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首页> 外文期刊>Journal of Bioscience and Bioengineering >Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of L-alanine in Escherichia coli
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Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of L-alanine in Escherichia coli

机译：alae基因的表达是通过全球调节剂LRP正面调节，响应于大肠杆菌的L-丙氨酸的细胞内积累

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The alaE gene in Escherichia coli encodes an L-alanine exporter that catalyzes the active export of L-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of talanyl-L-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less beta-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ-deficient and harbored this reporter plasmid showed significant induction of beta-galactosidase activity (approximately 17-fold) in the presence of 6 mM L-alanine, L-leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, Irp-deficient cells showed almost no beta-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide -161 to -83 with a physiologically relevant affinity (apparent K-D, 288.7 +/- 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis-element was increased by L-alanine and L-leucine, but not by Ala-Ala and a-alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of L-alanine, which eventually leads to intracellular homeostasis of L-alanine concentrations. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.
机译：大肠杆菌中的Alae基因编码了使用质子电化学电位催化L-丙氨酸的活性输出的L-丙氨酸出口。在我们以前的研究中，Alae表达被证明在替硝酸-L-丙氨酸（Ala-Ala）的存在下增加。在本研究中，将全局调节剂亮氨酸响应性调节蛋白（LRP）鉴定为ALAE基因的活化剂。将β-半乳糖苷酶基因的启动子融合给ALAE上游区域（240个核苷酸）。缺乏LacZ缺陷和患者的细胞本报告质粒显示出β-半乳糖苷酶活性（约17倍）的显着诱导在6mM L-丙氨酸，L-亮氨酸和Ala-Ala存在下。然而，具有较小的AlaE上游区域（180个核苷酸）的记者质粒产生在相同条件下具有惊显的低酶活性的转化体。相反，IRP缺陷的细胞显示出几乎没有β-半乳糖苷酶诱导，表明LRP积极调节ALAE表达。接下来，使用纯化的六三角氨酸标记的LRP（LRP-HIS）进行电泳迁移率移位测定（EMSA）和DNASE I的脚印测定。因此，我们发现LRP-HIS与生理相关亲和力（表观K-D，288.7 +/- 83.8nm）结合跨越核苷酸-161至-83的Alae上游区域。此外，L-丙氨酸和L-亮氨酸增加了LRP-HIS朝向其顺式元素的结合亲和力，但不通过Ala-Ala和A-丙氨酸。基于这些结果，我们得出结论，ALAE的基因表达是通过LRP对L-丙氨酸细胞内水平的调节，最终导致L-丙氨酸浓度的细胞内稳态。 （c）2016年，日本生物技术协会。版权所有。

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    Ihara Kohei; Sato Kazuki; Hori Hatsuhiro; Makino Yumiko; Shigenobu Shuji; Ando Tasuke; Isogai Emiko; Yoneyama Hiroshi;

  • 作者单位

    Tohoku Univ Grad Sch Agr Sci Dept Microbial Biotechnol Lab Anim Microbiol Aoba Ku 468-1 Aramaki Aza Aoba Sendai Miyagi 9800845 Japan;

    Tohoku Univ Grad Sch Agr Sci Dept Microbial Biotechnol Lab Anim Microbiol Aoba Ku 468-1 Aramaki Aza Aoba Sendai Miyagi 9800845 Japan;

    Tohoku Univ Grad Sch Agr Sci Dept Microbial Biotechnol Lab Anim Microbiol Aoba Ku 468-1 Aramaki Aza Aoba Sendai Miyagi 9800845 Japan;

    Natl Inst Basic Biol NIBB Core Res Facil Nishigonaka 38 Okazaki Aichi 4448585 Japan;

    Natl Inst Basic Biol NIBB Core Res Facil Nishigonaka 38 Okazaki Aichi 4448585 Japan;

    Tohoku Univ Grad Sch Agr Sci Dept Microbial Biotechnol Lab Anim Microbiol Aoba Ku 468-1 Aramaki Aza Aoba Sendai Miyagi 9800845 Japan;

    Tohoku Univ Grad Sch Agr Sci Dept Microbial Biotechnol Lab Anim Microbiol Aoba Ku 468-1 Aramaki Aza Aoba Sendai Miyagi 9800845 Japan;

    Tohoku Univ Grad Sch Agr Sci Dept Microbial Biotechnol Lab Anim Microbiol Aoba Ku 468-1 Aramaki Aza Aoba Sendai Miyagi 9800845 Japan;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;生物工程学（生物技术）;酿造工业;
  • 关键词

    Escherichia coli; L-Alanine; Exporter; AlaE; Lrp;

    机译：大肠杆菌;L-丙氨酸;出口商;ALAE;LRP;

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