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

在胞外蛋白酶从海洋细菌，互生单胞菌。菌株O-7， AprIV参与几丁质分解系统。AprIV和几丁质酶（中国）在n -乙酰氨基葡萄糖（GlcNAc）存在的情况下诱导ChiB、ChiC和ChiD。为了阐明在基因水平上对AprIV的调控，我们测定了与AprIV基因上游区域结合的蛋白n端氨基酸序列。结果表明，该调控蛋白为缺氧氧化还原控制蛋白（ArcA），属于arb /ArcA双组分信号转导系统的应答调控蛋白。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蛋白作为传感器激酶被GstArcBΔN蛋白转磷酸化。凝胶阻滞实验和BIAcore分析表明，与chiA、chiB、chiC和cbb1基因相比，磷酸化的ArcA （ArcA- p）更紧密地结合在aprIV和chiD基因的上游区域。这些结果表明，arb /ArcA双组分信号转导系统调控了菌株几丁质降解系统相关基因的表达。

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.