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Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

Kim S, Ihara K, Katsube S, Hori H, Ando T, Isogai E, Yoneyama H - Microbiologyopen (2015)

Bottom Line: 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 [(3) H]l-alanine accumulation occurred under either condition.This energy-dependent uphill accumulation of [(3) H]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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan.

No MeSH data available.


Related in: MedlinePlus

Effect of l-alanine and l-Ala-L-Ala on the growth of the wild-type strain and its alaE-deficient derivative. A fully grown cell suspension was serially diluted 10-fold and 5-μL aliquots were spotted onto minimal medium supplemented with 0.5–4.0 mmol/L l-Ala-l-Ala (left panel) or 2.5–20 mmol/L l-Ala (right panel). The top-left panel represents control medium without supplement. Plates were incubated at 37°C for 40 h. Strains used: MG1655, alaE-positive parent strain; MG1655ΔalaE, alaE-deficient isogenic derivative.
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fig02: Effect of l-alanine and l-Ala-L-Ala on the growth of the wild-type strain and its alaE-deficient derivative. A fully grown cell suspension was serially diluted 10-fold and 5-μL aliquots were spotted onto minimal medium supplemented with 0.5–4.0 mmol/L l-Ala-l-Ala (left panel) or 2.5–20 mmol/L l-Ala (right panel). The top-left panel represents control medium without supplement. Plates were incubated at 37°C for 40 h. Strains used: MG1655, alaE-positive parent strain; MG1655ΔalaE, alaE-deficient isogenic derivative.

Mentions: In addition, we assessed the effect of l-Ala-l-Ala and l-alanine on the growth of the alaE-deficient cells derived from an l-alanine-metabolizing wild-type strain by plating a 10-fold serially diluted cell suspension, and the results were compared with that of the alaE-positive parent cells (Fig.2). Both the alaE-deficient cells and the parent cells grew up to the 10−6 dilution in the control plate free from l-Ala-l-Ala and l-alanine. When the plate was impregnated with 4.0 mmol/L l-Ala-l-Ala, the parent cells grew up to a dilution of 10−4, a difference of two orders of magnitude from that on the l-Ala-l-Ala-free medium. In contrast, the alaE-deficient cells grew only at the 10−1 dilution, a difference of five orders of magnitude from the control plate. As the l-Ala-l-Ala concentration was decreased to 2.0, 1.0, and 0.5 mmol/L, the effect of l-Ala-l-Ala was reduced, as would be expected, however, the difference in growth when compared with the control plate was noticeable even at 0.5 mmol/L. Similar experiments were carried out in plates impregnated with l-alanine at concentrations of 2.5, 5.0, 10, and 20 mmol/L. The alaE-deficient cells showed higher susceptibility to l-alanine than the parent cells, yet the growth inhibitory effect was less significant when compared with that of l-Ala-l-Ala, suggesting that the primary substrate of the AlaE exporter may be alanine derivatives including l-Ala-l-Ala, but we cannot rule out a possibility that the primary substrate of the AlaE may be l-alanine.


Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

Kim S, Ihara K, Katsube S, Hori H, Ando T, Isogai E, Yoneyama H - Microbiologyopen (2015)

Effect of l-alanine and l-Ala-L-Ala on the growth of the wild-type strain and its alaE-deficient derivative. A fully grown cell suspension was serially diluted 10-fold and 5-μL aliquots were spotted onto minimal medium supplemented with 0.5–4.0 mmol/L l-Ala-l-Ala (left panel) or 2.5–20 mmol/L l-Ala (right panel). The top-left panel represents control medium without supplement. Plates were incubated at 37°C for 40 h. Strains used: MG1655, alaE-positive parent strain; MG1655ΔalaE, alaE-deficient isogenic derivative.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554458&req=5

fig02: Effect of l-alanine and l-Ala-L-Ala on the growth of the wild-type strain and its alaE-deficient derivative. A fully grown cell suspension was serially diluted 10-fold and 5-μL aliquots were spotted onto minimal medium supplemented with 0.5–4.0 mmol/L l-Ala-l-Ala (left panel) or 2.5–20 mmol/L l-Ala (right panel). The top-left panel represents control medium without supplement. Plates were incubated at 37°C for 40 h. Strains used: MG1655, alaE-positive parent strain; MG1655ΔalaE, alaE-deficient isogenic derivative.
Mentions: In addition, we assessed the effect of l-Ala-l-Ala and l-alanine on the growth of the alaE-deficient cells derived from an l-alanine-metabolizing wild-type strain by plating a 10-fold serially diluted cell suspension, and the results were compared with that of the alaE-positive parent cells (Fig.2). Both the alaE-deficient cells and the parent cells grew up to the 10−6 dilution in the control plate free from l-Ala-l-Ala and l-alanine. When the plate was impregnated with 4.0 mmol/L l-Ala-l-Ala, the parent cells grew up to a dilution of 10−4, a difference of two orders of magnitude from that on the l-Ala-l-Ala-free medium. In contrast, the alaE-deficient cells grew only at the 10−1 dilution, a difference of five orders of magnitude from the control plate. As the l-Ala-l-Ala concentration was decreased to 2.0, 1.0, and 0.5 mmol/L, the effect of l-Ala-l-Ala was reduced, as would be expected, however, the difference in growth when compared with the control plate was noticeable even at 0.5 mmol/L. Similar experiments were carried out in plates impregnated with l-alanine at concentrations of 2.5, 5.0, 10, and 20 mmol/L. The alaE-deficient cells showed higher susceptibility to l-alanine than the parent cells, yet the growth inhibitory effect was less significant when compared with that of l-Ala-l-Ala, suggesting that the primary substrate of the AlaE exporter may be alanine derivatives including l-Ala-l-Ala, but we cannot rule out a possibility that the primary substrate of the AlaE may be l-alanine.

Bottom Line: 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 [(3) H]l-alanine accumulation occurred under either condition.This energy-dependent uphill accumulation of [(3) H]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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan.

No MeSH data available.


Related in: MedlinePlus