Unique Plasmids Generated via pUC Replicon Mutagenesis in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426
Unique Plasmids Generated via pUC Replicon Mutagenesis in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426
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DOI：
10.1128/aem.01574-15
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发表时间：
2015-11-01
影响因子：
4.4
通讯作者：
Suzuki, Hirokazu
中科院分区：
文献类型：
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作者：
Kobayashi, Jyumpei;Tanabiki, Misaki;Suzuki, Hirokazu
质粒pGKE 75-cat（A138 T）包含pUC 18和编码具有A138 T氨基酸置换的热稳定氯霉素乙酰转移酶（CAT（A138 T））的cat（A138 T）基因，用作大肠杆菌-嗜热土芽孢杆菌穿梭质粒，其赋予G.嗜碱菌HTA 426。本研究检测了pGKE 75-cat（A138 T）在易错嗜热菌中的热适应定向诱变，产生了突变质粒pGKE 75（α β）-cat（A138 T），该突变质粒在65 ℃下导致了大量氯霉素抗性。pGKE 75（α β）-cat（A138 T）在cat（A138 T）基因中不含突变，但在pUC复制子中有两个突变，尽管该复制子在G.嗜碱菌生物化学表征表明，pGKE 75（α β）-cat（A138 T）赋予的有效氯霉素抗性可归因于在不完全形式的pGKE 75（α β）-cat（A138）T减少后细胞内CAT（A138 T）和乙酰辅酶A的增加。不完整质粒的减少可能是由于从突变型pUC复制子转录的RNA种类优化了质粒复制，这些RNA种类实际上是在G.嗜碱菌值得注意的是G.用pGKE 75（α β）-cat（A138 T）在60 ℃下用氯霉素选择转化嗜碱杆菌。此外，带有这两个突变的pUC 18衍生物在E.大肠杆菌中的高拷贝数，不依赖于培养温度和高质粒稳定性。由于这些特性在已知质粒中尚未观察到，因此这些结果扩展了G。kaustophilus和E.杆菌
The plasmid pGKE75-cat(A138T), which comprises pUC18 and the cat(A138T) gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CAT(A138T)), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-cat(A138T) in an error-prone thermophile, generating the mutant plasmid pGKE75(alpha beta)-cat(A138T) responsible for substantial chloramphenicol resistance at 65 degrees C. pGKE75(alpha beta)-cat(A138T) contained no mutation in the cat(A138T) gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75(alpha beta)-cat(A138T) is attributable to increases in intracellular CAT(A138T) and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75(alpha beta)-cat(A138)T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75(alpha beta)-cat(A138T) using chloramphenicol selection at 60 degrees C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli.