LoVullo Eric D, Molins-Schneekloth Claudia R, Schweizer Herbert P, Pavelka Martin S
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Microbiology (Reading). 2009 Apr;155(Pt 4):1152-1163. doi: 10.1099/mic.0.022491-0.
Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of F. tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first system is based upon the ability of transposon Tn7 to insert in both a site- and orientation-specific manner at high frequency into the attTn7 site located downstream of the highly conserved glmS gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of unmarked elements with the blaB gene, encoding a beta-lactamase, resulting in the replacement of blaB with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel d-glutamate auxotroph of F. tularensis LVS, created using an improved sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of F. tularensis in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.
土拉弗朗西斯菌是一种苛养革兰氏阴性菌，可引发人畜共患疾病兔热病。由于操控这种高致病性生物体存在困难且缺乏遗传工具，对土拉弗朗西斯菌的生物学和分子发病机制的研究受到了限制。然而，最近的进展极大地提高了研究人员对该生物体进行基因操作的能力。为了扩展分子工具箱，我们开发了两个系统，以单拷贝形式将遗传元件稳定整合到土拉弗朗西斯菌基因组中。第一个系统基于转座子Tn7能够以位点和方向特异性的方式高频插入高度保守的glmS基因下游的attTn7位点。第二个系统由一个基于sacB的自杀质粒组成，用于将无标记元件与编码β-内酰胺酶的blaB基因进行等位基因交换，从而导致blaB被该元件取代并失去氨苄青霉素抗性。为了测试这些新工具，我们用它们来补充使用改进的基于sacB的等位基因交换质粒创建的土拉弗朗西斯菌LVS新型d-谷氨酸营养缺陷型。这些新系统将有助于在兔热病生物学研究中对土拉弗朗西斯菌进行基因操作，特别是在使用多拷贝质粒或抗生素标记可能不合适的情况下。
