Development of Shuttle Vectors for Transformation of Diverse Rickettsia Species

Abstract

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.

Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection

“…gfp uv expression is driven by the R. prowazekii ompA promoter, and GFP uv fluoresces adequately into the FITC filter set (13). Accordingly, we hypothesized that transformed R. conorii(pRam18dRGA [AmTrCh]) should readily fluoresce into the FITC (GFP uv ) and TRITC (mCherry) channels as has been previously observed in other Rickettsia species (9,15). As shown in Fig.…”

“…However, the development of tools for chromosomal mutagenesis and selectively maintained extrachromosomal DNA in rickettsial species has been demonstrated (8). Plasmids isolated from nonpathogenic Rickettsia bacteria have been adapted for replication and selection in both Rickettsia and Escherichia species (9). These plasmids can potentially be useful for heterologous gene expression, reporter gene expression, and in trans complementation in diverse Rickettsia species.…”

“…The nonessential portion of the naturally occurring plasmid was removed, and the remaining portion was inserted into an Escherichia coli vector. The gfp uv and rpaar2 genes and a multiple cloning site (MCS) were introduced to produce the plasmid pRam18dRGA[MCS] (9,12,13). The 948-bp AmTrCh cassette encoding mCherry was subsequently added to create pRam18dRGA[AmTrCh] (9,14).…”

“…The gfp uv and rpaar2 genes and a multiple cloning site (MCS) were introduced to produce the plasmid pRam18dRGA[MCS] (9,12,13). The 948-bp AmTrCh cassette encoding mCherry was subsequently added to create pRam18dRGA[AmTrCh] (9,14). This plasmid has been successfully transformed into five pathogenic and nonpathogenic Rickettsia species (9,15).…”

“…The 948-bp AmTrCh cassette encoding mCherry was subsequently added to create pRam18dRGA[AmTrCh] (9,14). This plasmid has been successfully transformed into five pathogenic and nonpathogenic Rickettsia species (9,15).…”