MOLECULAR CHARACTERIZATION OF THE LACTOCOCCUS-LACTIS LLAKR2I RESTRICTION-MODIFICATION SYSTEM AND EFFECT OF AN IS982 ELEMENT POSITIONED BETWEEN THE RESTRICTION AND MODIFICATION GENES

The nucleotide sequence of the plasmid-encoded LlaKR2I restriction-mod ification (R-M) system of Lactococcus lactis subsp. lactis biovar diac etylactis KR2 was determined. This R-M system comprises divergently tr anscribed endonuclease (llaKR2IR) and methyltransferase (llaKR2IM) gen es; located in the intergenic region is a copy of the insertion elemen t IS982, whose putative transposase gene is codirectionally transcribe d with llaKR2IM. The deduced sequence of the LlaKR2I endonuclease shar ed homology with the type II endonuclease Sau3AI and with the MutH mis match repair protein, both of which recognize and cleave the sequence 5' GATC 3'. In addition, M . LlaKR2I displayed homology with the 5-met hylcytosine methyltransferase family of proteins, exhibiting greatest identity with M . Sau3AI. Both of these proteins shared notable homolo gy throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which enc ode M LlaKR2I, all remained undigested after treatment with Sau3AI des pite the presence of multiple 5' GATC 3' sites. The combination of the se data suggested that the specificity of the LlaKR2I R-M system was l ikely to be 5' GATC 3', with the cytosine residue being modified to 5- methylcytosine. The IS982 element located within the LlaKR2I R-M syste m contained at its extremities two 16-bp perfect inverted repeats flan ked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the llaKR2IR gene, was shown to overlap the direct repeat sequence on the other side of I S982, Specific deletion of IS982 and one of these direct repeats via a PCR strategy indicated that the LlaKR2I R-M determinants do not rely on elements within IS982 for expression and that the efficiency of bac teriophage restriction was not impaired.