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首页> 外文期刊>Journal of bacteriology >Helicobacter pylori ABC transporter: effect of allelic exchange mutagenesis on urease activity.
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Helicobacter pylori ABC transporter: effect of allelic exchange mutagenesis on urease activity.

机译：幽门螺杆菌ABC转运蛋白：等位基因交换诱变对脲酶活性的影响。

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Helicobacter pylori urease requires nickel ions in the enzyme active site for catalytic activity. Nickel ions must, therefore, be actively acquired by the bacterium. NixA (high-affinity nickel transport protein)-deficient mutants of H. pylori retain significant urease activity, suggesting the presence of alternate nickel transporters. Analysis of the nucleotide sequence of the H. pylori genome revealed a homolog of NikD, a component of an ATP-dependent nickel transport system in Escherichia coli. Based on this sequence, a 378-bp DNA fragment was PCR amplified from H. pylori genomic DNA and used as a probe to identify an H. pylori lambda ZAPII genomic library clone that carried these sequences. Four open reading frames of 621, 273, 984, and 642 bp (abcABCD) were revealed by sequencing and predicted polypeptides of 22.7, 9.9, 36.6, and 22.8 kDa, respectively. The 36.6-kDa polypeptide (AbcC) has significant homology (56% amino acid sequence identity) to an E. coli ATP-binding protein component of an ABC transport system, while none of the other putative proteins are significantly homologous to polypeptides in the available databases. To determine the possible contribution of these genes to urease activity, abcC and abcD were each insertionally inactivated with a kanamycin resistance (aphA) cassette and allelic exchange mutants of each gene were constructed in H. pylori UMAB41. Mutation of abcD resulted in an 88% decrease in urease activity to 27 +/- 31 mumol of NH3/min/mg of protein (P < 0.0001), and a double mutant of nixA and abcC resulted in the near abolishment of urease activity (1.1 +/- 1.4 mumol of NH3/min/mg of protein in the double mutant versus 228 +/- 92 mumol of NH3/min/mg of protein in the parent [P < 0.0001]). Synthesis of urease apoenzyme, however, was unaffected by mutations in any of the abc genes. We conclude that the abc gene cluster, in addition to nixA, is involved in production of a catalytically active urease.
机译：幽门螺杆菌脲酶在酶的活性位点需要镍离子以进行催化活性。因此，细菌必须积极地吸收镍离子。幽门螺杆菌的NixA（高亲和力镍转运蛋白）缺陷突变体保留了明显的脲酶活性，表明存在其他镍转运蛋白。幽门螺杆菌基因组核苷酸序列的分析揭示了NikD的同源物，NikD是大肠杆菌中ATP依赖性镍转运系统的组成部分。根据该序列，从幽门螺杆菌基因组DNA PCR扩增了一个378 bp的DNA片段，并用作探针来鉴定携带这些序列的幽门螺杆菌λZAPII基因组文库克隆。通过测序和预测的多肽分别为22.7、9.9、36.6和22.8 kDa，揭示了621、273、984和642 bp（abcABCD）的四个开放阅读框。 36.6 kDa多肽（AbcC）与ABC转运系统的大肠杆菌ATP结合蛋白成分具有显着同源性（56％氨基酸序列同一性），而其他推定蛋白均与现有多肽无显着同源性数据库。为了确定这些基因对脲酶活性的可能贡献，分别用卡那霉素抗性（aphA）盒将abcC和abcD插入失活，并在幽门螺杆菌UMAB41中构建每个基因的等位基因交换突变体。 abcD突变导致尿素酶活性降低88％，至NH3 / min / mg蛋白的27 +/- 31μmol（P <0.0001），而nixA和abcC的双重突变导致尿素酶活性几乎消失（在双突变体中，NH 3 / min / mg蛋白为1.1 +/-1.4μmolNH 3 / min / mg，而在亲本中为228 +/-92μmolNH 3 / min / mg蛋白[P ＜0.0001]。然而，脲酶脱辅酶的合成不受任何abc基因突变的影响。我们得出的结论是，除nixA外，abc基因簇还参与了催化活性脲酶的生产。

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