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Functional definition of regions necessary for replication and incompatibility in the Mycobacterium fortuitum plasmid
1Department of Clinical Sciences, London School of Hygiene & Tropical Medicine, UK. p.stolt@lshtm.ac.uk
Abstract:
Different parts of the Mycobacterium fortuitum plasmid pAL5000 necessary for plasmid replication and incompatibility were defined and studied. Two ORFs, named repA and repB, were defined which are necessary for replication. A pAL5000 derivative deleted in these genes can be made to replicate by providing the gene products in trans. A 435 bp fragment was defined which was necessary in cis for replication and which had an influence on copy number. This region (inc), which contains several repeated motifs, was also able to confer a degree of incompatibility when cloned into an otherwise unrelated mycobacterial replicon. pAL5000-derived plasmids carrying two copies of the inc region had a lower copy number and were less stable than the wild-type. These effects were only observed when the two regions were in the same orientation. Plasmids carrying only the inc region and no other parts of pAL5000 could be made to replicate if repA and repB were supplied in trans from another plasmid. Based on these findings, systems for selectively curing cells of one plasmid of a pair were designed and shown to be functional in Mycobacterium smegmatis. These have potential as a simple delivery system for achieving transposon mutagenesis or gene replacement in mycobacteria.
Insights
Researchers identified key genes (repA and repB) and a DNA region (inc) essential for Mycobacterium fortuitum plasmid pAL5000 replication and stability. This knowledge enables the development of novel tools for genetic manipulation in mycobacteria.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The plasmid pAL5000 from Mycobacterium fortuitum is a crucial tool in mycobacterial research.
- Understanding its replication and stability mechanisms is essential for developing genetic manipulation systems.
Purpose of the Study:
- To delineate the specific DNA regions and open reading frames (ORFs) of pAL5000 involved in its replication and incompatibility.
- To investigate the influence of these elements on plasmid copy number and stability.
- To design and validate plasmid-based systems for selective curing in mycobacteria.
Main Methods:
- Plasmid deletion analysis to identify essential replication and cis-acting elements.
- Complementation studies by providing gene products in trans.
- Cloning of specific regions into unrelated replicons to assess function.
- Orientation-dependent stability assays.
- Functional testing of plasmid curing systems in Mycobacterium smegmatis.
Main Results:
- Two ORFs, repA and repB, were identified as essential for pAL5000 replication.
- A 435 bp fragment (inc region) containing repeated motifs was found to be necessary in cis for replication and influenced copy number.
- The inc region conferred incompatibility and, when present in multiple copies or specific orientations, reduced plasmid copy number and stability.
- Functional plasmid curing systems were successfully demonstrated in Mycobacterium smegmatis.
Conclusions:
- The repA and repB genes and the inc region are critical determinants of pAL5000 replication, copy number, and stability.
- These findings facilitate the design of novel plasmid vectors for mycobacteria.
- The developed plasmid curing systems offer a promising platform for transposon mutagenesis and gene replacement in mycobacteria.