Transformation of Anaplasma marginale

Roderick F. Felsheim, Adela S.Oliva Chávez, Guy H. Palmer, Liliana Crosby, Anthony F. Barbet, Timothy J Kurtti, Ulrike G Munderloh

Research output: Contribution to journalArticle

31 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)167-174
Number of pages8
JournalVeterinary Parasitology
Volume167
Issue number2-4
DOIs
StatePublished - Feb 10 2010

Fingerprint

Anaplasma marginale
Anaplasma
Ticks
Transposases
ticks
plasmids
Plasmids
Ehrlichia
Anaplasmosis
transposons
Spectinomycin
spectinomycin
Streptomycin
streptomycin
bovine anaplasmosis
Bacteria
Rickettsia
Anaplasma phagocytophilum
anaplasmosis
Homologous Recombination

Keywords

  • Anaplasma marginale
  • Bovine anaplasmosis
  • Genetic transformation
  • Himar transposase
  • Homologous recombination
  • Ticks

Cite this

Felsheim, R. F., Chávez, A. S. O., Palmer, G. H., Crosby, L., Barbet, A. F., Kurtti, T. J., & Munderloh, U. G. (2010). Transformation of Anaplasma marginale. Veterinary Parasitology, 167(2-4), 167-174. https://doi.org/10.1016/j.vetpar.2009.09.018

Transformation of Anaplasma marginale. / Felsheim, Roderick F.; Chávez, Adela S.Oliva; Palmer, Guy H.; Crosby, Liliana; Barbet, Anthony F.; Kurtti, Timothy J; Munderloh, Ulrike G.

In: Veterinary Parasitology, Vol. 167, No. 2-4, 10.02.2010, p. 167-174.

Research output: Contribution to journalArticle

Felsheim, RF, Chávez, ASO, Palmer, GH, Crosby, L, Barbet, AF, Kurtti, TJ & Munderloh, UG 2010, 'Transformation of Anaplasma marginale', Veterinary Parasitology, vol. 167, no. 2-4, pp. 167-174. https://doi.org/10.1016/j.vetpar.2009.09.018
Felsheim RF, Chávez ASO, Palmer GH, Crosby L, Barbet AF, Kurtti TJ et al. Transformation of Anaplasma marginale. Veterinary Parasitology. 2010 Feb 10;167(2-4):167-174. https://doi.org/10.1016/j.vetpar.2009.09.018
Felsheim, Roderick F. ; Chávez, Adela S.Oliva ; Palmer, Guy H. ; Crosby, Liliana ; Barbet, Anthony F. ; Kurtti, Timothy J ; Munderloh, Ulrike G. / Transformation of Anaplasma marginale. In: Veterinary Parasitology. 2010 ; Vol. 167, No. 2-4. pp. 167-174.
@article{23adb5d922554491985d423dbfabf674,
title = "Transformation of Anaplasma marginale",
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.",
keywords = "Anaplasma marginale, Bovine anaplasmosis, Genetic transformation, Himar transposase, Homologous recombination, Ticks",
author = "Felsheim, {Roderick F.} and Ch{\'a}vez, {Adela S.Oliva} and Palmer, {Guy H.} and Liliana Crosby and Barbet, {Anthony F.} and Kurtti, {Timothy J} and Munderloh, {Ulrike G}",
year = "2010",
month = "2",
day = "10",
doi = "10.1016/j.vetpar.2009.09.018",
language = "English (US)",
volume = "167",
pages = "167--174",
journal = "Veterinary Parasitology",
issn = "0304-4017",
publisher = "Elsevier",
number = "2-4",

}

TY - JOUR

T1 - Transformation of Anaplasma marginale

AU - Felsheim, Roderick F.

AU - Chávez, Adela S.Oliva

AU - Palmer, Guy H.

AU - Crosby, Liliana

AU - Barbet, Anthony F.

AU - Kurtti, Timothy J

AU - Munderloh, Ulrike G

PY - 2010/2/10

Y1 - 2010/2/10

N2 - 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.

AB - 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.

KW - Anaplasma marginale

KW - Bovine anaplasmosis

KW - Genetic transformation

KW - Himar transposase

KW - Homologous recombination

KW - Ticks

UR - http://www.scopus.com/inward/record.url?scp=74449084826&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=74449084826&partnerID=8YFLogxK

U2 - 10.1016/j.vetpar.2009.09.018

DO - 10.1016/j.vetpar.2009.09.018

M3 - Article

VL - 167

SP - 167

EP - 174

JO - Veterinary Parasitology

JF - Veterinary Parasitology

SN - 0304-4017

IS - 2-4

ER -