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Published on: July 13, 2017
Development of oriC-plasmids for use in Mycoplasma hyorhinis
Hassan Z A Ishag1,2, Qiyan Xiong3, Maojun Liu1
1Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Research Center for Engineering and Technology of Veterinary Bio-products, Nanjing, 210014, China.
Abstract:
Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pig pathogen, belonging to the class Mollicutes. It causes polyserositis, arthritis and cancers in vitro, increasing attention of the researchers. Currently, there is no available genetic tool to manipulate its genome. This study describes a development of oriC-plasmids harboring either large (pGEMT-LoriC) or minimum (pGEMT-MoriC) origin of replication (oriC) of M. hyorhinis along with tetracycline resistance marker.These plasmids were successfully transformed into M. hyorhinis with average transformation frequency of 1.5 × 10-4 and 2.0 × 10-5 transformants/CFU for pGEMT-LoriC and pGEMT-MoriC respectively, and were integrated at the chromosomal oriC as well as remained freely replicating. We also constructed a Mini-oriC-HT1 targeting plasmid by inclusion of hlyC arms and was used to inactivate hlyC at average frequency of 50%. The efficiency of hlyC inactivation was further improved (by 90%) when Mini-oriC-HT2 that contains E. coli recA was used. In both cases, hemolysin mutant bacteria diminished the ability to lyse mouse RBCs compared to wild-type (P < 0.001). OriC-plasmids described in this study may, therefore open the way for functional genomics in M. hyorhinis. Furthermore, this is a first study demonstrated the gene associated with a hemolytic phenotype in mycoplasmas.
Insights
Researchers developed novel oriC-plasmids for Mycoplasma hyorhinis (M. hyorhinis), enabling genetic manipulation. This breakthrough facilitates functional genomics and gene studies in this important swine pathogen.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic swine pathogen causing significant health issues.
- Lack of genetic tools hinders research into M. hyorhinis pathogenesis and biology.
- Understanding M. hyorhinis requires methods for genome manipulation and gene function analysis.
Purpose of the Study:
- To develop and characterize oriC-plasmid-based tools for Mycoplasma hyorhinis genetic manipulation.
- To establish a system for targeted gene inactivation in M. hyorhinis.
- To investigate the hemolytic phenotype associated with a specific gene in M. hyorhinis.
Main Methods:
- Construction of oriC-plasmids (pGEMT-LoriC, pGEMT-MoriC) with tetracycline resistance marker.
- Transformation of M. hyorhinis and assessment of plasmid replication and integration.
- Development of targeting plasmids (Mini-oriC-HT1, Mini-oriC-HT2) for hlyC gene inactivation.
Main Results:
- Successful transformation of M. hyorhinis with oriC-plasmids, achieving transformation frequencies up to 1.5 × 10⁻⁴.
- Plasmids were found to integrate at the chromosomal oriC and replicate freely.
- Efficient inactivation of the hlyC gene (up to 90%) using the developed targeting system, resulting in reduced hemolytic activity.
Conclusions:
- The developed oriC-plasmids provide the first functional genetic tools for Mycoplasma hyorhinis.
- This study demonstrates the feasibility of targeted gene inactivation and functional genomics in M. hyorhinis.
- The gene responsible for the hemolytic phenotype in M. hyorhinis was identified and characterized.
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