Journal article
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, Vol.74(6), pp.1687-1695
03/2008
Handle:
https://hdl.handle.net/2376/105076
PMCID: PMC2268294
PMID: 18192416
Abstract
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 × 10
−7
to 2.9 × 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.
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Details
- Title
- 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
- Creators
- Kun Taek Park - Department of Veterinary Microbiology and Pathology, College of Veterinary MedicineJohn L Dahl - Department of Veterinary Microbiology and Pathology, College of Veterinary MedicineJohn P Bannantine - Department of Veterinary Microbiology and Pathology, College of Veterinary MedicineRaúl G Barletta - Department of Veterinary Microbiology and Pathology, College of Veterinary MedicineJongsam Ahn - Department of Veterinary Microbiology and Pathology, College of Veterinary MedicineAndrew J Allen - Department of Veterinary Microbiology and Pathology, College of Veterinary MedicineMary Jo Hamilton - Department of Veterinary Microbiology and Pathology, College of Veterinary MedicineWilliam C Davis - Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine
- Publication Details
- Applied and environmental microbiology, Vol.74(6), pp.1687-1695
- Academic Unit
- Veterinary Clinical Sciences, Department of; Veterinary Microbiology and Pathology, Department of
- Publisher
- American Society for Microbiology (ASM)
- Identifiers
- 99900546638501842
- Language
- English
- Resource Type
- Journal article