Evidence for Horizontal Transfer of the Eco T38I Restriction- Modification Gene to Chromosomal DNA by the P2 Phage and Diversity of Defective P2 Prophages in Escherichia coli TH38 Strains

Abstract

ABSTRACT A DNA fragment carrying the genes coding for a novel Eco T38I restriction endonuclease (R. Eco T38I) and Eco T38I methyltransferase (M. Eco T38I), which recognize G(A/G)GC(C/T)C, was cloned from the chromosomal DNA of Escherichia coli TH38. The endonuclease and methyltransferase genes were in a head-to-head orientation and were separated by a 330-nucleotide intergenic region. A third gene, the C. Eco T38I gene, was found in the intergenic region, partially overlapping the R. Eco T38I gene. The gene product, C. Eco T38I, acted as both a positive regulator of R. Eco T38I gene expression and a negative regulator of M. Eco T38I gene expression. M. Eco T38I purified from recombinant E . coli cells was shown to be a monomeric protein and to methylate the inner cytosines in the recognition sequence. R. Eco T38I was purified from E . coli HB101 expressing M. Eco T38I and formed a homodimer. The Eco T38I restriction (R)-modification (M) system (R-M system) was found to be inserted between the A and Q genes of defective bacteriophage P2, which was lysogenized in the chromosome at loc I, one of the P2 phage attachment sites observed in both E . coli K-12 MG1655 and TH38 chromosomal DNAs. Ten strains of E . coli TH38 were examined for the presence of the Eco T38I R-M gene on the P2 prophage. Conventional PCR analysis and assaying of R activity demonstrated that all strains carried a single copy of the Eco T38I R-M gene and expressed R activity but that diversity of excision in the ogr , D , H , I , and J genes in the defective P2 prophage had arisen.