Phase variation of Enterococcus faecalis pAD1 conjugation functions relates to changes in iteron sequence region

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• pAD1 is a 60 kb conjugative plasmid in Enterococcus faecalis, encoding hemolysin/bacteriocin.
• It mediates mating response to sex pheromone cAD1, facilitating efficient plasmid transfer.
• A reversible phase variation mechanism (Dryc/Dry+) controls conjugation functions, occurring at frequencies of 10^-4 to 10^-3.

Purpose of the Study:

• To elucidate the molecular basis of phase variation in pAD1 plasmid.
• To investigate the structural changes associated with the Dryc and Dry+ phenotypes.
• To understand the mechanism by which these changes affect plasmid conjugation.

Main Methods:

• Analysis of structural changes in the region between repA and repB in Dryc variants.
• Characterization of iteron clusters within this region.
• Introduction of iteron DNA on a multicopy plasmid into wild-type pAD1 derivatives to assess phenotype.

Main Results:

• Dryc variants show a 31- or 32-bp size increase in the repA-repB intergenic region.
• This increase involves one iteron cluster expanding from 13 to 17 copies.
• Introduction of increased iteron DNA into wild-type pAD1 induced the Dryc phenotype.

Conclusions:

• Structural alterations in iteron clusters within the repA-repB region are responsible for the Dryc phase variation phenotype.
• The expanded iteron region likely interferes with the binding of regulatory proteins (RepA/RepB) to traA.
• This interference may lead to the blocking of traA transcription, a key negative regulator of conjugation.