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Title: Knockout of an outer membrane protein operon of anaplasma marginale by transposon mutagenesis

item CROSBY, FRANCY - University Of Florida
item WAMSLEY, HEATHER - University Of Florida
item PATE, MELANIE - University Of Florida
item LUNDGREN, ANNA - University Of Florida
item Noh, Susan
item MUNDERLOH, ULRIKE - University Of Minnesota
item BARBET, ANTHONY - University Of Florida

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/31/2014
Publication Date: 4/11/2014
Citation: Crosby, F.L., Wamsley, H.L., Pate, M.G., Lundgren, A.M., Noh, S.M., Munderloh, U.G., Barbet, A.F. 2014. Knockout of an outer membrane protein operon of anaplasma marginale by transposon mutagenesis. Biomed Central (BMC) Genomics. 15:278.

Interpretive Summary: The surface proteins of infectious agents are functionally important because they mediate the interaction between the pathogen and its host and they are the targets of the host immune system. Overall little is known about the function of bacterial surface proteins and in particular those of obligate intracellular bacteria. In the case of A. marginale, an obligate intracellular pathogen of cattle, many surface proteins have been characterized and two, Msp2 and Msp3, are involved in evasion of the bovine immune system. Little is known about the function of the other >100 surface proteins. One of the best ways to determine gene function is through constructing mutations in the genes of interest. Toward this end, a mutant of A. marginale was recently produced that disrupts the gene that encodes the surface protein, Omp10. Additionally Omp7, 8 and 9 are adjacent to Omp10 and are transcribed along with Omp10. This mutant results in a marked reduction in the transcription of all of these genes and an absence of detectable protein from Omp9. This mutant will allow for the determination of the function of these conserved surface genes.

Technical Abstract: Large amounts of data generated by genomics, transcriptomics and proteomics technologies have increased our understanding of the biology of Anaplasma marginale. However, these data have also led to new assumptions that require testing, ideally through classic 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. 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. Transcripts 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. 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.