Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany.
FEMS Microbiol Lett. 2010 Jul;308(2):166-74. doi: 10.1111/j.1574-6968.2010.02005.x. Epub 2010 Apr 29.
The atuR-atuABCDEFGH gene cluster is essential for acyclic terpene utilization (Atu) in Pseudomonas aeruginosa and Pseudomonas citronellolis. The cluster encodes most proteins of the Atu pathway including the key enzyme, geranyl-CoA carboxylase. AtuR was identified as a repressor of the atu gene cluster expression by (1) amino acid similarity to TetR repressor family members, (2) constitutive expression of Atu proteins in the atuR insertion mutant and (3) specific binding of purified AtuR homodimers to the atuR-atuA intergenic region in electrophoretic mobility shift assay (EMSA). Two 13 bp inverted repeat sequences separated by 40 bp in the atuA operator/promoter region were identified to represent two sites of AtuR binding by EMSA. Changing of two or more bases within the inverted repeat sequences abolished the ability of AtuR to bind to its target. All EMSA experiments were sufficiently sensitive with ethidium bromide-stained DNA fragments after polyacrylamide gel electrophoresis.
atuR-atuABCDEFGH 基因簇是铜绿假单胞菌和香茅假单胞菌中环萜类化合物利用 (Atu) 的必需基因簇。该基因簇编码 Atu 途径的大多数蛋白质,包括关键酶香叶基-CoA 羧化酶。AtuR 通过以下方式被鉴定为 atu 基因簇表达的抑制剂:(1)与 TetR 阻遏物家族成员的氨基酸相似性,(2)atuR 插入突变体中 Atu 蛋白的组成型表达,以及 (3)纯化的 AtuR 同源二聚体在电泳迁移率变动分析 (EMSA) 中与 atuR-atuA 基因间区特异性结合。在 atuA 操纵子/启动子区域中,两个相隔 40 个碱基的 13 个碱基反向重复序列被鉴定为 AtuR 结合的两个位点。在反向重复序列中改变两个或更多碱基会使 AtuR 失去与靶标的结合能力。所有的 EMSA 实验都非常灵敏,经聚丙烯酰胺凝胶电泳后,溴化乙锭染色的 DNA 片段即可检测。