Molecular characterization of the restriction endonuclease gene (scrFIR) associated with the ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503

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Abstract
Summary: The nucleotide sequence of the chromosomally encoded type II ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503 was completed. The ScrFI restriction endonuclease (ENase) has previously been shown to specifically recognize 5’ CCNGG 3’ sites, cleaving after the second cytosine and the degenerate central base. The ENase gene (scrFIR; 862 bp) was located between, and co-directionally transcribed with, two formerly characterized 5-methylcytosine methyltransferase genes, which encode proteins that independently confer protection against ScrFI digestion. scrFIR codes for a protein of 272 amino acids with a predicted molecular mass of 31470 Da, which agrees favourably with a previously estimated molecular mass of 34 kDa for this enzyme. The deduced sequence of this protein did not show any significant homology with known protein sequences, including the isoschizomeric SsoII ENase from ShigeIIa sonnei. The ENase gene was cloned and expressed in Escherichia coli and Lactococcus; however, no in vivo restriction of phage was observed, suggesting that expression of the ENase gene may be repressed, or that the appropriate expression signals may be absent in the cloned constructs. The ability of ScrFI to cleave non-canonically modified 5’ CCNGG 3’ sequences suggested that some ScrFI sites may require complex modifications to fully impair digestion by this enzyme.
Funding Information
  • National and European Union funds
  • Food Sub-Programmes of the Operational Programme for Industrial Development, which is administered by the Irish Department of Agriculture Food and Forestry
  • BIOTECH (BIO2-CT94-3055)
  • European Community BRIDGE (BIOT-CT91-0263[SSMA])