The study on the conjugal transfer system of pheromone responsive plasmid in Enterococcus faecalis, pPD1.

1995年，用Tn917、Tn917lac和Tn916对pPD1进行了转座子诱变，以获得转移缺陷的插入突变体或构成交配聚集体的插入突变体，以确定与配偶转移和信息素反应有关的基因。测定了部分信息素应答基因的DNA序列，1996年测定了全部信息素应答基因的DNA序列，并在数据库中检索到与pPD1信息素应答基因在氨基酸序列上具有同源性的基因。其他信息素反应载体的信息素反应基因，pad1和pCF10。PPD1信息素应答基因的序列与pCF10相似，而与pAD1相似。PAD1控制区的所有基因都得到了较好的研究，其功能也越来越清晰。虽然在pCF10、PRGX中发现了TraA同源物，但它曾被认为是正调控因子，而TraA是负调节因子。本研究试图通过定点突变的方法来研究TraA基因在pPD1中的功能。通过聚合酶链式反应扩增出pPD1的TraA内区，并将其克隆到含有卡那霉素抗性基因的大肠杆菌载体中，使其在粪肠球菌中得到表达。通过同源重组将其整合到pPD1的TraA基因中，导致TraA基因失活。插入后的pPD1在交配期较短时，接合系统表现出结构性表达和较高的转移频率。这一结果表明，pPD1中的TraA和pAD1中的TraA也是负调控因子。综上所述，三个不同性质的信息素反应质粒在信息素反应系统中具有非常相似的基因组织和功能。此外，这些质粒很可能是从同一来源进化而来的。

In 1995, Transposon mutagenesis of pPD1 with Tn917, Tn917lac, and Tn916 was carried out to generate insertion mutants which were transfer-deficient or make mating aggregates constitutively, to identify genes responsible for conjugal transfer and pheromone response. A part of DNA sequence of pheromone responding genes was determined.In 1996, DNA sequence of all pheromone responding genes were determined and genes which had homology with pPD1 pheromone responding genes in amino acids sequence were searched in database. Pheromone responding genes of other pheromone responsive plasmids, pAD1 and pCF10. The order of pheromone responding genes of pPD1 was more similar to that of pCF10 than to that of pAD1.All genes in the control region of pAD1 was studied well and their functions were getting clear. Although traA homologue in pCF10, prgX was found, it once had been thought to be a positive regulator while traA was negative regulator. An attempt was made to understand the function of traA gene in pPD1 by site-directed mutagenesis. An internal region of traA of pPD1 was amplified by PCR and cloned into E.coli vector with knanamycin resistance gene which could be expressed in E.faecalis. This plasmid was integrated into traA gene of pPD1 by homologous recombination resulting in inactivation of traA gene. This pPD1 with the insertion showed constitutive expresssion of conjugation system and high transfer frequency during short mating period. This result indicated that traA in pPD1 was also negative regulator as well as that of pAD1.Summing up those results, three pheromone responsive plasmids with different characters were very similar in pheromone responding system, namely gene organization and their functions. In addition, it was quite likely that those plasmids had evolved from same origin.