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Applied and Environmental Microbiology, March 2008, p. 1687-1695, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.01208-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Kun Taek Park,1 John L. Dahl,2 John P. Bannantine,3 Raúl G. Barletta,4 Jongsam Ahn,5 Andrew J. Allen,6 Mary Jo Hamilton,1 and William C. Davis1*

Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine,1 School of Molecular Biosciences, Washington State University, Pullman, Washington 99164,2 National Animal Disease Center, USDA-ARS, Ames, Iowa 50010,3 Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583,4 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6495,5 Department of Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington 991646

Received 18 May 2007/ Accepted 30 December 2007

Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 x 10–7 to 2.9 x 10–7. Three genes were selected for mutagenesis: pknG and relA, which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis, and lsr2, a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis (M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp. The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.

* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, CVM, Washington State University, Pullman, WA 99164-7040. Phone: (509) 335-6051. Fax: (509) 335-8328. E-mail: davisw{at}vetmed.wsu.edu

{triangledown} Published ahead of print on 11 January 2008.

Applied and Environmental Microbiology, March 2008, p. 1687-1695, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.01208-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Allen, A. J., Park, K. T., Barrington, G. M., Lahmers, K. K., Hamilton, M. J., Davis, W. C. (2009). Development of a Bovine Ileal Cannulation Model To Study the Immune Response and Mechanisms of Pathogenesis of Paratuberculosis. CVI 16: 453-463 [Abstract] [Full Text]  


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