60NISR Research GRANT
2005 Research Grant

Glutathione Transporter of Escherichia coli: Aiming at theDirect Production of Glutathione
Hideyuki SUZUKI
Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University

Research aims
Like other organisms, E. coli synthesizes glutathione byg -glutamylcysteine synthetase and glutathione synthetase1).
E. coli excretes glutathione into the culture medium during
the exponential phase and glutathione concentration in theculture medium reaches maximum in the early stationary
phase5,10), but thereafter it is hydrolyzed by g -glutamyltranspeptidase (GGT) in the periplasm to liberate glutamic
acid and cysteinylglycine7,10). Glutamic acid and cysteinylglycine are taken up into the cytoplasm to be utilized assource of amino acids9). Even in the case of GGT-deficientstrain of E. coli, however, glutathione concentration in theculture medium decreased gradually after prolonged incubation. This finding prompted us to search for a glutathionetransporter, which had never been reported in bacteria.
The ybiK gene was reported as a member of the cysB
regulon and it was suggested to encode a protein involvedin glutathione transport or metabolism6), but its mechanism
was unclear. GenBank suggests that four genes, yliA, B, Cand D, locate downstream of ybiK, are transcribed withybiK (Figure 1). EcoCys3) suggests that YliA, B, C and D
constitute an uncharacterized member of the ATP-binding

cassette superfamily transporters. It suggests that yliA andB encode an ATP-binding component and a periplasmic
binding protein, respectively, and that yliC and D encode
plasma membrane components. From the above information, we speculated that YliA, B, C and D might encode aglutathione transporter.
Methods
Measurement of glutathione. Reduced glutathione wasmeasured with an HPLC (model LC-9A; Shimadzu)
equipped with a Shim-pack Amino-Na column and a fluorescence detector (model RF-535), with o-phthalaldehyde
as the detection reagent8). Reduced and oxidized glutathione could be measured separately by this method. Totalglutathione was measured with glutathione reductase by themethod of Fahey et al.2)
Transport assay. Transport assay was performed as described previously4) using [35S] glutathione (final 2 nM;
35.4 Tbq/mmol) except M9 glucose medium was used instead of M63 medium. When the effects of verapamil andcarbonylcyanide-m-chlorophenylhydrazone (CCCP) weredetermined, cells were preincubated in the presence of theseFig. 1. The structure of ybiK-yliABCD operon. (A) The order, size and products of the genes, and the location of the promoter suggested by GenBank andEcoCys were diagrammed. The region of DNA deleted in our D yli(A-B) mutation is shown by the bar above the genes. (B) The predicted structure of YliABCD transporter.

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chemicals for 30 min at 37°C prior to the addition of labeled glutathione.
Results
Effect of D ggt and D yli(A-B) mutations on the concentration of extracellular glutathione. Since the wild-type E.
coli accumulates only several m M glutathione at maximum
in the medium and it is difficult to measure such a low concentration of glutathione, the strains were transformed withplasmid pSH1391, which contains gshA and gshB genes on
pBR322, to overproduce glutathione-synthesizing enzymes.
There was little difference in the growth among the strainsused. The effects of D ggt and D yli(A-B) mutations on theconcentration of extracellular glutathione were compared(Figure 2). The glutathione concentration decreased afterreaching maximum during the early stationary phase wheneither ggt or yliAB was normal. On the other hand, the extracellular glutathione of the D ggt D yli(A-B) strain gradually increased even during the stationary phase. When theD ggt D yli(A-B) strain was complemented with pACYC177
containing ybiK-yliABCD operon, the extracellularglutathione was dramatically decreased.
Transport assay of YliABCD transporter. Transport
assay was performed using [35S] glutathione and GGT-deficient derivatives (Figure 3). The D yli(A-B) strain transported practically no glutathione, while its yliAB derivative obviously transported glutathione. Moreover, theD yli(A-B) strain transformed with pACYC177 containingybiK-yliABCD complemented the glutathione transport phenotype. However, the same strain transformed withpACYC177 containing ybiK-yliABC did not complement the phenotype (Figure 3).
Effect of D yli(A-B) mutation on the intracellular concentration of glutathione. Glutathione synthesis-deficient(D gshA) derivatives of the above strains were grownovernight in the minimal medium supplemented with andwithout 1 mM reduced glutathione. The cells were thenlysed by ultrasonication and the amount of glutathione accumulated inside the cells was measured by HPLC (FigureFig. 2. Glutathione concentration of the culture media. Strains SI37
(D ggt D yli(A-B); red square), SI49 (D ggt; blue square), SH1555(pACYC177::ybiK-yliABCD/D ggt D yli(A-B); yellow triangle),
SI103 (D yli(A-B); blue circle), and SI104 (ggt yliAB; green circle)
were grown in 100 ml minimal medium. At the time indicated, 2-ml of culture was subtracted. OD610 was measured using 1 ml of the 2 ml-culture.
Another 1 ml was centrifuged and the concentration of glutathione of theculture fluid was measured with glutathione reductase.

