Characterization of an active partition system for the Enterococcus faecalis pheromone-responding plasmid pAD1

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Enterococcus faecalis plasmid pAD1 is a conjugative plasmid involved in virulence.
• Previous studies identified regions for pAD1 replication and inheritance, but control mechanisms remain unclear.
• The pAD1 replication initiator (RepA) and origin (oriV) have been characterized.

Purpose of the Study:

• To investigate the roles of repB and repC genes in the maintenance of Enterococcus faecalis plasmid pAD1.
• To characterize the function of the iteron region and its interaction with RepB and RepC proteins.
• To elucidate the molecular mechanisms underlying pAD1 plasmid stability.

Main Methods:

• Mutagenesis and trans-complementation experiments were used to assess the function of RepB and RepC.
• DNA mobility shift and DNase I footprinting assays were employed to study protein-DNA interactions.
• The role of the iteron region as a centromere-like site was investigated.

Main Results:

• RepB and RepC proteins are essential for maximal stabilization of the pAD1 plasmid.
• The iteron region, comprising 25 direct repeats, functions as a centromere-like site for pAD1.
• RepC binds sequence-specifically to the iteron repeats, and RepB modulates this binding in an ATP-dependent manner.

Conclusions:

• RepB and RepC are key determinants for pAD1 plasmid stability in Enterococcus faecalis.
• The RepC-iteron interaction, modulated by RepB and ATP, is critical for pAD1 inheritance.
• This study provides new insights into the molecular mechanisms of low-copy-number plasmid maintenance.