Cloning and sequencing of LlaII restriction/modification genes from Lactococcus lactis and relatedness of this system to the Streptococcus pneumoniae DpnII system

Abstract

The natural 7.8-kb plasmid pSRQ700 was isolated from Lactococcus lactis subsp. cremoris DCH-4. It encodes a restriction/modification system named LlaII. When introduced into a phage-sensitive L. lactis strain, pSRQ700 confers strong phage resistance against the three most common lactococcal phage species, namely, 936, c2, and P335. The LlaII endonuclease was purified and found to cleave the palindromic sequence 5'-GATC-3'. It is an isoschizomer of Streptococcus pneumoniae DpnII. The plasmid pSRQ700 was mapped, and the genetic organization of LlaII was localized. Cloning and sequencing of the entire LlaII system allowed the identification of three open reading frames. The three genes (llaIIA, llaIIB, and llaIIC) overlapped and are under one putative promoter. A putative terminator was found at the end of llaIIC. The genes llaIIA and llaIIB coded for m6A methyltransferases, and llaIIC coded for an endonuclease. The LlaII system shares strong genetic similarities with the DpnII system. The deduced amino acid sequence of M.LlaIIA was 75% identical with that of M.DpnII, whereas M.LlaIIB was 88% identical with M.DpnA. However, R.LlaIII shared only 31% identity with R.DpnII.