論文 - 詳細
| RRC ID | 52869 |
|---|---|
| 著者 | Ihara K, Sato K, Hori H, Makino Y, Shigenobu S, Ando T, Isogai E, Yoneyama H. |
| タイトル | Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli. |
| ジャーナル | J Biosci Bioeng |
| Abstract |
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 l-alanyl-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 β-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 β-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, lrp-deficient cells showed almost no β-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 KD, 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 d-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. |
| 巻・号 | 123(4) |
| ページ | 444-450 |
| 公開日 | 2017-4-1 |
| DOI | 10.1016/j.jbiosc.2016.11.015 |
| PII | S1389-1723(16)30622-3 |
| PMID | 28057466 |
| MeSH | Alanine / metabolism* Alanine / pharmacology Amino Acid Transport Systems, Neutral / biosynthesis Amino Acid Transport Systems, Neutral / genetics Amino Acid Transport Systems, Neutral / metabolism Base Sequence DNA Footprinting Deoxyribonuclease I / metabolism Dipeptides / metabolism Dipeptides / pharmacology Electrophoretic Mobility Shift Assay Escherichia coli / drug effects Escherichia coli / genetics* Escherichia coli / metabolism* Escherichia coli Proteins / biosynthesis Escherichia coli Proteins / genetics Escherichia coli Proteins / metabolism Gene Expression Regulation, Bacterial* / drug effects Genes, Reporter / genetics Leucine / metabolism Leucine / pharmacology Leucine-Responsive Regulatory Protein / deficiency Leucine-Responsive Regulatory Protein / metabolism* Operon / drug effects Protein Binding / drug effects Regulatory Sequences, Nucleic Acid / genetics Up-Regulation / drug effects beta-Galactosidase / genetics beta-Galactosidase / metabolism |
| IF | 2.366 |
| 引用数 | 7 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
|
| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 2 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 遺伝子材料 | pQE-Lrp (RDB15924) MG1655 delta lrp (RDB15921) MG1655 delta lacZ (RDB15922) MG1655 delta lacZ delta lrp (RDB15923). |