Molecular Characterization of the Lactococcus lactis Lla KR2I Restriction-Modification System and Effect of an IS 982 Element Positioned between the Restriction and Modification Genes

Abstract

The nucleotide sequence of the plasmid-encoded Lla KR2I restriction-modification (R-M) system of Lactococcus lactis subsp. lactis biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease ( llaKR2IR ) and methyltransferase ( llaKR2IM ) genes; located in the intergenic region is a copy of the insertion element IS 982 , whose putative transposase gene is codirectionally transcribed with llaKR2IM . The deduced sequence of the Lla KR2I endonuclease shared homology with the type II endonuclease Sau 3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5′ GATC 3′. In addition, M · Lla KR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M · Sau 3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M · Lla KR2I, all remained undigested after treatment with Sau 3AI despite the presence of multiple 5′ GATC 3′ sites. The combination of these data suggested that the specificity of the Lla KR2I R-M system was likely to be 5′ GATC 3′, with the cytosine residue being modified to 5-methylcytosine. The IS 982 element located within the Lla KR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked 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 IS 982 . Specific deletion of IS 982 and one of these direct repeats via a PCR strategy indicated that the Lla KR2I R-M determinants do not rely on elements within IS 982 for expression and that the efficiency of bacteriophage restriction was not impaired.