Hu L T, Foxall P A, Russell R, Mobley H L
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201.
Infect Immun. 1992 Jul;60(7):2657-66. doi: 10.1128/iai.60.7.2657-2666.1992.
Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, synthesized at up to 6% of the soluble cell protein, hydrolyzes urea, thereby releasing ammonia, which may neutralize acid, allowing survival of the bacterium and initial colonization of the gastric mucosa. The urease protein is encoded by two subunit genes, ureA and ureB; however, accessory genes are necessary for enzyme activity. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.8-kb Sau3A partial fragment carrying ureCDAB, corresponding to four open reading frames described by A. Labigne, V. Cussac, and P. Courcoux (J. Bacteriol. 173:1920-1931, 1991). Clones were confirmed as ureas gene sequences by polymerase chain reaction amplification. The recombinant enzyme was purified from the soluble protein of French press lysates of Escherichia coli DH5 alpha(pHP402) by chromatography on DEAE-Sepharose, Phenyl-Sepharose, Mono-Q, and Superose 6 resins. Fractions containing a catalytically inactive apoenzyme were identified by an enzyme-linked immunosorbent assay (ELISA) by using antisera to native UreA (29.5 kDa) and UreB (66 kDa). Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column and on Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels. The protein reacted specifically on Western blots (immunoblots) with anti-UreA and anti-UreB antibodies and was recognized with an intensity equal to that of the native enzyme in an ELISA using human sera. Clones containing only ureA and ureB also produced an assembled but inactive enzyme. Enzyme activity was not restored by in trans complementation with cloned urease accessory gene sequences from Proteus mirabilis or Morganella morganii. H. pylori urease genes (ureCDAB) subcloned into pACYC184 were also not complemented with any of 1,000 cosmid clones containing H. pylori chromosomal sequences. However, larger clones containing 4.5 kb of DNA downstream of ureB synthesized catalytically active urease when grown in minimal medium. These data indicate that the ureA and ureB genes encoding H. pylori urease are transcribed and translated in E. coli and that these genes alone are sufficient for the synthesis and assembly of the native size enzyme. Genes downstream of ureB, however, are necessary for production of a catalytically active urease.
幽门螺杆菌是一种革兰氏阴性、微需氧、螺旋形细菌，是人类胃炎和消化性溃疡的病原体，高度局限于人类胃黏膜。脲酶的合成量高达可溶性细胞蛋白的6%，它水解尿素，从而释放氨，氨可中和酸，使细菌得以存活并初步定植于胃黏膜。脲酶蛋白由两个亚基基因ureA和ureB编码；然而，辅助基因对于酶活性是必需的。幽门螺杆菌脲酶基因从黏粒基因文库中分离出来，并亚克隆到一个5.8 kb的Sau3A部分片段上，该片段携带ureCDAB，对应于A. Labigne、V. Cussac和P. Courcoux描述的四个开放阅读框（《细菌学杂志》173:1920 - 1931, 1991）。通过聚合酶链反应扩增确认克隆为脲酶基因序列。重组酶通过在DEAE - 琼脂糖、苯基 - 琼脂糖、Mono - Q和Superose 6树脂上进行色谱分离，从大肠杆菌DH5α(pHP402)的法国压榨裂解物的可溶性蛋白中纯化得到。通过使用针对天然UreA（29.5 kDa）和UreB（66 kDa）的抗血清进行酶联免疫吸附测定（ELISA），鉴定出含有无催化活性的脱辅基酶的级分。纯化的重组脲酶在Superose 6柱上以及考马斯亮蓝染色的十二烷基硫酸钠 - 聚丙烯酰胺凝胶上与天然酶无法区分。该蛋白在蛋白质印迹（免疫印迹）上与抗UreA和抗UreB抗体发生特异性反应，并且在使用人血清的ELISA中与天然酶的识别强度相同。仅包含ureA和ureB的克隆也产生了组装好但无活性的酶。用奇异变形杆菌或摩根氏摩根菌的克隆脲酶辅助基因序列进行反式互补，酶活性未恢复。亚克隆到pACYC184中的幽门螺杆菌脲酶基因（ureCDAB）也未与包含幽门螺杆菌染色体序列的1000个黏粒克隆中的任何一个互补。然而，包含ureB下游4.5 kb DNA的更大克隆在基本培养基中生长时合成了具有催化活性的脲酶。这些数据表明，编码幽门螺杆菌脲酶的ureA和ureB基因在大肠杆菌中进行转录和翻译，并且仅这些基因就足以合成和组装天然大小的酶。然而，ureB下游的基因对于产生具有催化活性的脲酶是必需的。
