Creation and characterisation of a high-copy-number version of the pAL5000 mycobacterial replicon

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Mycobacterial plasmid vectors, typically derived from the pAL5000 replicon, are maintained at low copy numbers (approx. 5 copies/cell).
• Low copy numbers can limit the utility of plasmids for applications requiring high gene expression in mycobacteria.

Purpose of the Study:

• To develop a method for directly selecting high-copy-number plasmids in mycobacteria.
• To isolate and characterize an enhanced version of the pAL5000 replicon with increased copy number and stability.

Main Methods:

• A novel selection strategy was employed, involving iterative isolation and retransformation of plasmids from a mutant library to enrich for high-copy-number variants.
• The copy-up pAL5000 replicon variant was tested in *Mycobacterium smegmatis* and *Mycobacterium bovis* BCG.
• Gene expression was assessed using the green fluorescent protein (GFP) reporter.

Main Results:

• A copy-up mutant of the pAL5000 replicon was successfully selected, exhibiting a 7-fold increase in copy number (32–64 copies/cell) in *M. smegmatis*.
• The high-copy-number plasmid demonstrated stability over 100 generations without antibiotic selection.
• The high-copy-number phenotype was also observed in *M. bovis* BCG, and increased expression of cloned genes (GFP) was confirmed.

Conclusions:

• A novel method enables direct selection of high-copy-number mycobacterial plasmids.
• The identified RepA mutation (alanine deletion) enhances pAL5000 replicon copy number and gene expression in mycobacteria.
• The mutation likely affects RepA-RepB expression via altered RNA folding, offering a new tool for mycobacterial genetic engineering.