Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus
Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus
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超嗜热嗜酸菌岛硫化叶菌辛伐他汀选择标记的开发
DOI：
10.1128/aem.06095-11
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发表时间：
2012-01-01
影响因子：
4.4
通讯作者：
Shen, Yulong
中科院分区：
文献类型：
--
作者：
Zheng, Tao;Huang, Qihong;Shen, Yulong
摘要：我们在这里报告了一种针对超嗜热crearchaeon Sulfolobus islandicus 的新型选择标记。标记盒由 sac7d 启动子和编码 3-羟基-3-甲基戊二酰辅酶 A (HMG-CoA) 还原酶 (P sac7d -hmg) 的 hmg 基因组成，赋予辛伐他汀对此 crenarchaeon 的抗性。将表达载体pSeSD的pyrEF基因替换为构建硫化叶菌表达质粒pSSRlacS、pSSRAherA和pSSRNherA的P sac7d -hmg，构建基本质粒载体pSSR。携带 pSSRlacS 的硫化叶菌转化体的表征表明，该质粒在选择下得到了适当的维持。带有 pSSRAherA 的细胞获得了 His6 标记的 HerA 解旋酶的高水平表达。随后利用两个有效选择标记（pyrEF 和 hmg）的建立进行遗传分析。利用pyrEF标记构建岛链霉菌herA半二倍体菌株，并以辛伐他汀筛选获得pSSRNherA转化子。虽然从herA半二倍体细胞产生的基因敲除（ΔherA）细胞在5-氟乳清酸（5-FOA）存在下无法形成集落，但突变细胞可以通过从质粒（pSSRNherA）表达基因来拯救，因为它们的转化体在含有5-FOA和辛伐他汀的固体培养基上形成集落。这表明 HerA 对于岛链霉菌的细胞活力至关重要。据我们所知，这是抗生素选择标记在超嗜热嗜酸菌和天脑谱系的遗传研究中的首次应用。
ABSTRACT We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P sac7d -hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P sac7d -hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His6-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEF and hmg) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.