共有的 4 个基因仅在猫立克次体或祖先立克次体立克次体和加拿大立克次体的染色体上发现。我们获得了脉冲场凝胶电泳和 Southern blot 证据，证明 R. amblyommii 分离株 WB-8-2 中的质粒含有 pRM 和 pRF 之间保守的基因。 pRM质粒可为立克次体转化载体的开发提供基础。
Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.