IN-VIVO RESTRICTION BY LLAI IS ENCODED BY 3 GENES, ARRANGED IN AN OPERON WITH LLAIM, ON THE CONJUGATIVE LACTOCOCCUS PLASMID-PTR2030

The LlaI restriction and modification (R/M) system is encoded on pTR20 30, a 46.2-kb conjugative plasmid from Lactococcus lactis. The llaI me thylase gene, sequenced previously, encodes a functional type IIS meth ylase and is located similar to 5 kb upstream from the abiA gene, enco ding abortive phage resistance. In this study, the sequence of the reg ion between llaIM and abiA was determined and revealed four consecutiv e open reading frames (ORFs). Northern (RNA) analysis showed that the four ORFs were part of a 7-kb operon with llaIM and the downstream abi A gene on a separate transcriptional unit. The deduced protein sequenc e of ORF2 revealed a P-loop consensus moth for ATP/GTP-binding sites a nd a three-part consensus motif for GTP-binding proteins, Data bank se arches with the deduced protein sequences for all four ORFs revealed n o homology except for ORF2 with McrB, in three regions that coincided with the GTP-binding motifs in both proteins. To phenotypically analyz e the llaI operon, a 9.0-kb fragment was cloned into a high-copy-numbe r lactococcal shuttle vector, pTRKH2. The resulting construct, pTRK370 , exhibited a significantly higher level of in vivo restriction and mo dification in L. lactis NCK203 than the low-copy-number parental plasm id, pTR2030. A combination of deletion constructions and frameshift mu tations indicated that the first three ORFs were involved in LlaI rest riction, and they were therefore designated llaI.1, llaI.2, and llaI.3 , Mutating llal.1 completely abolished restriction, while disrupting l laI.2 or llaI.3 allowed an inefficient restriction of phage DNA to occ ur, manifested primarily by a variable plaque phenotype. ORF4 had no d iscernible effect on in vivo restriction. A frameshift mutation in lla IM proved lethal to L. lactis NCK203, implying that the restriction co mponent was active without the modification subunit. These results sug gested 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 enzym es by acquiring some characteristics reminiscent of type I enzymes.