The physical role of the proteases from a marine bacterium, Alteromonas sp. Strain 0-7

海洋细菌交替单胞菌蛋白酶的物理作用。

• 批准号: 15580069
• 负责人: MIYAMOTO Katsushiro
• 金额: $ 2.3万
• 依托单位: Osaka University of Pharmaceutical Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580069/
• 关键词: marine bacteria; response regulator; histidine kinase; chitin; protease; ArcA; ArcB

Within the extracellularproteases from the marine bacterium, Alteromonassp.strain O-7, AprIV is involved in the chitinolytic system. AprIV and chitinases (ChiA.ChiB,ChiC, and ChiD) were induced in the presence of N-acetylglucosamine (GlcNAc). To clarify the regulation of AprIV at the gene level, N-terminal amino acid sequence of the protein binding the upstream region of aprIV gene was determined. The result suggest that the regulatory protein is an anoxic redox control protein (ArcA), which is classified into the response regulator of ArcB/ArcA two-component signal transduction system. Thus, we cloned the areA and arcB gene of the strain, and expression plasmids ArcA and ArcB lacking the N-terminal membrane binding region (ArcBAN) were constructed by using the GST fusion protein expression vector pGEX-6P-1. The ArcA protein was transphosphorylated by the GstArcBΔN protein as a sensor kinase. Gel retardation assay and BIAcore analysis demonstrated that phosphorylated ArcA (ArcA-P) more tightly bound to the upstream region of aprIV and chiD genes compared to those of chiA,chiB,chiC, and cpb1 genes. These results suggest that ArcB/ArcA two-component signal transduction system regulates the expression of genes involved in chitin degradation system of the strain.

在来自海洋细菌Alteromonassp.菌株O-7的胞外蛋白酶中，AprIV参与几丁质分解系统。在N-乙酰葡糖胺（GlcNAc）存在下诱导AprIV和几丁质酶（ChiA、ChiB、ChiC和ChiD）。为了阐明AprIV在基因水平上的调控，测定了与aprIV基因上游区结合的蛋白质的N-末端氨基酸序列。结果表明，该调节蛋白是一种缺氧氧化还原控制蛋白（ArcA），属于ArcB/ArcA双组分信号转导系统的反应调节因子。因此，我们克隆了该菌株的areA和arcB基因，并利用GST融合蛋白表达载体pGEX-6P-1构建了缺失N端膜结合区（ArcBAN）的表达质粒ArcA和ArcB。ArcA蛋白被作为传感激酶的GstArcBΔN蛋白转磷酸化。凝胶阻滞试验和BIAcore分析表明，磷酸化的ArcA（ArcA-P）与aprIV和chiD基因的上游区域的结合比chiA，chiB，chiC和cpb 1基因的结合更紧密。这些结果表明，ArcB/ArcA双组分信号转导系统调控着该菌株几丁质降解系统相关基因的表达。