Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of L-alanine in Escherichia coli

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.