Cloning and characterization of the MamI restriction-modification system from Microbacterium ammoniaphilum in Escherichia coli

The genes encoding a class-IIN restriction-modification (R-M) system (MamI, sequence specificity [GRAPHICS] from Microbacterium ammoniaphilum have been cloned in Escherichia coli. The vector used for cloning was plasmid pUC18 modified by the inclusion of three MamI recognition sites. Recombinant clones containing the mamIM gene in its genomic context became fully methylated in vivo and remained completely resistant against digestion with the R . MamI restriction endonuclease (ENase). Determination of the nucleotide (nt) sequence revealed three open reading frames with lengths of 1089 bp (ORF1), 276 bp (ORFc) and 927 bp (ORF2). On the basis of expression and deletion experiments, the 1089-bp ORF1 was assigned to mamIM encoding the M . MamI DNA methyltransferase (MTase). By amino acid sequencing of the N terminus of R . MamI and comparison of the deduced nt sequence with ORF2, the 927-bp ORF2 was identified as the mamIR gene encoding R . MamI. The 276-bp ORFc, located between mamIR and mamIM, is part of the DNA sequence downstream from mamIM shown to be necessary for controlled mamIM expression.