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Construction of mini-Tn4001tet and its use in Mycoplasma gallisepticum
Ina Pour-El1, Cary Adams, F Chris Minion
1Veterinary Medical Research Institute, Ames, Iowa 50011, USA.
Abstract:
The Mollicutes are a group of cell-wall-less bacteria and are important plant and animal pathogens. Progress toward analyzing their pathogenic mechanisms has been hampered by the few available genetic tools. Of the two transposons shown to function in mycoplasmas, only Tn4001 is readily amenable to modification and development. One disadvantage of using Tn4001 in mycoplasmas has been independent insertion of the insertion sequence, IS256, probably as a result of inadequate control of the transposase expression in mycoplasmas. In this study, we describe the construction of a mini-Tn4001 containing the tetM antibiotic resistance gene from Tn916. The transposase gene was placed outside the inverted repeats to lower the frequency of independent transposition events. Transposition of mini-Tn4001tet in Mycoplasma gallisepticum occurred at a frequency of 1-8 x 10(-6), a frequency similar to that of the parent transposon. Insertions of mini-Tn4001tet were random and only single insertions were observed. Several unique restriction sites between the inverted repeat sequences provide for further development of mini-Tn4001.
Insights
Researchers developed a new genetic tool, mini-Tn4001tet, for studying Mollicutes bacteria. This tool improves genetic analysis of these important plant and animal pathogens by enabling controlled transposition.
Area of Science:
- Microbiology
- Bacteriology
- Molecular Biology
Background:
- Mollicutes are cell-wall-less bacteria causing significant plant and animal diseases.
- Limited genetic tools hinder the study of Mollicutes pathogenicity.
- Tn4001 is a transposon suitable for modification, but IS256 independent insertion is problematic.
Purpose of the Study:
- To construct a modified transposon, mini-Tn4001tet, for improved genetic manipulation of Mollicutes.
- To reduce independent transposition events of the insertion sequence IS256.
- To facilitate the analysis of Mollicutes pathogenic mechanisms.
Main Methods:
- Construction of a mini-Tn4001 derivative containing the tetM antibiotic resistance gene.
- Relocation of the transposase gene outside the inverted repeats to control expression.
- Transposition experiments of mini-Tn4001tet in Mycoplasma gallisepticum.
Main Results:
- Mini-Tn4001tet transposition occurred at a frequency of 1-8 x 10(-6) in Mycoplasma gallisepticum.
- Transposition events resulted in random, single insertions of the mini-transposon.
- The modified transposon demonstrated controlled transposition, reducing independent insertion events.
Conclusions:
- The developed mini-Tn4001tet is a valuable and stable genetic tool for Mollicutes research.
- This tool enables more efficient and reliable genetic analysis of Mollicutes pathogens.
- Further development of mini-Tn4001 is possible due to unique restriction sites.