RRC ID 52869
Author Ihara K, Sato K, Hori H, Makino Y, Shigenobu S, Ando T, Isogai E, Yoneyama H.
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.
Journal 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.
Volume 123(4)
Pages 444-450
Published 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
Times Cited 7
DNA material pQE-Lrp (RDB15924) MG1655 delta lrp (RDB15921) MG1655 delta lacZ (RDB15922) MG1655 delta lacZ delta lrp (RDB15923).