Jonuscheit Melanie, Martusewitsch Erika, Stedman Kenneth M, Schleper Christa
Institute of Microbiology and Genetics, Darmstadt University of Technology, Schnittspahnstr. 10, Germany.
Mol Microbiol. 2003 Jun;48(5):1241-52. doi: 10.1046/j.1365-2958.2003.03509.x.
Sulfolobus solfataricus has developed into an important model organism for molecular and biochemical studies of hyperthermophilic archaea. Although a number of in vitro systems have been established for the organism, efficient tools for genetic manipulations have not yet been available for any hyperthermophile. In this work, we have developed a stable and selectable shuttle vector based on the virus SSV1 of Sulfolobus shibatae. We have introduced pUC18 for propagation in Escherichia coli and the genes pyrEF coding for orotidine-5'-monophosphate pyrophosphorylase and orotidine-5'-monophosphate decarboxylase of Sulfolobus solfataricus as selectable marker to complement pyrimidine auxotrophic mutants. Furthermore, the beta-galactosidase gene (lacS) was introduced into this vector as a reporter under the control of the strong and heat-inducible promoter of the Sulfolobus chaperonin (thermosome). After transformation of a S. solfataricus pyrEF/lacS double mutant, the vector was found to reside as a single-copy vector, stably integrated into the host chromosome via the site-specific recombination system of SSV1. Specific beta-galactosidase activities in transformants were found to be fourfold higher than in wild-type S. solfataricus cells, and increased to more than 10-fold after heat shock. Greatly increased levels of lacS mRNA were detected in Northern analyses, demonstrating that this reporter gene system is suitable for the study of regulated promoters in Sulfolobus and that the vector can also be used for the high-level expression of genes from hyperthermophilic archaea.
嗜热栖热菌已发展成为嗜热古菌分子和生化研究的重要模式生物。尽管已为该生物体建立了许多体外系统，但尚未有任何嗜热菌可利用的高效遗传操作工具。在这项工作中，我们基于柴田硫化叶菌的病毒SSV1开发了一种稳定且可选择的穿梭载体。我们引入了pUC18以便在大肠杆菌中繁殖，并引入了嗜热栖热菌的乳清苷-5'-单磷酸焦磷酸化酶和乳清苷-5'-单磷酸脱羧酶的编码基因pyrEF作为选择标记，以补充嘧啶营养缺陷型突变体。此外，β-半乳糖苷酶基因（lacS）在嗜热栖热菌伴侣蛋白（热体）的强热诱导启动子的控制下作为报告基因被引入该载体。在转化嗜热栖热菌pyrEF/lacS双突变体后，发现该载体以单拷贝载体形式存在，通过SSV1的位点特异性重组系统稳定整合到宿主染色体中。发现转化体中的特异性β-半乳糖苷酶活性比野生型嗜热栖热菌细胞高四倍，热休克后增加到十倍以上。在Northern分析中检测到lacS mRNA水平大幅增加，表明该报告基因系统适用于研究嗜热栖热菌中受调控的启动子，并且该载体还可用于嗜热古菌基因的高水平表达。
