Molecular characterization and regulatory analysis of dnaK operon of halophilic lactic acid bacterium Tetragenococcus halophila
Molecular characterization and regulatory analysis of dnaK operon of halophilic lactic acid bacterium Tetragenococcus halophila
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DOI：
10.1263/jbb.93.388
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发表时间：
2002-04-01
影响因子：
2.8
通讯作者：
Ishizaki, A
中科院分区：
文献类型：
--
作者：
Fukuda, D;Watanabe, M;Ishizaki, A
我们克隆并鉴定了嗜盐四联球菌 JCM5888 的 dnaK 操纵子。克隆片段的核苷酸序列分析表明dnaK操纵子由四个开放阅读框组成，组织结构为hrcA-grpE-dnaK-dnaJ。在 hrcA 上游区域发现了两个调节性 CIRCE（控制伴侣表达反向重复）元件。 T halophila dnaK 编码 618 个氨基酸的蛋白质，计算分子量为 67 kDa。推导的嗜盐菌DnaK氨基酸序列与乳酸乳球菌、清酒乳杆菌和枯草芽孢杆菌相应的DnaK同源物的氨基酸序列高度相似。使用 pET 表达系统，嗜盐杆菌 DnaK 在大肠杆菌中过表达，并且发现纯化的 DnaK 具有 ATP 酶和重折叠活性。 Northern杂交分析表明，dnaK基因的转录是由热休克诱导的，并且检测到多个转录本，其中包括最大大小为4.9-kb的四顺反子mRNA，其对应于完整dnaK操纵子的转录本。在 3-4 M 的高 NaCl 浓度下，dnaK 转录物的数量增加了约 3.5 倍，但在相同的 KCl 浓度下则没有增加。这些结果表明，克隆的 DnaK 作为功能性分子伴侣，在盐度适应中发挥重要作用。
We have cloned and characterized the dnaK operon of Tetragenococcus halophila JCM5888. Nucleotide sequence analysis of cloned fragments showed that the dnaK operon consists of four open reading frames with the organization hrcA-grpE-dnaK-dnaJ. Two regulatory CIRCE (Controlling Inverted Repeat of Chaperone Expression) elements were identified in the region upstream of hrcA. The T halophila dnaK encoded a protein of 618 amino acids with a calculated molecular mass of 67 kDa. The deduced amino acid sequence of T halophila DnaK showed high similarities with those of the corresponding DnaK homologues of Lactococcus lactis, Lactobacillus sakei and Bacillus subtilis. Using a pET expression system, the T halophila DnaK was overexpressed in Escherichia coli and the purified DnaK was found to exhibit ATPase and refolding activities. Northern hybridization analysis revealed that the transcription of the dnaK gene was induced by heat shock, and several transcripts were detected including a tetra-cistronic mRNA with a maximum size of 4.9-kb which corresponds to the transcript of the complete dnaK operon. The amount of dnaK transcripts increased about 3.5-fold at high NaCl concentration of 3-4 M, but not at the same KCl concentrations. These results suggest that the cloned DnaK acts as a functional molecular chaperone and plays an important role in salinity adaptation.