The archaeal halophilic virus-encoded Dam-like methyltransferase M. phiCh1-I methylates adenine residues and complements dam mutants in the low salt environment of Escherichia coli

Mol Microbiol. 2000 Mar;35(5):1168-79. doi: 10.1046/j.1365-2958.2000.01786.x.

Abstract

The genome of the archaeal virus phiCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA. Virus particles contain several RNA species in sizes of 100-800 nucleotides. A fraction of phiCh1 genomes is modified within 5'-GATC-3' and related sequences, as determined by various restriction enzyme digestion analyses. High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6-methyladenosine. Genetic analyses and subsequent sequencing led to the identification of a DNA (N6-adenine) methyltransferase (mtase) gene. The protein product was designated M.phiCh1-I. By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the beta-subgroup of the (N6-adenine) methyltransferase class. The mtase gene of phiCh1 was classified as a 'late' gene, as determined by measuring the kinetics of mRNA and protein expression in N. magadii during the lytic cycle of phiCh1. After infection of cells, M.phiCh1-I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells. Consequently, only about 5% of the phiCh1 progeny genomes after infection of N. magadii carry the M.phiCh1-I methylation in contrast to 50% of virus genomes generated by induction of phiCh1-lysogenic N. magadii cells. Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo. Interestingly, it seemed to exhibit a higher sequence specificity in E. coli that resulted in adenine methylation exclusively in the sequence 5'-GATC-3'. Additionally, expression of M.phiCh1-I in dam- E. coli cells led to a complete substitution of the function of M. Dam in DNA mismatch repair.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Archaea / virology*
  • Bacteriophages / enzymology*
  • DNA Methylation
  • Escherichia coli / genetics*
  • Escherichia coli Proteins
  • Genetic Complementation Test*
  • Molecular Sequence Data
  • Osmolar Concentration
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Site-Specific DNA-Methyltransferase (Adenine-Specific) / chemistry
  • Site-Specific DNA-Methyltransferase (Adenine-Specific) / genetics
  • Site-Specific DNA-Methyltransferase (Adenine-Specific) / metabolism*
  • Sodium Chloride
  • Transcription, Genetic

Substances

  • Escherichia coli Proteins
  • Sodium Chloride
  • Dam methyltransferase
  • Site-Specific DNA-Methyltransferase (Adenine-Specific)
  • dam protein, E coli