title: Knockout of an outer membrane protein operon of Anaplasma marginale by transposon mutagenesis.
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.
authors.affiliation: College of Veterinary Medicine, University of Florida, Department of Infectious Diseases and Pathology, 2015 SW 16th avenue, Gainesville, FL 32610, USA. crosbyl@ufl.edu.
authors.forename: Francy L
authors.initials: FL
authors.lastname: Crosby
authors.forename: Heather L
authors.initials: HL
authors.lastname: Wamsley
authors.forename: Melanie G
authors.initials: MG
authors.lastname: Pate
authors.forename: Anna M
authors.initials: AM
authors.lastname: Lundgren
authors.forename: Susan M
authors.initials: SM
authors.lastname: Noh
authors.forename: Ulrike G
authors.initials: UG
authors.lastname: Munderloh
authors.forename: Anthony F
authors.initials: AF
authors.lastname: Barbet
mesh_terms: D042322:Anaplasma marginale / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002876:Chromosomes, Bacterial; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D055785:Gene Knockdown Techniques; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009876:Operon
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial
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