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Updated: May 26, 2026
An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines
Published on: October 11, 2022
Development of shuttle vectors for transformation of diverse Rickettsia species
Nicole Y Burkhardt1, Gerald D Baldridge, Phillip C Williamson
1Department of Entomology, University of Minnesota, St. Paul, Minnesota, USA.
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.
Insights
Researchers developed a novel shuttle vector system using Rickettsia amblyommii plasmids for efficient genetic manipulation of various Rickettsia species. This tool enhances future rickettsial research and biological studies.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Plasmids are common in Rickettsia species, with some harboring multiple.
- Rickettsia amblyommii AaR/SC possesses three distinct plasmids: pRAM18, pRAM23, and pRAM32.
- Understanding Rickettsia plasmid biology is crucial for developing genetic tools.
Purpose of the Study:
- To develop a shuttle vector system for Rickettsia transformation.
- To create tools for genetic and biological studies of rickettsiae.
- To utilize R. amblyommii plasmids for creating novel vectors.
Main Methods:
- Southern analysis and real-time PCR were used to characterize R. amblyommii plasmids.
- Shuttle vectors were constructed by inserting selection cassettes into R. amblyommii plasmids.
- Electroporation was employed to transform Rickettsia species with the developed vectors.
Main Results:
- A recombinant plasmid, pRAM18/Rif/GFPuv, successfully transformed Rickettsia parkeri and Rickettsia bellii.
- Smaller shuttle vectors (pRAM18dRG, pRAM18dRGA, pRAM32dRGA) were created and tested.
- Stable transformation and maintenance of shuttle vectors were observed in multiple Rickettsia species, including R. bellii and R. parkeri.
Conclusions:
- A stable shuttle vector system based on R. amblyommii plasmids was successfully established.
- This system enables efficient transformation of diverse Rickettsia species.
- The developed vectors represent a significant advancement for rickettsial research.
