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LlaFI, a type III restriction and modification system in Lactococcus lactis

P Su1, H Im, H Hsieh

  • 1Gist-Brocades Australia, Moorebank NSW 2170, Australia. p.su@unsw.edu.au

Applied and Environmental Microbiology
|January 30, 1999
PubMed
Summary
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This study characterizes the LlaFI type III restriction and modification system in Lactococcus lactis, the first identified in gram-positive bacteria. It details the system

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Restriction and modification (R/M) systems are crucial for bacterial defense and genome maintenance.
  • Type III R/M systems, characterized by their unique DNA recognition and cleavage mechanisms, are less understood compared to other types.
  • Lactococcus lactis is a significant bacterium in food fermentation and probiotic applications.

Purpose of the Study:

  • To identify and characterize a novel type III restriction and modification system in Lactococcus lactis.
  • To elucidate the genetic organization and molecular features of this new system, named LlaFI.
  • To determine the cofactor requirements for the enzymatic activity of LlaFI.

Main Methods:

  • Plasmid DNA isolation and sequencing of pND801 from Lactococcus lactis LL42-1.

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  • Bioinformatic analysis including open reading frame (ORF) identification and homology searches.
  • Amino acid sequence alignment of LlaFI subunits with known type III R/M enzymes.
  • Biochemical assays to determine cofactor dependency (ATP, Mg2+, S-adenosyl methionine) for endonuclease activity.

    • Main Results:

    • Identification of the LlaFI type III R/M system encoded by the 12-kb plasmid pND801.
    • Discovery of two adjacent ORFs encoding putative restriction (Res) and modification (Mod) subunits with significant homology to known type III enzymes.
    • Characterization of conserved motifs in the Mod (S-adenosyl methionine binding) and Res (helicase) subunits.
    • LlaFI activity requires ATP and Mg2+, and is stimulated by S-adenosyl methionine.

    Conclusions:

    • LlaFI represents the first characterized type III restriction and modification system in lactic acid bacteria and gram-positive bacteria.
    • The genetic and molecular features of LlaFI are consistent with other type III R/M systems, with conserved functional domains.
    • This discovery expands the known diversity of R/M systems in bacteria and provides a new tool for genetic manipulation in L. lactis.