L-Alanine Exporter, AlaE, of Escherichia coli Functions as a Safety Valve to Enhance Survival under Feast Conditions.

Satoshi Katsube,Tasuke Ando,Hiroshi Yoneyama

doi:10.3390/ijms20194942

Abstract

The intracellular level of amino acids is determined by the balance between their anabolic and catabolic pathways. L-alanine is anabolized by three L-alanine synthesizing enzymes and catabolized by two racemases and D-amino acid dehydrogenase (DadA). In addition, its level is regulated by L-alanine movement across the inner membrane. We identified the novel gene alaE, encoding an L-alanine exporter. To elucidate the physiological function of L-Alanine exporter, AlaE, we determined the susceptibility of alaE-, dadA-, and alaE/dadA-deficient mutants, derived from the wild-type strain MG1655, to L-alanyl-L-alanine (Ala-Ala), which shows toxicity to the L-alanine-nonmetabolizing variant lacking alaE. The dadA-deficient mutant has a similar minimum inhibitory concentration (MIC) (>1.25 mg/mL) to that observed in MG1655. However, alaE- and alaE/dadA-deficient mutants had MICs of 0.04 and 0.0025 mg/mL, respectively. The results suggested that the efficacy of AlaE to relieve stress caused by toxic intracellular accumulation of L-alanine was higher than that of DadA. Consistent with this, the intracellular level of alanine in the alaE-mutant was much higher than that in MG1655 and the dadA-mutant. We, therefore, conclude that AlaE functions as a ‘safety-valve’ to prevent the toxic level accumulation of intracellular L-alanine under a peptide-rich environment, such as within the animal intestine.

Highlights

Bacteria live in environments where nutritional conditions change extensively, such as within the animal intestine
We further investigated the physiological function of alanine exporter (AlaE) by comparing the impact of its capacity to relieve stress caused by high accumulation of intracellular L-alanine with that of D-amino acid dehydrogenase (DadA), by employing isogenic alaE, dadA, and alaE/dadA-deficient mutants, derived from the wild-type strain MG1655
The double mutant MG1655∆alaE∆dadA was strikingly susceptible to Ala-Ala (MIC, 0.0025 mg/mL), the level of which was the same as that of MLA301∆alaE, which completely lacks both L-alanine synthetic and alanine racemase activities [27]

Summary

Introduction

Bacteria live in environments where nutritional conditions change extensively (both temporally and spatially), such as within the animal intestine. The expression of the alaE gene has been found to be regulated by the global regulator Lrp (leucine responsive protein) in a positive manner in the presence of L-alanine and L-leucine, but not D-alanine [28], suggesting that AlaE could function as a “safety valve” to prevent an abrupt increase of an intracellular L-alanine level that could lead to growth inhibition when E. coli faces a feast environmental condition Still, this hypothesis is not conclusive because (i) the L-alanine non-metabolizing strain used in the previous studies [27,28,29] does not exist in the natural environment and (ii) the alanine catabolic pathway involving DadA has not been taken into account in the earlier studies. We further investigated the physiological function of AlaE by comparing the impact of its capacity to relieve stress caused by high accumulation of intracellular L-alanine with that of DadA, by employing isogenic alaE-, dadA-, and alaE/dadA-deficient mutants, derived from the wild-type strain MG1655

Results and Discussion

Accumulation of Intracellular Alanine in MG1655 and its Derivatives

Materials and Methods

Determination of Intracellular Amino Acid Concentration

Coculture Assay