Genetic Organization and Molecular Analysis of the Eco VIII Restriction-Modification System of Escherichia coli E1585-68 and Its Comparison with Isospecific Homologs

Abstract

ABSTRACT The Eco VIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the Eco VIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the Eco VIII R-M system was recently acquired by the genome. The 921-bp Eco VIII endonuclease (R · Eco VIII) gene ( ecoVIIIR ) encodes a 307-amino-acid protein with an M r of 35,554. The convergently oriented Eco VIII methyltransferase (M · Eco VIII) gene ( ecoVIIIM ) consists of 912 bp that code for a 304-amino-acid protein with an M r of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5′-AAGCTT-3′. Preparations of Eco VIII R-M enzymes purified to homogeneity were characterized. R · Eco VIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5′ protruding ends. M · Eco VIII functions as a monomer and modifies the first adenine residue at the 5′ end of the specific sequence to N 6 -methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the Hin dIII ( Haemophilus influenzae Rd) and Lla CI ( Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M · Eco VIII with M · Hin dIII and M · Lla CI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m 6 N -adenine β-class methyltransferases. The deduced amino acid sequence of R · Eco VIII shows weak homology with its two isoschizomers, R · Hin dIII (26%) and R · Lla CI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R · Eco VIII (D 108 X 12 DXK 123 ), as well as in the primary structures of R · Lla CI and R · Hin dIII. Polyclonal antibodies raised against R · Eco VIII did not react with R · Hin dIII, while anti-M · Eco VIII antibodies cross-reacted with M · Lla CI but not with M · Hin dIII. R · Eco VIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M · Eco VIII enzyme. The biological implications of this finding are discussed.