EXPRESSION OF HELICOBACTER-PYLORI UREASE GENES IN ESCHERICHIA-COLI GROWN UNDER NITROGEN-LIMITING CONDITIONS
EXPRESSION OF HELICOBACTER-PYLORI UREASE GENES IN ESCHERICHIA-COLI GROWN UNDER NITROGEN-LIMITING CONDITIONS
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DOI：
10.1128/jb.174.8.2466-2473.1992
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发表时间：
1992-04-01
影响因子：
3.2
通讯作者：
LABIGNE, A
中科院分区：
文献类型：
--
作者：
CUSSAC, V;FERRERO, RL;LABIGNE, A
幽门螺杆菌产生一种强效脲酶，据信在胃十二指肠疾病的发病机制中起作用。四种基因（ureA, ureB， ureC和ureD）在引入空肠弯曲杆菌时能够实现脲酶阳性表型，而携带这些基因的大肠杆菌细胞不表达脲酶活性（a . Labigne， V. Cussac, and P. Courcoux， J. Bacteriol. 173:1920-1931, 1991）。结果表明，幽门螺杆菌脲酶基因可以在大肠杆菌中表达。发现这种表达依赖于迄今未描述的辅助脲酶基因的存在。对重组cosmid pILL585进行亚克隆，并进行酶切分析，克隆出一个1.2 kb的片段（pILL753），该片段可以在限氮条件下培养的大肠杆菌细胞中检测到脲酶活性（每分钟每毫克蛋白质水解0.83 +/- 0.39 μ mol尿素）。用mini-Tn3-Km对pILL753进行转座子诱变，除了先前鉴定的4.2 kb区域外，还鉴定了3.3 kb的DNA区域，该区域对大肠杆菌脲酶活性至关重要。对这段3.3 kb的DNA片段进行测序，发现有5个开放阅读框编码多肽，预测分子量分别为20,701 （UreE）、28,530 （UreF）、21,744 （UreG）、29,650 （UreH）和19,819 （UreI）。在所鉴定的9个脲酶基因中，尿素、ureB、ureF、ureG和ureH被证明是大肠杆菌中脲酶表达所必需的，因为这些基因中的每一个突变都会导致阴性表型。ureC、ureD和ureI基因虽然属于脲酶基因簇，但在大肠杆菌中脲酶的表达并不是必需的。预测的UreE和UreG多肽与产气克雷伯菌脲酶操纵子的辅助基因编码的多肽具有一定程度的相似性（考虑到保守的氨基酸变化，相似性分别为33%和92%），而这种同源性仅限于UreF多肽的一个结构域（产气克雷伯菌UreF多肽的最后73个氨基酸相似性为44%）。除了该酶的两个尿素和UreB结构多肽外，尚未确定由幽门螺杆菌脲酶基因簇编码的9个蛋白的作用。
Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39-mu-mol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.