Plasmid replication-associated single-strand-specific methyltransferases

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Pathogenic bacteria like Burkholderia cenocepacia and Escherichia coli harbor unique genes on plasmids.
• Unusual DNA methyltransferase (MTase) genes, M.BceJIII and M.EcoGIX, were identified on plasmids pBCA072 and pESBL, respectively.

Purpose of the Study:

• To investigate the enzymatic activity and in vivo behavior of M.BceJIII and M.EcoGIX.
• To understand the factors influencing the asymmetric DNA methylation patterns observed.
• To explore the role of M.EcoGIX in plasmid establishment and host interaction.

Main Methods:

• Pacific Biosciences Single-Molecule Real-Time (SMRT) sequencing was employed to analyze DNA methyltransferases.
• Artificial constructs were used to study enzyme activity.
• Genetic and biochemical assays were performed to assess enzyme function and interactions.
• Mating experiments with engineered plasmid derivatives were conducted.

Main Results:

• Both M.BceJIII and M.EcoGIX exhibit promiscuous N6-methyladenine (m6A) modification of single DNA strands in the SAY motif.
• In vivo, methylation is asymmetric, occurring on only one DNA strand and is incomplete (~40% modification).
• Enzyme activity is dependent on plasmid replication mode; DNA Polymerase I (PolI)-dependent origins support asymmetric modification, while PolI-independent origins do not.
• M.EcoGIX facilitates plasmid pESBL establishment by blocking restriction enzymes in an orientation-dependent manner.

Conclusions:

• A potential MTase-PolI complex may distinguish between different plasmid replication origins.
• Asymmetric single-strand DNA methylation by these MTases is influenced by replication machinery.
• M.EcoGIX plays a role in conjugal transfer and plasmid establishment by interacting with the host DNA replication and restriction systems.