Functional localization of Enterococcus faecalis pheromone receptor TraA.

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1)Functional localization of TraA of Enterococcus pheromone responsive conjugative plasmid (EPRCP) was investigated using its binding specificity to corresponding peptide pheromones and DNA fragments, and with chimera or mosaic TraA proteins in vitro. DPAC (DNA associated Protein-tag Affinity Chromatography) and PPAC (peptide associated Protein-tag Affinity Chromatography) were used for the assay.2)Binding sites (DNA) of pAD1 TraA were reinvestigated using PCR amplified DNA fragments and DPAC. The originally reported DNA binding site which was determined by DNA foot-printing did not bind TraA by itself and required spare binding site for binding. Two more DNA binding sites were identified and each of them could bind TraA by itself.3)Clinically isolated vancomycin resistant enterococci were investigated for the plasmid contents. It contained several plasmids and among the plasmid two EPRCP were identified. One of them was seemingly integrated to chromosome of the strain and became evident in transconjugants. Epidemiological study using synthetic pheromones revealed that numerous VREs were harboring EPRCPs of known class.4)With an aim to investigate relationship between conjugation and replication, RepA and RepB of pAD1 were purified and tested for the interaction with TraA. Not obvious positive results were obtained, however the DNA binding properties of RepA and RepB were revealed. The property of pAD1 RepA and the replication of pAD1 was elucidated. (in collaboration with Maria Victoria Francia and Don B. Clewell.)