Characterization of thel‐alanine exporter AlaE ofEscherichia coliand its potential role in protecting cells from a toxic‐level accumulation ofl‐alanine and its derivatives

Abstract

We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [3H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [3H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [3H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.

Secretion of d-alanine by Escherichia coli

“…In our previous study, E. coli was found to possess L-alanine export systems where the alaE gene was identified to encode the major L-alanine exporter AlaE (Hori et al, 2011b;Kim et al, 2015). Since wild-type E. coli cells usually do not secrete alanine under normal culture conditions (Hori et al, 2011a;Kinoshita et al, 1957), a physiological function of the L-alanine exporter, AlaE, was assumed to be a 'safety valve' that protects the cells from toxic levels of accumulated L-alanine and its derivatives (Hori et al, 2011b).…”

“…We have recently found that activities of an L-alanine exporter, AlaE, were inhibited by CCCP, but not by DCCD, suggesting that proton-electrochemical potential is the energy source for AlaE transport activity (Kim et al, 2015). Furthermore, AlaE was found to catalyse the L-alanineexchange reaction using inverted membrane vesicles that had been preloaded with unlabelled L-alanine in the absence of an energy source (Kim et al, 2015).…”

“…Furthermore, AlaE was found to catalyse the L-alanineexchange reaction using inverted membrane vesicles that had been preloaded with unlabelled L-alanine in the absence of an energy source (Kim et al, 2015). Thus, we assumed that an increase in extracellular L-alanine levels upon treatment of L-Ala-L-Ala-loaded JW1178 cells with CCCP was likely caused by an enhanced rate of L-alanine efflux through AlaE.…”

“…Subsequently, four genes (ygaW, ytfF, yddG and yeaS) were identified that encode the L-alanine exporter, with the ygaW gene being renamed alaE because it encodes the major L-alanine exporter (Hori et al, 2011b). Furthermore, AlaE has recently been found to export L-alanine using protonelectrochemical potential (Kim et al, 2015).…”

More than stoichiometry: the molecular composition of inorganic and organic substrates controls ammonium regeneration by bacteria

“…Hence, we are drawn to conclude that our bacterial community could not grow 410 on L-alanine as its only organic substrate, although previous researchers have grown bacterial 411 strains on this substrate (Franklin & Venables 1976;Goldman et al 1987). It seems that high 412 concentrations of L-alanine may be toxic for some bacteria as found by Kim et al ( 2015), while 413 other bacteria have the necessary enzymes to use the amino-acid for growth (Coudert 1975). An 414 alternative explanation could be that growing on amino acids requires deamination in order to use 415 the carbon backbone for respiration or further cell building.…”