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Stimulation of transposition of the Mycobacterium tuberculosis insertion sequence IS6110 by exposure to a
K Ghanekar1, A McBride, O Dellagostin
1Molecular Microbiology Group, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK.
Abstract:
The Mycobacterium tuberculosis-specific insertion sequence IS6110/986 has been widely used as a probe because of the multiple polymorphism observed among different strains. To investigate transposition of IS6110, a series of artificially constructed composite transposons containing IS6110 and a kanamycin resistance marker were constructed. The composite transposons were inserted into a conditionally replicating, thermosensitive, Escherichia coli-mycobacterial shuttle vector and introduced into M. smegmatis mc2155. Lawns of transformants were grown at the permissive temperature on kanamycin-supplemented agar and subsequently prevented from further growth by shifting to the non-permissive temperature. Under normal atmospheric conditions, kanamycin-resistant papillae appeared after only about 5-6 weeks of incubation. However, these events were not associated with transposon mobilization. In contrast, lawns that were exposed to a 48 h microaerobic shock generated kanamycin-resistant papillae after only 6-14 days. These events were generated by conservative transposition of the IS6110 composite transposon into the M. smegmatis chromosome, with loss of the shuttle vector. In common with other IS3 family elements, transposition of IS6110 is thought to be controlled by translational frameshifting. However, we were unable to detect any significant frameshifting within the putative frameshifting site of IS6110, and the level of frameshifting was not affected by microaerobic incubation. The finding that transposition of IS6110 is stimulated by incubation at reduced oxygen tensions may be relevant to transposition of IS6110 in M. tuberculosis harboured within TB lesions.
Insights
Microaerobic conditions stimulate the transposition of Mycobacterium tuberculosis insertion sequence IS6110 in M. smegmatis. This finding is crucial for understanding IS6110 activity within tuberculosis lesions.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The Mycobacterium tuberculosis-specific insertion sequence IS6110 is a key genetic element used for strain typing due to its polymorphic nature.
- Understanding the transposition mechanisms of IS6110 is critical for its application in diagnostics and for comprehending its role in bacterial evolution.
Purpose of the Study:
- To investigate the transposition activity of the IS6110 insertion sequence under varying atmospheric conditions.
- To determine the influence of microaerobic environments on IS6110 mobilization and its potential relevance to Mycobacterium tuberculosis pathogenesis.
Main Methods:
- Construction of artificial composite transposons containing IS6110 and a kanamycin resistance marker.
- Insertion of these transposons into a shuttle vector and introduction into Mycobacterium smegmatis mc2155.
- Incubation of transformants under normal atmospheric conditions and microaerobic shock, followed by analysis of kanamycin resistance and transposon integration.
Main Results:
- Microaerobic shock significantly accelerated the appearance of kanamycin-resistant papillae in M. smegmatis, indicating enhanced transposition.
- Kanamycin resistance under microaerobic conditions was linked to conservative transposition of the IS6110 composite transposon into the host chromosome.
- No significant translational frameshifting was detected within the putative frameshifting site of IS6110, and microaerobic conditions did not affect frameshifting levels.
Conclusions:
- Transposition of IS6110 is stimulated by reduced oxygen tensions, a condition relevant to Mycobacterium tuberculosis residing in hypoxic TB lesions.
- The study provides insights into the environmental regulation of IS6110 transposition, potentially impacting its behavior in vivo.
- Further research is warranted to elucidate the precise mechanisms controlling IS6110 transposition under microaerobic conditions.