Structure and function of the shufflon in plasmid r64

Area of Science:

• Molecular Biology
• Microbial Genetics
• Bacterial Conjugation

Background:

• Site-specific recombination is crucial for prokaryotic biological diversity.
• The R64 plasmid's shufflon system, unlike typical inversion systems, utilizes seven recombination sites (sfx) and a recombinase (Rci) to invert four DNA segments.
• This inversion generates diverse pilV genes encoding adhesins that determine bacterial recipient specificity during liquid mating.

Purpose of the Study:

• To elucidate the mechanism of shufflon recombination mediated by Rci protein.
• To investigate the role of sfx sequence asymmetry in recombination frequency and specificity.
• To explore the function of the Rci C-terminal domain in binding and recombination.

Main Methods:

• Overexpression and purification of Rci protein from plasmid R64.
• In vitro recombination assays using purified Rci and shufflon DNA.
• Analysis of DNA cleavage patterns (staggered cuts) and sequence conservation within sfx sites.
• Construction and testing of artificial symmetric sfx sequences.
• Investigation of Rci binding to sfx sequences and the effect of the C-terminal domain.

Main Results:

• Shufflon recombination involves a 5' protruding 7-bp staggered cut within sfx sequences.
• sfx sequences are asymmetric, with conserved spacer and right arm sequences, but variable left arm sequences.
• Rci binds to sfx sites non-specifically to left arms and specifically to right arms; left arm sequence significantly impacts inversion frequency.
• Artificial symmetric sfx sequences exhibit the highest inversion frequency.
• Rci-dependent deletion occurs between directly oriented symmetric sfx sites, suggesting asymmetry prevents such recombination.
• The Rci C-terminal domain is not essential for recombination with symmetric sfx sites.

Conclusions:

• The asymmetry of R64 sfx sequences is critical for regulating shufflon inversion and preventing unwanted deletions.
• The Rci C-terminal domain likely mediates sequence-specific and non-specific binding to asymmetric sfx sites.
• Modifications to sfx sequences, particularly creating symmetry, can enhance shufflon inversion efficiency.