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Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7
M L Beggs1, J T Crawford, K D Eisenach
1Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, USA.
Abstract:
The Mycobacterium avium plasmid pLR7 is representative of a group of small plasmids that are common in isolates from AIDS patients with disseminated M. avium infections. Determination of the functions of these and other plasmids has been hampered by the lack of methods for genetic manipulation of M. avium. In this study, the region of pLR7 capable of replication was identified and sequenced. Fragments of pLR7 were cloned into a pUC18 derivative carrying a kanamycin resistance marker and introduced into a plasmid-free M. avium strain by electroporation. The origin of replication was located on a 1.8-kb PvuII-to-SmaI fragment. An open reading frame encoding a putative Rep protein was identified. Two other open reading frames were identified in this region. A shuttle vector, pMB351, was constructed with the pLR7 origin of replication, pUC18, and the kanamycin resistance gene from Tn5. This vector was successfully transformed into M. avium, Mycobacterium tuberculosis, and Mycobacterium bovis.
Insights
Researchers identified the replication region of the Mycobacterium avium plasmid pLR7. This breakthrough enables genetic manipulation of M. avium, crucial for understanding infections in AIDS patients.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Mycobacterium avium plasmid pLR7 is common in AIDS patients with disseminated infections.
- Lack of genetic manipulation methods hinders understanding of M. avium plasmids.
Purpose of the Study:
- Identify and sequence the replication region of the pLR7 plasmid.
- Develop tools for genetic manipulation of M. avium.
Main Methods:
- Cloning pLR7 fragments into a pUC18 derivative with a kanamycin resistance marker.
- Electroporation to introduce plasmids into M. avium.
- DNA sequencing to identify open reading frames.
Main Results:
- The replication origin was localized to a 1.8-kb PvuII-to-SmaI fragment.
- A putative Rep protein-encoding open reading frame was identified.
- A functional shuttle vector, pMB351, was constructed and transformed into M. avium, M. tuberculosis, and M. bovis.
Conclusions:
- The replication region of pLR7 has been successfully identified.
- A novel shuttle vector facilitates genetic studies in mycobacteria.
- This research paves the way for understanding M. avium pathogenesis and developing new therapies.