Lla FI, a Type III Restriction and Modification System in Lactococcus lactis

Abstract

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, harbored in L. lactisLL42-1. Sequencing revealed two adjacent open reading frames (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 that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence.LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.

Spreading antibiotic resistance through spread manure: characteristics of a novel plasmid type with low %G+C content

“…The region upstream of this tnpR ‐like gene varied between the plasmids. On plasmid pHHV35, a restriction‐modification (RM) system was identified based on homology to type III RM systems found in some Firmicutes (Su et al ., 1999). In contrast, plasmids pHHV216 and pHH1107 carried two orfs of unknown function in the corresponding region, as well as a putative integrase gene, which was homologous to that on the Acinetobacter plasmid pAV2 (Mengoni et al ., 2007).…”

Novel type I restriction specificities through domain shuffling of HsdS subunits in Lactococcus lactis

“…The presence of restriction/modification (R/M) systems in dairy starter bacteria, such as Lactococcus lactis, has industrial significance, as they can be exploited to control bacteriophage inhibition of dairy fermentations. These phage defence systems are widespread in Lactococcus (for a recent review, see Forde and Fitzgerald, 1999) and, while much of the early genetic and biochemical characterization of R/M systems in this genus was primarily associated with type II systems (Klaenhammer and Fitzgerald, 1994;Fitzgerald et al, 1995), more recent work has detected type I (Schouler et al, 1998a, b;McGrath et al, 1999;Seegers et al 2000) and type III systems (Su et al, 1999). Indeed type I systems are particularly widespread and are likely to contribute significantly to phage resistance in this genus (Schouler et al, 1998b).…”

BceS1, a new addition to the type III restriction and modification family

“…The seven conserved helicase motifs were observed in all the type III Res sequences ( Table 2) and it can be seen that the motifs indeed are very conserved. The reported type III LlaFI Res subunit in L. lactis [9] showed only weak similarity to these motifs. These results might indicate that there are various subgroups of the type III R/M system, the EcoP1, StyLT1, B. cereus system and the system in Pasteurella haemolytica [13], representing one subgroup and the system in HinfIII, LlaF1 and some of the Res enzymes in H. pylori representing another subgroup.…”

“…CFEs were isolated as described by Su et al [9], from the cultures in the late-exponential phase of B. cereus ATCC 10987.…”