Characterization of LlaCI, a new restriction-modification system from Lactococcus lactis subsp. cremoris W15

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Naturally occurring plasmids in Lactococcus lactis subsp. cremoris harbor genetic systems.
• Restriction-modification (R/M) systems provide defense against foreign DNA, including bacteriophages.

Purpose of the Study:

• To characterize the LlaCI R/M system found on the pAW153 plasmid in L. lactis.
• To investigate the phage resistance conferred by the LlaCI system.

Main Methods:

• Isolation of the R/M system on a chloramphenicol-resistant derivative plasmid (pAW153cat).
• Cloning of a specific DNA fragment containing the R/M genes into expression vectors.
• Assessing phage sensitivity in L. lactis strains harboring the cloned R/M system.
• DNA sequencing of the R/M genes and comparative analysis with known R/M systems.

Main Results:

• The LlaCI R/M system, when present on pAW153cat or cloned fragments, reduced sensitivity to specific L. lactis phages (936 and P335 species).
• Higher plasmid copy numbers correlated with increased phage restriction efficacy.
• Sequencing identified two open reading frames encoding the LlaCI methylase (IlaCIM) and endonuclease (IlaCIR).
• Sequence analysis revealed significant homology of IlaCIM to HindIII methylase and lower homology of IlaCIR to HindIII endonuclease.
• The LlaCI operon organization, with convergent transcription and gene separation, differs from the HindIII operon.

Conclusions:

• The LlaCI R/M system provides a defense mechanism against specific bacteriophages in L. lactis.
• The characterized LlaCI genes (ilaCIM and ilaCIR) are functional and contribute to phage resistance.
• The distinct genetic organization of the LlaCI operon compared to other R/M systems highlights evolutionary diversity.