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

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

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)444-450
Number of pages7
JournalJournal of Bioscience and Bioengineering
Volume123
Issue number4
DOIs
Publication statusPublished - 2017 Apr 1

Keywords

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

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Fingerprint Dive into the research topics of 'Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of L-alanine in Escherichia coli'. Together they form a unique fingerprint.

Cite this