Abstract
BackgroundBacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.Methodology/Principal FindingsWe have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.Conclusions/SignificanceThe results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.
Highlights
Bacterial conjugation is a mechanism for horizontal gene transfer among bacteria
Optimization of the integration assay In earlier work, the ability of TrwC to catalyze site-specific integration was assayed by mobilization of a suicide plasmid to an oriTw-containing recipient strain, in a recA background [6]
The integrants are selected in Cm plates, the resistance conferred by the suicide plasmid, and integration subsequently confirmed by PCR amplification of a specific region of the cointegrate molecule with primers P1 and P2 (Fig. 1A)
Summary
Bacterial conjugation is a mechanism for horizontal gene transfer among bacteria. By this process, a DNA molecule of any origin and length can be transferred to a recipient cell, if it contains an origin of transfer (oriT); the conjugative machinery can be provided in trans. There are three functional modules in the conjugative machinery [5]: i) the relaxosome, a nucleoprotein complex required for plasmid DNA processing, which is related to rolling-circle replication systems. TrwC, the relaxase of conjugative plasmid R388, is able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering
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