|Park, Kun - WASHINGTON STATE UNIV.|
|Dahl, John - WASHINGTON STATE UNIV.|
|Barletta, Raul - UNIV. OF NE, LINCOLN|
|Ahn, Johngsam - UNIV. OF NE MED. CTR.|
|Allen, Andrew - WASHINTON STATE UNIV.|
|Hamilton, Mary - WASHINGTON STATE UNIV.|
|Davis, William - WASHINGTON STATE UNIV.|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 30, 2007
Publication Date: March 1, 2008
Citation: Park, K.T., Dahl, J.L., Bannantine, J.P., Barletta, R.G., Ahn, J., Allen, A.J., Hamilton, M.J., Davis, W.C. 2008. Demonstration of Allelic Exchange in the Slow-Growing Bacterium Mycobacterium avium subsp. paratuberculosis, and Generation of Mutants with Deletions at the pknG, relA and lsr2 Loci. Applied and Environmental Microbiology. 74(6):1687-1695. Interpretive Summary: Mycobacterium avium subsp. paratuberculosis (Map) causes Johne’s disease, an economically significant disease that impacts the US dairy industry. This bacterium has been extremely difficult to manipulate genetically, with regard to adding DNA or changing the DNA by adding mutations. This study demonstrates the ability to generate mutations in the bacterium’s chromosome using a transposon, which is a piece of DNA that can insert into other DNAs. This transposon is introduced into the bacterium using a recombinant virus called phAE87. This study demonstrates this technique on three different genes (pknG, relA and lsr2). The ability to create transposon mutations in Map opens the door to many functional biology studies including understanding function of specific genes, creating attenuated (nonvirulent) vaccine strains and understanding the pathogenesis.
Technical Abstract: Mycobacterium avium subsp. paratuberculosis (Map) is the causative pathogen of Johne’s disease, a chronic inflammatory wasting disease in ruminants. The disease has been difficult to control because of the lack of an effective vaccine. To address this need, we developed an efficient allelic exchange method to generate directed mutations within preselected Map genes. With our novel optimized method, the allelic exchange frequency was 78 - 100 % for a transduction frequency of 9.5 x 10**-8 - 1.6 x 10**-7. Three genes were selected for mutagenesis: pknG and relA, genes known to be important virulence factors in mycobacteria and lsr2, a gene regulating lipid biosynthesis. Mutants were successfully generated in the sequencing project virulent strain K10 and in a recombinant strain expressing the green fluorescent protein gene, gfp. The improved efficiency of disrupting selected genes in Map should accelerate development of additional mutants for vaccine testing and functional studies.