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.
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.
Park
Kun Taek
KT
Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-7040, USA.
Dahl
John L
JL
Bannantine
John P
JP
Barletta
Raúl G
RG
Ahn
Jongsam
J
Allen
Andrew J
AJ
Hamilton
Mary Jo
MJ
Davis
William C
WC
D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D017353:Gene Deletion; D005798:Genes, Bacterial; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics* / Q000472:pathogenicity; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins