Cloning vectors for lactococci based on a plasmid encoding resistance to cadmium

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Lactococcus lactis is a key bacterium in food fermentation.
• Developing efficient cloning vectors for L. lactis is crucial for genetic manipulation.
• Cadmium resistance (CdR) can serve as a selectable marker in lactococci.

Purpose of the Study:

• To identify and characterize a plasmid conferring cadmium resistance in L. lactis.
• To construct and evaluate novel food-grade cloning vectors for L. lactis.
• To assess the utility of CdR as a selectable marker in lactococcal systems.

Main Methods:

• Plasmid identification and characterization using restriction enzyme mapping.
• Construction of shuttle and food-grade cloning vectors by subcloning genes.
• Transformation of L. lactis using electroporation and assessment of vector stability.
• Evaluation of cadmium resistance and nisin resistance (NisR) expression.

Main Results:

• A 8.8-kb plasmid, pND302, encoding cadmium resistance (CdR) was isolated from L. lactis M71.
• Two shuttle cloning vectors (pND304, pND624) and a food-grade vector (pND625) were successfully constructed.
• pND625, derived solely from lactococcal DNA, expresses both NisR and CdR, featuring versatile polylinkers.
• Both pND302 and pND625 demonstrated stable transformation and maintenance in L. lactis LMO230.

Conclusions:

• pND302 provides a valuable selectable marker for L. lactis.
• pND302 and pND625 are promising food-grade cloning vectors for lactococcal genetic engineering.
• The developed vectors facilitate versatile cloning applications in lactic acid bacteria.