Unidirectional theta replication of the structurally stable Enterococcus faecalis plasmid pAM beta 1

Area of Science:

• Bacterial genetics
• Molecular biology
• Plasmid replication

Background:

• Many bacterial replicons are poorly understood due to challenges in identifying their replication origins.
• Plasmid pAM beta 1 is a key replicon in Gram-positive bacteria, but its origin of replication requires precise characterization.

Purpose of the Study:

• To characterize and fine-map the replication origin of plasmid pAM beta 1.
• To investigate the utility of bacterial termination signals for localizing replication origins.
• To elucidate the replication mechanism of plasmid pAM beta 1.

Main Methods:

• Exploitation of the Bacillus subtilis termination signal, terC, in plasmid derivatives.
• Analysis of replication intermediates using two-dimensional gel electrophoresis.
• Single-nucleotide precision mapping of replication endpoints.

Main Results:

• Accumulation of theta-form molecules with invariant endpoints in terC-containing derivatives.
• Precise localization of the plasmid origin and terC at single-nucleotide resolution.
• Confirmation of the plasmid origin's location through analysis of wild-type replication intermediates.

Conclusions:

• Plasmid pAM beta 1 replication proceeds unidirectionally via a theta mechanism.
• Bacterial termination signals are effective tools for localizing replication origins.
• Termination mechanisms in Bacillus subtilis may resemble those in Escherichia coli.
• Gram-positive bacteria harbor plasmids that replicate by theta mechanisms, in addition to rolling circle mechanisms.