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Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

机译:使用基于抗药性的宿主标记系统破坏嗜热古细菌中糖转运蛋白基因簇

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摘要

We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmgTk) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apuTk) or a gene cluster which includes apuTk and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 μM simvastatin were isolated. The transformants exhibited growth in the presence of 20 μM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmgTk locus when the endogenous hmgTk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmgPf) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ΔapuTk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that ApuTk is a major polysaccharide-degrading enzyme in T. kodakaraensis.
机译:我们已经开发了一种使用抗生素辛伐他汀的超嗜热古生嗜热球菌嗜热球菌的基因破坏系统,该融合基因旨在利用谷氨酸脱氢酶启动子过表达3-羟-3-甲基戊二酰辅酶A(HMG-CoA)还原酶基因(hmgTk)。使用该系统,我们破坏了柯达氏锥虫淀粉酶基因(apuTk)或包括apuTk和编码假定糖转运蛋白成分的基因的基因簇。将干扰质粒引入野生型柯达氏疟原虫KOD1细胞,并分离出对4μM辛伐他汀有抗性的转化体。转化子在20μM辛伐他汀存在下表现出生长,并且我们观察到细胞内HMG-CoA还原酶活性增加了30倍。通过双交换重组的预期基因破坏发生在目标基因座,但是当使用内源性hmgTk基因时,我们还观察到了hmgTk基因座的重组事件。可以通过使用激烈热球菌(hmgPf)的相应基因或在转化前线性化质粒来避免这种情况。尽管两种基因破坏菌株在氨基酸或丙酮酸上均显示正常生长,但没有糖转运蛋白基因的细胞却无法在麦芽低聚糖或多糖上生长,这表明基因簇编码参与这些化合物摄取的唯一糖转运蛋白。 ΔapuTk菌株不能在支链淀粉上生长,而在直链淀粉上仅表现出低水平的生长,这表明ApuTk是柯达氏衣原体中主要的多糖降解酶。

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