Development of shuttle vectors for transformation of diverse rickettsia species

Nicole Y. Burkhardt, Gerald D Baldridge, Phillip C. Williamson, Peggy M. Billingsley, Chan C. Heu, Roderick F. Felsheim, Timothy J Kurtti, Ulrike G Munderloh

Research output: Contribution to journalArticle

33 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere29511
JournalPloS one
Volume6
Issue number12
DOIs
StatePublished - Dec 21 2011

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genetic vectors
Rickettsia
Genetic Vectors
plasmids
Plasmids
Rickettsia bellii
Rickettsia parkeri
Bearings (structural)
Rickettsia montanensis
R Factors
Cloning
rifampicin
Rifampin
Chromosomes
electroporation
Electroporation
probes (equipment)
Genes
molecular cloning
quantitative polymerase chain reaction

Cite this

Burkhardt, N. Y., Baldridge, G. D., Williamson, P. C., Billingsley, P. M., Heu, C. C., Felsheim, R. F., ... Munderloh, U. G. (2011). Development of shuttle vectors for transformation of diverse rickettsia species. PloS one, 6(12), [e29511]. https://doi.org/10.1371/journal.pone.0029511

Development of shuttle vectors for transformation of diverse rickettsia species. / Burkhardt, Nicole Y.; Baldridge, Gerald D; Williamson, Phillip C.; Billingsley, Peggy M.; Heu, Chan C.; Felsheim, Roderick F.; Kurtti, Timothy J; Munderloh, Ulrike G.

In: PloS one, Vol. 6, No. 12, e29511, 21.12.2011.

Research output: Contribution to journalArticle

Burkhardt, NY, Baldridge, GD, Williamson, PC, Billingsley, PM, Heu, CC, Felsheim, RF, Kurtti, TJ & Munderloh, UG 2011, 'Development of shuttle vectors for transformation of diverse rickettsia species', PloS one, vol. 6, no. 12, e29511. https://doi.org/10.1371/journal.pone.0029511
Burkhardt NY, Baldridge GD, Williamson PC, Billingsley PM, Heu CC, Felsheim RF et al. Development of shuttle vectors for transformation of diverse rickettsia species. PloS one. 2011 Dec 21;6(12). e29511. https://doi.org/10.1371/journal.pone.0029511
Burkhardt, Nicole Y. ; Baldridge, Gerald D ; Williamson, Phillip C. ; Billingsley, Peggy M. ; Heu, Chan C. ; Felsheim, Roderick F. ; Kurtti, Timothy J ; Munderloh, Ulrike G. / Development of shuttle vectors for transformation of diverse rickettsia species. In: PloS one. 2011 ; Vol. 6, No. 12.
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