BIochemical and Molecular Genetics on the Components of Respiratory Chain of Bacteria as a Juvenile Periodontal Pathogen.

放线菌是青少年牙周炎的病原之一。这种生物可以在好氧环境或厌氧环境中生长。一般来说，好氧生物利用好氧电子传递系统（呼吸链）产生的电化学电位作为生长的能量。在本项目中，我利用分子生物学和生化技术研究了a.a acuinomycetemcomkans呼吸成分，以阐明呼吸链的生理作用。最初，我纯化了NADH-泛醌氧化还原酶（NADH脱氢酶）和泛醇-氧氧化还原酶（泛醇氧化酶），几乎达到均匀性。纯化得到的NADH脱氢酶由单个多肽的同聚体组成，分子量为47000，表明该酶属于NDH-1型脱氢酶。泛醇氧化酶由分子量为50,000和29,000的两个亚基组成。还原-负氧化差谱和分子组成表明该氧化酶属于细胞色素bd型喹啉氧化酶。我通过比对其他细菌bd型氧化酶的氨基酸序列，获得了保守区域的信息。用PCR方法扩增了a亚基部分编码区域的DNA，序列数据表明与已发表的流感嗜血杆菌序列具有高度同源性。开放阅读框核苷酸全序列的测定正在进行中。我们发现了这种细菌呼吸链的独特抑制剂。这些单宁是从植物中分离出来的。对这些化合物的抑制作用机制进行了研究。

Acti no bacillus actinoinycetemcomitans is one of the etiological pathogens in juvenile periodontitis. This organism can grow in the aerobic atmosphere or anaerobic conditions. In general, aerobic organism uses the electrochemical potential produced by aerobic electron transferring system (respiratory chain) as energy for the growth. In this project, I studied the respiratory components of A.acuinomycetemcomkans by use of molecular biological and biochemical techniques in order to clarify the physiological role of the respiratory chain. Initially I purified almost to homogeneity the NADH-ubiquinone oxidoreductase (NADH dehydrogenase) and ubiquinol-oxygen oxidoreductase (ubiquinol oxidase). The purified NADH dehydrogenase was composed of homotrimer of single polypeptide with a molecular weight of 47,000, indicating that this enzyme belongs to NDH-1 type dehydrogenase. The ubiquinol oxidase was composed of two subunits with the molecular weights of 50,000 and 29,000. The results of reduced-minus-oxidized difference spectrum and the molecular composition suggest that this oxidase belongs to cytochrome bd type quinol oxidase. I obtained the information for the conserved regions, by alignment of the amino acid sequences of other bacterial bd type oxidases. By PCR methods, we amplified the DNA for the partial region encoding a part of subunit a. The DNA sequence data indicated the high homology to that of Huemophilus influenzae all sequence of which was already published. The determination of complete sequence of nucleotide of the open reading frame is now in progress.We found the unique inhibitors for the respiratory chain of this bacteria. These are tannins which was isolated from plant. The mechanism of the inhibition of these compounds has been studying.