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

We describe a type III restriction and modification (R/M) system, LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, ha rbored in L. lactis LL42-1, Sequencing revealed two adjacent open reading f rames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon, A homology search revealed th at the two ORFs exhibited significant similarity to type III restriction (R es) and modification (Mod) subunits, The complete amino acid sequence of th e Mod subunit of LlaFI was aligned with the amino acid sequences of four pr eviously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the ce ntral regions are more variable, An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-termi nal region of the Mod protein. The seven conserved helicase moths found in the previously described type III R/M systems were found at the same relati ve positions in the LlaFI Res sequence. LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzy mes. ATP and Mg2+ are required for endonucleolytic activity; however, the a ctivity is not strictly dependent on the presence of Ado-Met but, is stimul ated by it. To our knowledge, this is the first type III R/M system that ha s been characterized not just in lactic acid bacteria but also in gram-posi tive bacteria.