The yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a novel glutathione importer with an ATP-binding cassette

Abstract

Glutathione protects cells and organisms from oxygen species and peroxides and is indispensable for aerobically living organisms. Moreover, it acts against xenobiotics and drugs by the formation and excretion of glutathione S conjugates. In this study, we show that the yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a glutathione transporter with the ATP-binding cassette. The transporter imports extracellular glutathione into the cytoplasm in an ATP-dependent manner. This transporter, along with gamma-glutamyltranspeptidase, has an important role in E. coli growth with glutathione as a sole sulfur source.

FIG. 1.

The structure of the ybiK-yliABCD operon. The order, size, and products of the genes and the location of the promoter suggested by GenBank and EcoCys were diagrammed. The region of DNA deleted in our ΔyliAB mutation is shown by the bar above the genes. Gene names in parentheses are the new names proposed, after glutathione importer.

FIG. 2.

Glutathione concentration of the culture media. Strains SI37 (Δggt ΔyliAB) (filled square), SI49 (Δggt) (open square), SH1555 (pACYC177::ybiK⁺-yliA⁺ yliB⁺ yliC⁺ yliD⁺/Δggt ΔyliAB) (filled triangle), SI103 (ΔyliAB) (filled circle), and SI104 (ggt⁺ yliA⁺ yliB⁺) (open circle) were grown in 100 ml minimal medium. At the times indicated, 2 ml of culture was subtracted. An optical density at 610 nm was measured using 1 ml of the 2-ml culture. Another 1 ml was centrifuged, and the concentration of glutathione of the culture fluid was measured with glutathione reductase.

FIG. 3.

Glutathione uptake in a transporter assay. (A) Strains SI35 (Δggt ΔyliAB) (filled triangles), SH703 (Δggt) (open circles), SH1552 (pACYC177::ybiK⁺-yliA⁺ yliB⁺ yliC⁺ yliD⁺/Δggt ΔyliAB) (filled circles), and SH1554 (pACYC177::ybiK⁺-yliA⁺ yliB⁺ yliC⁺/Δggt ΔyliAB) (open triangles). (B) Strains SH1617 (pACYC177::ybiK⁺-yliA⁺ yliB⁺ yliC⁺ yliD⁺-lacZ⁺/Δggt ΔyliAB) (filled circles), SH1618 (pACYC177::ybiK⁺-yliB⁺ yliC⁺ yliD⁺-lacZ⁺/Δggt ΔyliAB) (open circles), SH1619 (pACYC177::ybiK⁺-yliA⁺ yliC⁺ yliD⁺-lacZ⁺/Δggt ΔyliAB) (filled triangles), and SH1620 (pACYC177::ybiK⁺-yliA⁺ yliB⁺ yliD⁺-lacZ⁺/Δggt ΔyliAB) (open triangles).

FIG.4.

Accumulation of glutathione in the cells grown in minimal medium supplemented with 1 mM glutathione. Strains SI100 (pACYC177/ΔgshA Δggt ΔyliAB), SI109 (pACYC177/ΔgshA Δggt), SI153 (pACYC177::ybiK⁺-yliA⁺ yliB⁺ yliC⁺/ΔgshA Δggt ΔyliAB), and SI154 (pACYC177::ybiK⁺-yliA⁺ yliB⁺ yliC⁺ yliD⁺/ΔgshA Δggt ΔyliAB) were grown in minimal medium supplemented with 1 mM reduced glutathione for 12 h. The amount of glutathione found in the cells was expressed as relative to that for strain SI154. Seventy-four nanomoles of glutathione per mg of cells was found in strain SI154.

FIG. 5.

Effect of verapamil on glutathione uptake. Glutathione uptake of strain SH1552 was measured in the absence of verapamil (filled circles) and in the presence of 1 mM (open circles) and 10 mM verapamil (open triangles).

FIG. 6.

Growth of E. coli strains on a minimal medium plate supplemented with glutathione as the sole sulfur source. Strains were grown overnight in 5 ml LB medium, washed twice, and suspended with 5 ml of 1× M9 buffer. One μl of cell suspension was plotted on plates and grown overnight at 37°C. Strains 1 (SH1535; cysA ΔyliAB::Kan^r), 2 (SH1527; cysA), 3 (SH1525; cysA Δggt ΔyliAB::Kan^r), and 4 (SH1504; cysA Δggt) were grown on minimal medium supplemented with 0.05 mM thiamine without any sulfur source (column a) or with 0.3 mM glutathione (column b) or 0.3 mM cysteine (column c) as a sole sulfur source.