Lee Seok-Woo, Sabet Mojgan, Um Heung-Sik, Yang Jun, Kim Hyeong C, Zhu Weidong
Division of Periodontics, School of Dental and Oral Surgery, Columbia University, 630 W. 168th Street, PH-7E-110, New York, New York 10032, USA.
Gene. 2006 Apr 12;371(1):102-11. doi: 10.1016/j.gene.2005.11.027. Epub 2006 Feb 20.
A newly emerged periodontopathic pathogen Tannerella forsythia (formerly Bacteroides forsythus), a Gram-negative, filament-shaped, strict anaerobic, non-pigmented oral bacterium, possesses a surface (S-) layer. In our previous studies, the S-layer has been isolated, and shown to mediate hemagglutination, adhesion/invasion of epithelial cell, and murine subcutaneous abscess formation. In the present study, biochemical and molecular genetic characterization of the S-layer are reported. Amino acid sequencing and mass spectrometry indicated that the S-layer is composed of two different proteins, termed 200 and 210 kDa proteins. It was also shown that these proteins are glycosylated. The genes encoding the core proteins of these glycoproteins, designated as tfsA and tfsB, have been identified in silico, cloned, and their sequences have been determined. The tfsA (3.5 kb) and tfsB (4.1 kb) genes are located in tandem, and encode for 135 and 152 kDa proteins, respectively. An apparent discrepancy in molecular weights, 135 vs. 200 kDa and 152 vs. 210 kDa, is accounted for carbohydrate residues attached to the core proteins. Amino acid sequence comparison exhibited a 24% similarity between the 200 and 210 kDa proteins. Further sequence analyses showed that TfsA and TfsB possess putative signal peptide sequences with cleavage sites at alanine residues, and transmembrane domains on the C-terminal region. Northern blot and RT-PCR analyses confirmed an operon structure of tfsAB, suggesting co-regulation of these genes in producing the S-layer. Putative promoter sequences and transcription termination sequences for this operon have also been identified. Comparison with database indicates that the S-layer of T. forsythia has a unique structure exhibiting no homology to other known S-layers of prokaryotic organisms. The present study shows that the T. forsythia S-layer is very unique, since it appears to be composed of two large glycoproteins, and it does not reveal any homology to other known S-layer proteins or glycoproteins of prokaryotic organisms.
一种新出现的牙周病原菌福赛斯坦纳菌（原名福赛拟杆菌），是一种革兰氏阴性、丝状、严格厌氧、无色素的口腔细菌，具有表面（S-）层。在我们之前的研究中，S-层已被分离出来，并显示出介导血凝、上皮细胞黏附/侵袭以及小鼠皮下脓肿形成的作用。在本研究中，报告了S-层的生化和分子遗传学特征。氨基酸测序和质谱分析表明，S-层由两种不同的蛋白质组成，分别称为200 kDa和210 kDa蛋白质。还表明这些蛋白质是糖基化的。在计算机上已鉴定出编码这些糖蛋白核心蛋白的基因，分别命名为tfsA和tfsB，进行了克隆并确定了其序列。tfsA（3.5 kb）和tfsB（4.1 kb）基因串联排列，分别编码135 kDa和152 kDa的蛋白质。分子量上明显的差异，即135 kDa与200 kDa以及152 kDa与210 kDa，是由附着在核心蛋白上的碳水化合物残基造成的。氨基酸序列比较显示200 kDa和210 kDa蛋白质之间有24%的相似性。进一步的序列分析表明，TfsA和TfsB具有推定的信号肽序列，在丙氨酸残基处有切割位点，并且在C端区域有跨膜结构域。Northern印迹和RT-PCR分析证实了tfsAB的操纵子结构，表明这些基因在产生S-层过程中存在共同调控。还鉴定出了该操纵子的推定启动子序列和转录终止序列。与数据库比较表明，福赛斯坦纳菌的S-层具有独特的结构，与原核生物其他已知的S-层没有同源性。本研究表明，福赛斯坦纳菌的S-层非常独特，因为它似乎由两种大的糖蛋白组成，并且与原核生物其他已知的S-层蛋白或糖蛋白没有任何同源性。
