Conchas R F, Carniel E
Unité d'Ecologie Bactérienne, Institut Pasteur, Paris, France.
Gene. 1990 Mar 1;87(1):133-7. doi: 10.1016/0378-1119(90)90505-l.
The various pathogenic Yersinia species are not readily and efficiently transformed by classical methods. For this reason, the electroporation technique was applied for genetic transformation of these species. Using optimal conditions, we were able to transform the six Yersinia strains studied with the two most widely used groups of plasmids: pSU2718 (a pACYC184 derivative) and pK19 (a pUC19 derivative). Only Yersinia enterocolitica (Y. e.) serotype 0:8 gave poor results (less than 5 x 10(2) transformants/microgram) DNA). Electrical transformation of the other species resulted in high efficiencies, up to 10(5) transformants/microgram DNA for Y. e. serotypes 0:3 and 0:9, 10(6) for Y. pseudotuberculosis and 10(7) for Y. pestis. The results varied for each strain with the type of plasmid used. Neither the introduced foreign plasmid nor the resident 72-kb virulence plasmid underwent detectable deletions. Transformation was most efficient with supercoiled DNA, decreasing by one and four orders of magnitude for relaxed circular and linearized plasmids, respectively. The ability to easily and efficiently transfer plasmid DNA via electroporation will greatly facilitate the application of recombinant DNA technology for direct cloning and analysis of significant genes into Yersinia.
各种致病性耶尔森氏菌属物种不易通过传统方法进行有效转化。因此，电穿孔技术被应用于这些物种的遗传转化。在优化条件下，我们能够用两组最常用的质粒对所研究的六种耶尔森氏菌菌株进行转化：pSU2718（pACYC184衍生物）和pK19（pUC19衍生物）。只有小肠结肠炎耶尔森氏菌（Y. e.）血清型0:8的转化效果较差（每微克DNA的转化子少于5×10²个）。其他物种的电转化效率很高，小肠结肠炎耶尔森氏菌血清型0:3和0:9每微克DNA的转化子高达10⁵个，假结核耶尔森氏菌为10⁶个，鼠疫耶尔森氏菌为10⁷个。每个菌株的结果因所用质粒类型而异。导入的外源质粒和常驻的72 kb毒力质粒均未发生可检测到的缺失。超螺旋DNA的转化效率最高，松弛环状质粒和线性化质粒的转化效率分别降低一个和四个数量级。通过电穿孔轻松高效地转移质粒DNA的能力将极大地促进重组DNA技术在耶尔森氏菌中直接克隆和分析重要基因的应用。