4). All the glutathione found was reduced form and no oxidized form was observed. The amount of total glutathione
measured with glutathione reductase agreed well with thatof reduced glutathione found by HPLC (data not shown).
When these four strains were grown in the minimal
medium without glutathione, no detectable glutathione wasfound inside these strains. Although the D yli(A-B) mutation
decreased the accumulation of glutathione inside the cells,
non-negligible accumulation of glutathione was observed
even in the strain with D yli(A-B) D ggt D gshA (strainSI100). The D yli(A-B) mutation was complemented withpACYC177 containing ybiK-yliABCD (strainSI154), but not with pACYC177 containing ybiK yliABC (strain SI153).
Transport of glutathione by YliABCD transporter depends on ATPase activity. Effect of ATPase inhibitor verapamil on glutathione transport by YliABCD transporter
was determined. Transport of glutathione by YliABCD
transporter was strongly inhibited in the presence of 10 mM
verapamil (Figure 5). Membrane potential inhibitor, CCCP,

Fig. 3. Glutathione uptake in a transporter assay. Strain SI35 (D ggt
D yli(A-B); green circles), SH703 (D ggt; yellow circles), SH1552(pACYC177::ybiK-yliABCD/D ggt D yli(A-B); pink squares).

Fig. 4. Accumulation of glutathione in the cells grown in minimal
medium supplemented with 1 mM glutathione. Strains SI100(pACYC177/D gshA D ggt D yli(A-B)), SI109 (pACYC177/D gshA D ggt),SI153 (pACYC177::ybiK-yliABC/D gshA D ggt D yli(A-B)), SI154(pACYC177::ybiK-yliABCD/D gshA D ggt D yli(A-B)) were grown inminimal medium supplemented with 1 mM reduced glutathione for 12 h.
The amount of glutathione found in the cells was expressed as relative tostrain SI154. Seventy four nmole of glutathione per mg-cells were foundin strain SI154.

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ing protein, respectively, and YliC and D being plasma
membrane components.
This is the first report not only of bacterial glutathionetransporter, but also of a glutathione importer with ATPbinding cassette among all organisms. The homology
search suggests that Escherichia coli O157 : H7, Shigellaflexneri, Salmonella enterica serovar Typhi, and Salmonella
enterica serovar Typhimurium have homologues. Our finding of a new glutathione importer with ATP-binding cassette indicates that there is more diversity in the mechanismof glutathione transport across cell membranes than previously considered.
References

Fig. 5. Effect of verapamil on glutathione uptake. Glutathione uptake ofstrain SH1552 was measured in the absence of verapamil (pink circles),and in the presence of 10 mM verapamil (blue triangles).

had no effect at 100 m M (data not shown).
Effect of D yli(A-B) mutation on the cell growth. The
ability of YliABCD transporter to utilize glutathione as asole sulfur source was also investigated. The cysA gene encodes a sulfate permease and cysA mutants cannot growwith SO42 in the medium; they are cysteine auxotrophs.
The cysA D ggt (sulfate transport and GGT deficient) straingrew weakly on minimal medium with 0.3 mM glutathione
as a sole sulfur source, while almost no growth was observed for its D yli(A-B) derivative on the same plate. The
doubling times of cysA, cysA D yli(A-B), cysA D ggt, andcysA D yli(A-B) D ggt strains in minimal medium supplemented with 0.3 mM glutathione at 37°C were 1.7, 2.1, 3.2,
and 12 h, respectively.
Conclusion
All of the results indicate that YliABCD compose anATP-binding cassette superfamily transporter, YliA and B
being an ATP-binding component and a periplasmic bind-1) P. Apontoweil and W. Berends. 1975. Glutathione biosynthesis in Escherichia coli K 12. Properties of the enzymes and regulation.
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