Watanabe-Kato T, Hayashi J I, Terazawa Y, Hoover C I, Nakayama K, Hibi E, Kawakami N, Ikeda T, Nakamura H, Noguchi T, Yoshimura F
Department of Endodontics, School of Dentistry, Aichi-Gakuin University, Nagoya, 464, Japan.
Microb Pathog. 1998 Jan;24(1):25-35. doi: 10.1006/mpat.1997.0170.
Fimbriae are considered to be an important virulence factor of Porphyromonas gingivalis. In order to identify genes essential for fimbriation, other than fimA which encodes the major subunit protein of fimbriae, transposon mutagenesis and immunological screening techniques were used to isolate fimbria-deficient mutants. R751::*Omega4, a suicide vector that carries Tn4351, was transferred from Escherichia coli to P. gingivalis by conjugation. Twenty-two independent fimbria-deficient mutants were identified among the resulting transformants. Southern hybridization analysis with pBlue 4351, a transposon-specific probe, and R751 indicated that 45% of the mutants resulted from single transposon insertions and that the remaining 55% of the mutants resulted from cointegration of R751 sequences. Southern hybridization analysis with pUCBg12.1, a probe for the fimA region, indicated that nine of the mutants contained insertions within the 2.5 kb SacI DNA fragment of P. gingivalis that contains fimA, ORF1 (which encodes a 15 kDa protein), and the C-terminal portion of ORF5 (which encodes a 63 kDa protein). Polymerase chain reaction (PCR) analysis and further Southern hybridization analysis indicated that the insertion site(s) for all nine of these mutants was within the fimA gene. Southern hybridization analysis also indicated that the remaining thirteen mutants contained insertions somewhere outside the 10 kb fimA region. Analysis by pulsed field gel electrophoresis (PFGE) revealed that insertions for most of the thirteen mutants mapped to a 300 kb NotI fragment and are located at least approximately 200 kb away from fimA. These results identify genetic loci other than fimA, that are required for fimbriation of P. gingivalis. Future cloning and characterization of these genetic loci should be straightforward since they are now marked by antibiotic resistance genes carried by the transposon.
菌毛被认为是牙龈卟啉单胞菌的一个重要毒力因子。为了鉴定菌毛形成所必需的基因，除了编码菌毛主要亚基蛋白的fimA之外，还利用转座子诱变和免疫筛选技术分离菌毛缺陷型突变体。携带Tn4351的自杀载体R751::*Omega4通过接合作用从大肠杆菌转移至牙龈卟啉单胞菌。在所得的转化体中鉴定出22个独立的菌毛缺陷型突变体。用转座子特异性探针pBlue 4351和R751进行的Southern杂交分析表明，45%的突变体是由单个转座子插入导致的，其余55%的突变体是由R751序列的共整合导致的。用fimA区域的探针pUCBg12.1进行的Southern杂交分析表明，其中9个突变体在牙龈卟啉单胞菌2.5 kb的SacI DNA片段内含有插入序列，该片段包含fimA、ORF1（编码一种15 kDa的蛋白质）和ORF5的C末端部分（编码一种63 kDa的蛋白质）。聚合酶链反应（PCR）分析和进一步的Southern杂交分析表明，所有这9个突变体的插入位点都在fimA基因内。Southern杂交分析还表明，其余13个突变体在10 kb的fimA区域之外的某个位置含有插入序列。脉冲场凝胶电泳（PFGE）分析显示，这13个突变体中的大多数插入序列定位于一个300 kb的NotI片段，并且距离fimA至少约200 kb。这些结果鉴定出了除fimA之外的、牙龈卟啉单胞菌菌毛形成所需的基因位点。由于这些基因位点现在由转座子携带的抗生素抗性基因标记，因此未来对这些基因位点的克隆和表征应该很简单。
