DEVELOPMENT OF A HOST-VECTOR SYSTEM FOR PERIODONTO-PATHOGENIC ANAEROBIC BACTERIA

牙龈卟啉单胞菌(P.G.)一直被普遍认为是不可能用质粒DNA转化的。在本研究中解释其原因是由于该物种中存在限制性内切酶(S)，因为当尝试使用从P.G.提取的质粒DNA时，转化是可能的。施特雷恩本人。在了解到这一点后，我们试图获得一个缺乏限制性内切酶的突变体，作为转化实验的良好受体菌株。为此，对经N-甲基-N‘-硝基-N-亚硝基(NTG)与Pe5-2 DNA诱变的菌株进行了转化试验，并在没有抗生素的情况下进行了转化子(红霉素抗性菌落)的培养，以允许质粒分离。通过这种方法，可以获得一些具有整合外源DNA能力的菌株。随后，我们进一步努力寻找能在P.G.中复制并稳定维持的质粒。细胞。已知的唯一可转移到P.G.的pE5-2。在该物种中，通过接合或转化的细胞是非常不稳定的；我们推测，Pe5-2的rep基因起源的蛋类杆菌与P.G.来自较近物种的质粒的rep基因可能会增加稳定性。在这方面，我们使用了几个重组质粒，它们是由从黑色素口腔厌氧菌中检测到的质粒片段与Pe5-2红霉素抗性片段连接而成的。其中，我们成功地找到了一种重组载体pYH400，它与解糖紫球藻的rep基因重组，并能在P.g.细胞中非常稳定地维持。它有可能成为该物种很好的克隆载体。

Porphyromonas gingivalis (P.g.) has been generally thought that its transformation by plasmid DNA is impossible. The reason for it was elucidated in this study to be attributable to the presence of restriction enzyme (s) in this species, because transformation was possible when it was tried using plasmid DNAs extracted from the P.g. straine itself. Upon this knowlege, we then tried to obtain a mutant that lacks the restriction enzyme, usable as a good recipient strain for transformation experiments. For this, trials of transformation of strains that had been mutagenized with N-methyle-N'-nitro-N-nitrosoguanidin (NTG) with pE5-2 DNA were performed, and the transformants (erythromycin resistant colonies) thus obtained were cultured in the absence of the antibiotic to allow the plasmid segregation. By this means some strains that exhibited a capacity of incorporating foreign DNAs could be obtained. Subsequently, we made further efforts to find plasmids that can replicate and be stably maintained in P.g. cells. The only known plasmid pE5-2 that can be transferred into P.g. cells by either conjugation or transformation is extremely unstable in this species ; we presume that Bacteroides eggerthii, in which the rep gene of pE5-2 originated, is rather distant from P.g. A rep gene from a plasmid of a closer species would likely increase the stability. In this respect, we employed several recombinant plasmids that were constructed by ligating fragments of the plasmids detected from the black-pigmented oral anaerobic species and the erythromycin-resistnce fragment of pE5-2. Among them, we could successfuly found a plasmid, pYH400, a recombinant with the rep gene of a plasmid from Porphyromonas asaccharolytica, which could be maintained in the P.g.cells very stably. It would possibly be usable as a good cloning vector for this species.