Journal article
Transformation of Anaplasma marginale
Veterinary parasitology, Vol.167(2-4), pp.167-174
02/10/2010
Handle:
https://hdl.handle.net/2376/114614
PMCID: PMC2817780
PMID: 19837516
Abstract
The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, ≤1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80–90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination.
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Details
- Title
- Transformation of Anaplasma marginale
- Creators
- Roderick F Felsheim - Department of Entomology, University of Minnesota, 1980 Folwell Avenue, St. Paul, MN 55108, United StatesAdela S. Oliva Chávez - Department of Entomology, University of Minnesota, 1980 Folwell Avenue, St. Paul, MN 55108, United StatesGuy H Palmer - Washington State University, Department of Veterinary Pathology & Microbiology, 402 Bustad Hall, Pullman, WA 99164, United StatesLiliana Crosby - Department of Infectious Diseases and Pathology, College of Veterinary Medicine, 2015 SW 16th Avenue, University of Florida, Gainesville, FL 32610, United StatesAnthony F Barbet - Department of Infectious Diseases and Pathology, College of Veterinary Medicine, 2015 SW 16th Avenue, University of Florida, Gainesville, FL 32610, United StatesTimothy J Kurtti - Department of Entomology, University of Minnesota, 1980 Folwell Avenue, St. Paul, MN 55108, United StatesUlrike G Munderloh - Department of Entomology, University of Minnesota, 1980 Folwell Avenue, St. Paul, MN 55108, United States
- Publication Details
- Veterinary parasitology, Vol.167(2-4), pp.167-174
- Academic Unit
- Paul G. Allen School for Global Animal Health
- Publisher
- Elsevier B.V
- Identifiers
- 99900547834701842
- Language
- English
- Resource Type
- Journal article