Antisense mRNA-Mediated Bacteriophage Resistance in Lactococcus lactis subsp. lactis

Area of Science:

• Microbiology
• Molecular Biology
• Dairy Science

Background:

• Bacteriophage infection poses a significant threat to dairy starter cultures, impacting lactic acid production.
• Lactococcus lactis is a crucial bacterium in dairy fermentation, susceptible to various bacteriophages.
• Developing robust phage resistance is essential for maintaining efficient dairy production.

Purpose of the Study:

• To engineer broad-spectrum resistance against bacteriophages in Lactococcus lactis.
• To investigate the efficacy of antisense mRNA technology for phage resistance.
• To assess the impact of antisense gene expression on dairy starter culture performance.

Main Methods:

• Expression of antisense mRNA targeting a conserved bacteriophage gene in Lactococcus lactis.
• Utilizing a 1,654-bp coding sequence for a 51-kDa protein in antisense orientation.
• Assessing phage resistance by plaque-forming unit (PFU) reduction and plaque morphology analysis.
• Southern hybridization to confirm plasmid stability and antisense construct maintenance.

Main Results:

• Antisense mRNA expression achieved over 99% reduction in bacteriophage PFU.
• Maximum resistance required the entire 1,654-bp coding sequence in antisense orientation.
• Truncated genes or sense orientation failed to confer significant resistance.
• Antisense plasmid constructs were stably maintained in Lactococcus lactis.
• Antisense expression did not impede bacterial growth or acid production.

Conclusions:

• Antisense mRNA technology is a viable strategy for engineering broad-spectrum bacteriophage resistance in Lactococcus lactis.
• The specific 51-kDa protein's coding sequence in antisense orientation is critical for effective phage inhibition.
• This approach offers a promising solution for protecting dairy starter cultures without compromising their metabolic function.