Zhang Changyi, She Qunxin, Bi Hongkai, Whitaker Rachel J
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Appl Environ Microbiol. 2016 May 2;82(10):3070-3081. doi: 10.1128/AEM.00455-16. Print 2016 May 15.
Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus
Currently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains.
冰岛硫化叶菌是研究古菌生物学以及利用功能群体基因组学将新生物学与进化生态学联系起来的模型。在本研究中，我们在冰岛硫化叶菌中开发了一种新的可反向选择的遗传标记，以扩展该物种的遗传工具箱。我们表明，冰岛硫化叶菌M.16.4对嘌呤类似物6-甲基嘌呤（6-MP）的抗性是由于由M164_0158（apt）编码的推定腺嘌呤磷酸核糖转移酶失活所致。通过构建无标记的α-淀粉酶缺失突变体，首次说明了apt基因作为一种新型可反向选择标记的应用。此外，6-MP反向选择特性被用于正向（功能丧失）突变试验，以揭示冰岛硫化叶菌M.16.4在apt位点的自发突变谱。此外，嗜热栖热菌中apt基因的普遍保守性表明，相同的策略可广泛应用于其他嗜热栖热菌模式生物。这些结果表明，apt位点代表了一种用于嗜热栖热古菌冰岛硫化叶菌遗传操作和序列分析的新工具。
目前，pyrEF/5-氟乳清酸（5-FOA）反向选择系统仍然是嗜热栖热菌遗传学中唯一的反向选择标记。由于研究界构建的大多数硫化叶菌突变体都来自缺乏pyrEF基因的遗传宿主，因此pyrEF/5-FOA系统不再可用于正向突变试验。为硫化叶菌模型展示的apt/6-MP反向选择系统使得再次研究使用pyrEF缺陷背景菌株构建的突变体中的突变谱成为可能。此外，额外的可反向选择标记将使我们能够进行更复杂的遗传研究，即研究染色体DNA转移机制并量化冰岛硫化叶菌菌株之间的重组频率。
