Evidence for horizontal transfer of the EcoT38I restriction-modification gene to chromosomal DNA by the P2 phage and diversity of defective P2 prophages in Escherichia coli TH38 strains.
A DNA fragment carrying the genes coding for a novel EcoT38I restriction endonuclease (R.EcoT38I) and EcoT38I methyltransferase (M.EcoT38I), 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.EcoT38I gene, was found in the intergenic region, partially overlapping the R.EcoT38I gene. The gene product, C.EcoT38I, acted as both a positive regulator of R.EcoT38I gene expression and a negative regulator of M.EcoT38I gene expression. M.EcoT38I purified from recombinant E. coli cells was shown to be a monomeric protein and to methylate the inner cytosines in the recognition sequence. R.EcoT38I was purified from E. coli HB101 expressing M.EcoT38I and formed a homodimer. The EcoT38I 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 locI, 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 EcoT38I 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 EcoT38I 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.
Kita
Keiko
K
Department of Biotechnology, Tottori University, Tottori, Japan. kita@kais.kyoto-u.ac.jp
Kawakami
Hideaki
H
Tanaka
Hiroaki
H
D000595:Amino Acid Sequence; D017901:Bacteriophage P2 / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial; D021901:DNA, Intergenic; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D022761:Gene Transfer, Horizontal; D005798:Genes, Bacterial; D014644:Genetic Variation; D008242:Lysogeny; D008969:Molecular Sequence Data; D039002:Prophages / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D021901:DNA, Intergenic; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D015257:DNA-Cytosine Methylases; C473325:DNA modification methylase EcoT38I, E coli; D004262:DNA Restriction Enzymes; C473324:DNA restriction enzyme EcoT38I, E coli; D015252:Deoxyribonucleases, Type II Site-Specific