Lee K S, Song S B, Kim K E, Kim Y H, Kim S K, Kho B H, Ko D K, Choi Y K, Lee Y K, Kim C K, Kim Y C, Lim J Y, Kim Y, Min K H, Wanner B L
Research Center for Biomedicinal Resources (Bio-Med RRC), Pai-Chai University, Taejon, 302-735, Korea.
Biochem Biophys Res Commun. 2000 Mar 16;269(2):526-31. doi: 10.1006/bbrc.2000.2328.
A bacterial alkaline phosphatase (BAP, the phoA gene product) is primarily responsible for the hydrolysis of the substrates 5-bromo-4-chloro-3-indolylphosphate-p-toluidine (XP) and p-nitrophenyl phosphate (pNPP). Using these substrates and an E. coli phoA mutant, we have cloned Enterobacter aerogenes genes conferring an XP(+) phenotype. Two types of clones were identified based on phenotypic tests and DNA sequences. One of them is a E. aerogenes phoA gene (XP(+), pNPP(+)) as expected; surprisingly the other one was found to be a ushA gene (XP(+), pNPP(-)), which encodes an UDP (uridine 5'-diphosphate)-sugar hydrolase. The E. aerogenes ushA gene shares high sequence identity with ushA of E. coli and the mutationally silent ushA0 gene of Salmonella typhimurium at both the nucleotide (over 79%) and amino acid (over 93%) levels. Expression of the E. aerogenes ushA gene in E. coli produced high level of UDP-sugar hydrolase, as confirmed by TLC (thin layer chromatography) analysis together with a presence of a strong band due to a XP hydrolysis on a polyacrylamide gel.
一种细菌碱性磷酸酶（BAP，phoA基因产物）主要负责底物5-溴-4-氯-3-吲哚磷酸对甲苯胺盐（XP）和对硝基苯磷酸酯（pNPP）的水解。利用这些底物和大肠杆菌phoA突变体，我们克隆了产气肠杆菌中赋予XP（+）表型的基因。基于表型测试和DNA序列鉴定出了两种类型的克隆。其中之一正如预期的那样是产气肠杆菌phoA基因（XP（+），pNPP（+））；令人惊讶的是，另一个是ushA基因（XP（+），pNPP（-）），它编码一种UDP（尿苷5'-二磷酸）-糖水解酶。产气肠杆菌ushA基因与大肠杆菌的ushA基因以及鼠伤寒沙门氏菌的无突变沉默ushA0基因在核苷酸水平（超过79%）和氨基酸水平（超过93%）均具有高度的序列同一性。产气肠杆菌ushA基因在大肠杆菌中的表达产生了高水平的UDP-糖水解酶，这通过薄层色谱（TLC）分析以及聚丙烯酰胺凝胶上因XP水解出现的强条带得以证实。
