Conjugative mobilization as an alternative vector delivery system for lactic streptococci.

Area of Science:

• Microbiology
• Molecular Biology
• Genetic Engineering

Background:

• Polyethylene glycol-mediated protoplast transformation in lactic streptococci exhibits significant variability.
• There is a need for alternative, reliable methods for vector delivery in these bacteria.

Purpose of the Study:

• To evaluate conjugative mobilization as a feasible alternative for vector delivery in lactic streptococci.
• To assess the efficiency and integrity of plasmid transfer using this method.

Main Methods:

• Utilized the broad-host-range conjugative plasmid pVA797 for mobilizing other plasmids.
• Introduced plasmids into various Streptococcus species, including Streptococcus lactis, Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, Streptococcus thermophilus, and Streptococcus faecalis.
• Employed Streptococcus lactis LM2301 as an intermediate donor for recipient differentiation.

Main Results:

• Successfully mobilized partially homologous cloning vector pVA838 and shuttle vector pSA3 into target Streptococcus strains.
• Achieved conjugation frequencies ranging from 10(-2) to 10(-6) transconjugants per recipient.
• Confirmed that transferred plasmids (pVA797, pVA838, pSA3) were maintained intact and functional.

Conclusions:

• Conjugative mobilization is a viable and efficient method for genetic manipulation of diverse lactic streptococci strains.
• This technique provides a robust alternative to protoplast transformation for introducing vectors.
• The study also reports on vector construction, cointegrate resolution, segregation, and stability.