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

牙周病原性厌氧细菌宿主载体系统的开发

基本信息

批准号：
05671525
负责人：
YOSHIMOTO Hisashi
金额：
$ 1.15万
依托单位：
KANAGAWA DENTAL COLLEGE
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671525/
关键词：
Porphyromonas gingivalis Transformation Plasmid Restriction Endonuclease P.gingivalis

项目摘要

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.

牙龈卟啉单胞菌（P.g.）一般认为用质粒DNA转化它是不可能的。在本研究中阐明了其原因，可归因于该物种中存在限制性内切酶，因为当使用从P.g.自己紧张。在此基础上，我们试图获得一个缺乏限制酶的突变体，可作为转化实验的良好受体菌株。为此，用pE 5 -2 DNA转化已用N-甲基-N ′-硝基-N-亚硝基胍（NTG）诱变的菌株，并在不存在抗生素的情况下培养由此获得的转化体（红霉素抗性菌落）以允许质粒分离。通过这种方法，可以获得一些表现出整合外源DNA的能力的菌株。随后，我们做了进一步的努力，以找到质粒，可以复制和稳定地维持在P. g。细胞目前已知的唯一一个能转入P.g.细胞通过接合或转化是非常不稳定的，在这个物种中，我们推测，Bacteroides eggerthii，其中的rep基因的pE 5 -2的起源，是相当遥远的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，我们采用了几种重组质粒，这些质粒是通过连接从黑色着色的口腔厌氧菌中检测到的质粒片段和pE 5 -2的红霉素抗性片段而构建的。其中，我们成功地找到了一个质粒pYH 400，它是一个与解砷卟啉单胞菌质粒的rep基因重组的质粒，它能在P.g.细胞中非常稳定地维持。它可能是一个很好的克隆载体。