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

1995年，用Tn917、Tn917lac和Tn916对pPD1进行转座子诱变，产生转移缺陷或组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年测定了所有信息素应答基因的DNA序列，并在数据库中检索氨基酸序列中与pPD1信息素应答基因同源的基因。其他信息素响应质粒的信息素响应基因pAD1和pCF10。与pAD1相比，pPD1的信息素响应基因顺序与pCF10更为相似。pAD1控制区的所有基因都得到了很好的研究，它们的功能也越来越清楚。虽然在pCF10中发现了traA的同源物prgX，但曾经认为它是正调控物，而traA是负调控物。试图通过定点诱变了解traA基因在pPD1中的功能。通过PCR扩增pPD1的traA内部区域，并将其克隆到含有克那霉素耐药基因的大肠杆菌载体中，该载体可在粪肠杆菌中表达。该质粒通过同源重组整合到pPD1的traA基因中，导致traA基因失活。插入后的pPD1具有偶联系统的本构表达和短交期内高的传递频率。这表明pPD1中的traA与pAD1一样也是负调节因子。综上所述，三种不同性状的信息素响应质粒在信息素响应系统，即基因组织及其功能上非常相似。此外，这些质粒很可能是从同一个起源进化而来的。

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.