In vivo restriction by LlaI is encoded by three genes, arranged in an operon with llaIM, on the conjugative Lactococcus plasmid pTR2030.

The LlaI restriction and modification (R/M) system is encoded on pTR2030, a 46.2-kb conjugative plasmid from Lactococcus lactis. The llaI methylase gene, sequenced previously, encodes a functional type IIS methylase and is located approximately 5 kb upstream from the abiA gene, encoding abortive phage resistance. In this study, the sequence of the region between llaIM and abiA was determined and revealed four consecutive open reading frames (ORFs). Northern (RNA) analysis showed that the four ORFs were part of a 7-kb operon with llaIM and the downstream abiA gene on a separate transcriptional unit. The deduced protein sequence of ORF2 revealed a P-loop consensus motif for ATP/GTP-binding sites and a three-part consensus motif for GTP-binding proteins. Data bank searches with the deduced protein sequences for all four ORFs revealed no homology except for ORF2 with MerB, in three regions that coincided with the GTP-binding motifs in both proteins. To phenotypically analyze the llaI operon, a 9.0-kb fragment was cloned into a high-copy-number lactococcal shuttle vector, pTRKH2. The resulting construct, pTRK370, exhibited a significantly higher level of in vivo restriction and modification in L. lactis NCK203 than the low-copy-number parental plasmid, pTR2030. A combination of deletion constructions and frameshift mutations indicated that the first three ORFs were involved in LlaI restriction, and they were therefore designated llaI.1, llaI.2, and llaI.3. Mutating llaI.1 completely abolished restriction, while disrupting llaI.2 or llaI.3 allowed an inefficient restriction of phage DNA to occur, manifested primarily by a variable plaque phenotype. ORF4 had no discernible effect on in vivo restriction. A frameshift mutation in llaIM proved lethal to L. lactis NCK203, implying that the restriction component was active without the modification subunit. These results suggested that the LlaI R/M system is unlike any other R/M system studied to date and has diverged from the type IIS class of restriction enzymes by acquiring some characteristics reminiscent of type I enzymes.