Su Hongsheng, Shao Zhongqi, Tkalec Lydia, Blain Françoise, Zimmermann Joe
IBEX Pharmaceuticals Inc., 5485 Pare, Montreal, Quebec H4P 1P7, Canada1.
Microbiology (Reading). 2001 Mar;147(Pt 3):581-589. doi: 10.1099/00221287-147-3-581.
Flavobacterium heparinum (now Pedobacter heparinus) is a Gram-negative soil bacterium which can produce yellow pigments. It synthesizes five enzymes that degrade glycosoaminoglycan molecules. The study of this unique bacterium has been limited by the absence of a genetic manipulation system. In this paper, the construction of a conjugation/integration plasmid system and a broad-host-range plasmid, both of which contain a F. heparinum functional selective marker created by placing the trimethoprim resistance gene, dhfrII, under the control of the hepA regulatory region is described. Both plasmids were introduced into F. heparinum by conjugation and/or electroporation, and trimethoprim resistant colonies were obtained. Fifty electroporants were obtained per microgram covalently closed circular plasmid DNA. The existence of integrated plasmid DNA was confirmed by Southern hybridization and PCR. The existence of a derivative of the broad-host-range plasmid pBBR1 in F. heparinum was demonstrated by plasmid digestion and Southern hybridization, and by transformation of Escherichia coli.
肝素黄杆菌（现称肝素Pedobacter菌）是一种革兰氏阴性土壤细菌，可产生黄色色素。它能合成五种降解糖胺聚糖分子的酶。由于缺乏遗传操作体系，对这种独特细菌的研究受到了限制。本文描述了一种接合/整合质粒系统和一种广宿主范围质粒的构建，这两种质粒都含有一个通过将甲氧苄啶抗性基因dhfrII置于hepA调控区域控制下而创建的肝素黄杆菌功能选择标记。两种质粒都通过接合和/或电穿孔导入肝素黄杆菌，并获得了对甲氧苄啶有抗性的菌落。每微克共价闭合环状质粒DNA可获得50个电穿孔转化子。通过Southern杂交和PCR证实了整合质粒DNA的存在。通过质粒酶切、Southern杂交以及转化大肠杆菌，证明了广宿主范围质粒pBBR1的衍生物在肝素黄杆菌中的存在。
