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Open Access Research article

Knockout of an outer membrane protein operon of Anaplasma marginale by transposon mutagenesis

Francy L Crosby1*, Heather L Wamsley2, Melanie G Pate2, Anna M Lundgren1, Susan M Noh3, Ulrike G Munderloh4 and Anthony F Barbet1

Author Affiliations

1 College of Veterinary Medicine, University of Florida, Department of Infectious Diseases and Pathology, 2015 SW 16th avenue, Gainesville, FL 32610, USA

2 Physiological Sciences, 2015 SW 16th avenue, Gainesville, FL 32610, USA

3 USDA-ARS Animal Disease Research Unit, Animal Disease Research Unit, 3003 ADBF, Pullman, WA 99164, USA

4 Department of Entomology, University of Minnesota, 219 Hodson Hall 1980 Folwell avenue, St. Paul, Minneapolis, MN 55108, USA

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BMC Genomics 2014, 15:278  doi:10.1186/1471-2164-15-278

Published: 11 April 2014

Abstract

Background

The large amounts of data generated by genomics, transcriptomics and proteomics have increased our understanding of the biology of Anaplasma marginale. However, these data have also led to new assumptions that require testing, ideally through classical genetic mutation. One example is the definition of genes associated with virulence. Here we describe the molecular characterization of a red fluorescent and spectinomycin and streptomycin resistant A. marginale mutant generated by Himar1 transposon mutagenesis.

Results

High throughput genome sequencing to determine the Himar1-A. marginale genome junctions established that the transposon sequences were integrated within the coding region of the omp10 gene. This gene is arranged within an operon with AM1225 at the 5’ end and with omp9, omp8, omp7 and omp6 arranged in tandem at the 3’ end. RNA analysis to determine the effects of the transposon insertion on the expression of omp10 and downstream genes revealed that the Himar1 insertion not only reduced the expression of omp10 but also that of downstream genes. Transcript expression from omp9, and omp8 dropped by more than 90% in comparison with their counterparts in wild-type A. marginale. Immunoblot analysis showed a reduction in the production of Omp9 protein in these mutants compared to wild-type A. marginale.

Conclusions

These results demonstrate that transposon mutagenesis in A. marginale is possible and that this technology can be used for the creation of insertional gene knockouts that can be evaluated in natural host-vector systems.