Department of Entomology, University of Minnesota, St. Paul, Minnesota, USA.
PLoS One. 2011;6(12):e29511. doi: 10.1371/journal.pone.0029511. Epub 2011 Dec 21.
Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 - 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.
质粒已在大多数研究的立克次体物种中被鉴定出来，其中一些物种保持着多种不同的质粒。通过使用质粒特异性探针的 Southern 分析，在 Rickettsia amblyommii AaR/SC 中证明了三种不同的质粒。通过实时 PCR 估计 AaR/SC 中 pRAM18、pRAM23 和 pRAM32 每个染色体的拷贝数分别为 2.0、1.9 和 1.3。R. amblyommii AaR/SC 质粒的克隆和测序为立克次体的转化提供了开发穿梭载体的机会。在 pRAM18 中插入了一个编码利福平抗性和荧光标记的选择盒，得到了一个 27.6 kbp 的重组质粒，pRAM18/Rif/GFPuv。用 pRAM18/Rif/GFPuv 电穿孔 Rickettsia parkeri 和 Rickettsia bellii 可在 2 周内获得 GFPuv 表达的立克次体。较小的载体 pRAM18dRG、pRAM18dRGA 和 pRAM32dRGA 分别携带相同的选择盒，它们通过将 parA 和 dnaA 样基因从 pRAM18 或 pRAM32 转移到载体骨架中制成。R. bellii 维持了最高数量的 pRAM18dRGA（13.3-28.1 个拷贝），而 R. parkeri、Rickettsia monacensis 和 Rickettsia montanensis 分别含有 9.9、5.5 和 7.5 个拷贝。用 pRAM32dRGA 转化的相同物种分别维持 2.6、2.5、3.2 和 3.6 个拷贝。R. monacensis 天然存在的质粒 pRM 在经过 15 次传代后，仍以与野生型 R. monacensis 相似的水平存在于穿梭载体转化的 R. monacensis 中。基于 R. amblyommii 质粒 pRAM18 和 pRAM32 的穿梭载体系统实现了多种立克次体的稳定转化，为立克次体的遗传和生物学研究提供了一种新的研究工具，将极大地促进这方面的研究。
