title: Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli.
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.
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza Aoba, Aoba-ku, Sendai 980-0845, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Ihara
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza Aoba, Aoba-ku, Sendai 980-0845, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Sato
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza Aoba, Aoba-ku, Sendai 980-0845, Japan.
authors.forename: Hatsuhiro
authors.initials: H
authors.lastname: Hori
authors.affiliation: NIBB Core Research Facilities, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Aichi, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Makino
authors.affiliation: NIBB Core Research Facilities, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Aichi, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Shigenobu
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza Aoba, Aoba-ku, Sendai 980-0845, Japan.
authors.forename: Tasuke
authors.initials: T
authors.lastname: Ando
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza Aoba, Aoba-ku, Sendai 980-0845, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Isogai
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza Aoba, Aoba-ku, Sendai 980-0845, Japan. Electronic address: hiroshi.yoneyama.a4@tohoku.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yoneyama
mesh_terms: D000409:Alanine / Q000378:metabolism* / Q000494:pharmacology; D026921:Amino Acid Transport Systems, Neutral / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D007930:Leucine / Q000378:metabolism / Q000494:pharmacology; D051756:Leucine-Responsive Regulatory Protein / Q000172:deficiency / Q000378:metabolism*; D009876:Operon / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: C560941:AlaE protein, E coli; D026921:Amino Acid Transport Systems, Neutral; D004151:Dipeptides; D029968:Escherichia coli Proteins; D051756:Leucine-Responsive Regulatory Protein; C002956:alanylalanine; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase; D007930:Leucine; D000409:Alanine
keywords: AlaE; Escherichia coli; Exporter; Lrp; l-Alanine