Teughels W, Sliepen I, De Keersmaecker S, Quirynen M, Lippmann J, Pauwels M, Fives-Taylor P
Catholic University Leuven, Department of Periodontology, Leuven, Belgium.
Oral Microbiol Immunol. 2005 Oct;20(5):274-81. doi: 10.1111/j.1399-302X.2005.00224.x.
BACKGROUND/AIMS: The development of an electro-transformation system and the construction of shuttle plasmids for Actinobacillus actinomycetemcomitans have enhanced the molecular analysis of virulence factors. However, inefficient transformation is frequently encountered. This study investigated the efficiency of electro-transformation and expression of Green Fluorescent Protein (GFP) in 12 different A. actinomycetemcomitans strains. The influence of the plasmid vector, serotype, and phenotype were the major factors taken into consideration.
Twelve serotyped A. actinomycetemcomitans strains were independently electro-transformed with two different Escherichia coli-A. actinomycetemcomitans shuttle plasmids (pVT1303 and pVT1304), both containing an identical ltx-GFPmut2 gene construct but a different backbone (pDMG4 and pPK1, respectively). The transformation efficiency, transformation frequency, and electro-transformation survival rate were determined by culture techniques. GFP expression was observed at the colony level by fluorescence microscopy.
All strains could be transformed with both plasmids. However, major differences were observed for the transformation efficiency, transformation frequency, and electro-transformation survival rate between strains. The data demonstrated that plasmid vector, serotype, and phenotype are key players for obtaining a successful transformation. An inverted relationship between the electro-transformation survival rate and tranformation frequency was also observed. GFP expression was also influenced by phenotype, serotype and plasmid vector.
The serotype of A. actinomycetemcomitans has an important influence on its survival after electro-transformation and on transformation frequency. The expression of GFP is strain and plasmid vector dependent.
背景/目的：放线共生放线杆菌电转化系统的开发以及穿梭质粒的构建增强了对毒力因子的分子分析。然而，经常会遇到转化效率低下的问题。本研究调查了12种不同的放线共生放线杆菌菌株中电转化效率和绿色荧光蛋白（GFP）的表达情况。质粒载体、血清型和表型的影响是主要考虑因素。
用两种不同的大肠杆菌-放线共生放线杆菌穿梭质粒（pVT1303和pVT1304）分别对12种血清型的放线共生放线杆菌菌株进行电转化，这两种质粒都含有相同的ltx-GFPmut2基因构建体，但骨架不同（分别为pDMG4和pPK1）。通过培养技术测定转化效率、转化频率和电转化存活率。通过荧光显微镜在菌落水平观察GFP表达。
所有菌株都能用这两种质粒进行转化。然而，菌株之间在转化效率、转化频率和电转化存活率方面存在显著差异。数据表明，质粒载体、血清型和表型是成功转化的关键因素。还观察到电转化存活率与转化频率之间呈负相关。GFP表达也受表型、血清型和质粒载体的影响。
放线共生放线杆菌的血清型对其电转化后的存活率和转化频率有重要影响。GFP的表达取决于菌株和质粒载体。
