Mobley H L, Garner R M, Bauerfeind P
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201, USA.
Mol Microbiol. 1995 Apr;16(1):97-109. doi: 10.1111/j.1365-2958.1995.tb02395.x.
Urease is a virulence determinant, a taxonomic and diagnostic marker, and immunogen for Helicobacter pylori, an aetiologic agent of gastritis and peptic ulceration. This enzyme requires Ni2+ ions in the active site for successful hydrolysis of urea. When expressed in Escherichia coli, recombinant urease is only weakly active unless urease structural subunits are overexpressed, exogenous NiCl2 is added, and the host strain is grown in medium that does not chelate free Ni2+. As wild-type H. pylori does not require such conditions for very high levels of urease expression, we reasoned that additional genes were required to accumulate the metal ion. To isolate such genes, E. coli SE5000 (pHP808), which carries the H. pylori urease gene cluster, was complemented with a lambda ZAP-derived plasmid library of the H. pylori chromosome. One of 1000 ampicillin-resistant clones, plated onto urea segregation agar, produced detectable urease. Urease activity of this co-transformant, grown in Luria broth containing 1 microM NiCl2, was 36 mumol NH3 min-1 mg-1 protein. Urease-enhancing activity, which is not directly linked to the urease gene cluster, was localized by subcloning and nucleotide sequencing. The largest open reading frame, designated nixA, predicted a polypeptide of 34,317 Da that displayed characteristics of an integral membrane protein. In vitro transcription-translation of nixA sequences yielded a polypeptide estimated to be 32 kDa in size. An in-frame Bal31 deletion within nixA abolished urease-enhancing activity. At 50 nM NiCl2, E. coli containing the nixA clone transported 1250 +/- 460 pmol Ni2+ min-1 10(-8) cells, whereas the vector control transported only 140 +/- 85 pmol Ni2+ min-1 10(8) cells, i.e. significantly less (P = 0.01). We conclude that NixA confers upon E. coli a high-affinity nickel-transport system (KT = 11.3 +/- 2.4 nM; Vmax = 1750 +/- 220 pmol Ni2+ min-1 10(-8) cells) and is necessary for expression of catalytically active urease, regardless of growth conditions.
脲酶是幽门螺杆菌的一种毒力决定因素、分类和诊断标志物以及免疫原，幽门螺杆菌是胃炎和消化性溃疡的病原体。这种酶在活性位点需要Ni2+离子才能成功水解尿素。当在大肠杆菌中表达时，重组脲酶活性很低，除非脲酶结构亚基过表达、添加外源NiCl2，并且宿主菌株在不螯合游离Ni2+的培养基中生长。由于野生型幽门螺杆菌在非常高水平的脲酶表达时不需要这些条件，我们推测需要额外的基因来积累金属离子。为了分离这些基因，携带幽门螺杆菌脲酶基因簇的大肠杆菌SE5000（pHP808）用来自幽门螺杆菌染色体的λZAP衍生质粒文库进行了互补。在尿素分离琼脂平板上接种的1000个氨苄青霉素抗性克隆中，有一个产生了可检测到的脲酶。在含有1μM NiCl2的Luria肉汤中生长的这种共转化体的脲酶活性为36μmol NH3 min-1 mg-1蛋白质。通过亚克隆和核苷酸测序定位了与脲酶基因簇没有直接联系的脲酶增强活性。最大的开放阅读框，命名为nixA，预测了一个34317 Da的多肽，显示出整合膜蛋白的特征。nixA序列的体外转录-翻译产生了一个估计大小为32 kDa的多肽。nixA内的框内Bal31缺失消除了脲酶增强活性。在50 nM NiCl2时，含有nixA克隆的大肠杆菌每分钟每10(-8)个细胞转运1250±460 pmol Ni2+，而载体对照每分钟每10(8)个细胞仅转运仅140±85 pmol Ni2+，即明显更少（P = 0.0）我们得出结论，NixA赋予大肠杆菌一个高亲和力的镍转运系统（KT = 11.3±2.4 nM；Vmax = 1750±220 pmol Ni2+ min-1 10(-8)个细胞），并且无论生长条件如何，对于催化活性脲酶的表达都是必需的。
