Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations

Veterinary Research
Gareth A MaglennonBRaDP1T Consortium

Abstract

Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain...Continue Reading

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Citations

Dec 3, 2014·International Journal of Medical Microbiology : IJMM·Ali F AboklaishO Brad Spiller
Oct 18, 2014·Veterinary Research·Ivana IndikovaMichael P Szostak
May 6, 2015·Veterinary Microbiology·Benjamin B A Raymond, Steven Djordjevic
Mar 15, 2015·Genome Announcements·Michael J CalcuttMark A McIntosh
Dec 3, 2014·Journal of Microbiological Methods·Jiahe LiJinxiang Li
Oct 25, 2016·Veterinary Microbiology·Beth S CookAndrew N Rycroft
Jun 27, 2019·Veterinary Research·Alannah S DeeneyAndrew N Rycroft
May 11, 2021·Frontiers in Microbiology·Jeannett M ClampittF Chris Minion

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Datasets Mentioned

BETA
KF861545

Methods Mentioned

BETA
PCR
electrophoresis
GAM

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