Klimpel K W, Clark V L
Department of Microbiology and Immunology, University of Rochester, School of Medicine and Dentistry, NY 14642.
J Dent Res. 1990 Sep;69(9):1567-72. doi: 10.1177/00220345900690090601.
Western blot analysis that used antisera to the E. coli core enzyme and sigma factors was used for examination of the RNA polymerase of Actinobacillus actinomycetemcomitans, Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis. Both antisera reacted with proteins in A. actinomycetemcomitans and S. mutans whole-cell extracts. Reactions were seen with some F. nucleatum proteins when the anti-core RNA polymerase antisera were used, but the cross-reacting proteins were not of an expected molecular weight for beta or beta'. No reaction with F. nucleatum proteins was seen when extracts were reacted with antisera to E. coli sigma factor. There were no cross-reacting proteins detected in P. gingivalis extracts with either antisera. These results suggest that E. coli RNA polymerase may not be sufficiently similar to P. gingivalis and F. nucleatum RNA polymerase for E. coli RNA polymerase to recognize P. gingivalis or F. nucleatum promoters. Partially purified P. gingivalis and F. nucleatum RNA polymerase exhibited a specificity for a P. gingivalis DNA template, while having a decreased activity from an E. coli DNA template. The antibiotic sensitivity profile of P. gingivalis and F. nucleatum RNA polymerase activity was shown to differ from that of E. coli, with these activities not being affected by rifampicin, streptovaricin, or streptolydigin. We conclude that the efficient cloning and expression of P. gingivalis and F. nucleatum genes in E. coli will require the use of promoter-containing expression vectors.
利用抗大肠杆菌核心酶和σ因子的抗血清进行的蛋白质印迹分析，用于检测伴放线放线杆菌、变形链球菌、具核梭杆菌和牙龈卟啉单胞菌的RNA聚合酶。两种抗血清均与伴放线放线杆菌和变形链球菌全细胞提取物中的蛋白质发生反应。当使用抗核心RNA聚合酶抗血清时，可观察到与一些具核梭杆菌蛋白质的反应，但交叉反应的蛋白质分子量并非β或β′的预期分子量。当提取物与抗大肠杆菌σ因子的抗血清反应时，未观察到与具核梭杆菌蛋白质的反应。在牙龈卟啉单胞菌提取物中，两种抗血清均未检测到交叉反应蛋白。这些结果表明，大肠杆菌RNA聚合酶与牙龈卟啉单胞菌和具核梭杆菌RNA聚合酶的相似性可能不足以使大肠杆菌RNA聚合酶识别牙龈卟啉单胞菌或具核梭杆菌的启动子。部分纯化的牙龈卟啉单胞菌和具核梭杆菌RNA聚合酶对牙龈卟啉单胞菌DNA模板具有特异性，而对大肠杆菌DNA模板的活性则降低。牙龈卟啉单胞菌和具核梭杆菌RNA聚合酶活性的抗生素敏感性谱显示与大肠杆菌不同，这些活性不受利福平、链黑菌素或链球菌溶素的影响。我们得出结论，要在大肠杆菌中高效克隆和表达牙龈卟啉单胞菌和具核梭杆菌基因，需要使用含启动子的表达载体。
