Identification of the recognition sequence for the M.StyLTI methyltransferase of Salmonella typhimurium LT7: an asymmetric site typical of type-III enzymes

The StyLTI restriction-modification (R-M) system is encoded by chromosomal genes of Salmonella typhimurium LT7. We report here the identification of the nucleotide (nt) sequence methylated by the StyLTI modification methyltransferase (M.StyLTI). This enzyme was partially purified from an Escherichia coli strain expressing the cloned M.StyLTI-encoding gene, but lacking StyLTI restriction activity, and used to methylate DNAs of known sequence, using S-adenosyl-[methyl-3H]-methionine as the methyl donor. The [3H]methylated DNA was then digested with various endonucleases. Examination of labelled and unlabelled restriction fragments allowed us to map the M.StyLTI sites in perfectly defined regions of the DNA. Comparison of the nt sequences of DNA segments with or without M.StyLTI sites permitted us to identify the asymmetric and nondegenerate pentanucleotide, 5'-CAGAG-3', 3'-GTCTC-5' as the StyLTI sequence. M.StyLTI was found to methylate only the 3' A (see asterisk) in the upper strand of this sequence. Thus, M.StyLTI recognises and methylates the DNA in a manner very similar to that of the three known type-III MTases, M.EcoPI, M.EcoP15, and M.HinfIII. This strongly suggests that StyLTI constitutes a fourth type-III R-M system.