Functional localization of Enterococcus faecalis pheromone receptor TraA.

1)利用肠球菌信息素应答接合质粒（EPRCP）的TraA与相应肽信息素和DNA片段的结合特异性，以及与嵌合体或嵌合体TraA蛋白的体外结合特异性，研究了肠球菌信息素应答接合质粒（EPRCP）TraA的功能定位。采用DPAC（DNA associated Protein-tag Affinity Chromatography）和PPAC（peptide associated Protein-tag Affinity Chromatography）技术对pAD 1 TraA的结合位点进行了研究。最初报道的DNA结合位点是通过DNA足迹法确定的，它本身不与TraA结合，需要备用的结合位点才能结合。3）对临床分离的万古霉素耐药肠球菌进行质粒含量检测。它含有几个质粒，并在质粒中鉴定出两个EPRCP。其中一个似乎整合到菌株的染色体上，并在接合子中变得明显。利用人工合成的信息素进行的流行病学研究表明，许多VRE中含有已知类别的EPRCPs。4）为了研究接合与复制之间的关系，纯化了pAD 1的RepA和RepB，并检测了它们与TraA的相互作用。未得到明显的阳性结果，但显示了RepA和RepB的DNA结合特性。阐明了pAD 1 RepA的性质及复制。(in与Maria维多利亚Francia和Don B合作。Clewell.）

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.)