Cleavage and Rejoining of DNA in the Shufflon-Specific Recombinase

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The pilV gene, the last gene of the pil operon of plasmid R64 is under the control of shufflon multiple inversion. R64 shufflon consists of four DNA segments, separated and flanked by seven sfx sequences. Site-specific recombinations mediated by the rci product occur between any inverted, sfx sequences, resulting. in inversions of the four segments independently or in groups. Thus the C-terminal segments of PilV adhesins interconvert among seven types, which determine the recipient specificity in R64 liquid matings.Rci protein was overproduced and purified. Specific cleavages were introduced into the sfx sequence when a sfx DNA fragment was incubated with Rci. Cleavages occurred in the form of a 5' protruding 7-bp staggered cut, suggesting that DNA cleavage and rejoining in the shufflon recombination take place at these positions. The sfx sequences were shown to consist of central 7-bp spacer sequence and left and right 12-bp arms, where the sfx left arm sequence are not conserved. Rci tein was shown to bind to entire sfx sequences, suggesting that one Rci molecule binds to the conserved sfx right arm in a sequence-specific manner and the second Rci molecule binds to the sfx left arm in a non-sequence-specific manner. Different sfx left arm sequences exhibited different inversion frequencies. When a symmetric sfx sequence, where the left and right arm sequences are the same, was constructed, recombination between symmetric sfx sequences in direct orientation was observed. Asymmetry of the sfx sequence may be the reason why Rci protein acts on only the inverted sfx sequences. R64 liquid matings using various waa mutants of E. coli K-12 and S. enterica serovar Typhimurium LT2 as recipient strains indicated that the specific receptors for the PilV adhesins are LPS molecules. Binding of PilV adhesins to respective LPS molecules was demonstrated.