Glutathione (GSH) and its derivative phytochelatin are important binding factors in transition-metal homeostasis in many eukaryotes. Here, we demonstrate that GSH is also involved in chromate, Zn(II), Cd(II), and Cu(II) homeostasis and resistance in Escherichia coli. While the loss of the ability to synthesize GSH influenced metal tolerance in wild-type cells only slightly, GSH was important for residual metal resistance in cells without metal efflux systems. In mutant cells without the P-type ATPase ZntA, the additional deletion of the GSH biosynthesis system led to a strong decrease in resistance to Cd(II) and Zn(II). Likewise, in mutant cells without the P-type ATPase CopA, the removal of GSH led to a strong decrease of Cu(II) resistance. The precursor of GSH, ␥-glutamylcysteine (␥EC), was not able to compensate for a lack of GSH. On the contrary, ␥EC-containing cells were less copper and cadmium tolerant than cells that contained neither ␥EC nor GSH. Thus, GSH may play an important role in trace-element metabolism not only in higher organisms but also in bacteria.Under aerobic growth conditions, either glutathione (GSH; L-␥-glutamyl-L-cysteine-glycine) or the small 12-kDa protein thioredoxin (TrxB) is essential to maintain a reduced environment in the cytosol of Escherichia coli cells (5,27,60,65). Since E. coli thioredoxin reductase can transfer electrons from NADH to glutaredoxin 4 (Grx4, GrxD, or YdhD) and Grx4 can reduce Grx1 (GrxA) and Grx3 (GrxC) (14), E. coli is able to catalyze the reduction of disulfides without GSH. Thus, GSH by itself is not essential for the survival of this bacterium (17).The cellular GSH is kept almost completely reduced (2, 30): the reduced GSH-oxidized GSH (GSSG) couple has a standard redox potential at pH 7.0 of Ϫ240 mV (66). Using a potential of about Ϫ260 mV in vivo (29) and the Nernst equation results in the calculation of a GSH concentration of about 5 mM and a GSSG concentration of about 5 M. Therefore, any change in the GSH concentration is likely to influence the cellular metabolism by changing the redox potential of the cytoplasm and maybe also that of the periplasm (59). A decrease of the GSH concentration by half would increase the cytoplasmic redox potential by 18 mV.GSH is also involved in the osmoadaptation of E. coli (39). As a response to highly osmotic conditions, a mutant strain unable to synthesize trehalose as an osmoprotectant accumulates GSH to a concentration about 10-fold that under normal conditions. The first and quickest response of E. coli to changing osmotic conditions is to change the cytoplasmic potassium concentration (39), and indeed, GSH is needed for the regulation of this pool (13), probably through interaction with the GSH-gated potassium efflux system KefCYabF (43). Moreover, GSH is involved in the detoxification of methylglyoxal (13), although there are GSH-independent pathways of methylglyoxal degradation (44), and resistance to chlorine compounds (7, 67). In bacteria other than E. coli, GSH is essential for thiamine synthesis (19) and ...

Escherichia coli excretes the catecholate siderophore enterobactin in response to iron deprivation. While the mechanisms underlying enterobactin biosynthesis and ferric enterobactin uptake and utilization are widely understood, nearly nothing is known about how enterobactin is exported from the cell. Mutant and highperformance liquid chromatography analyses demonstrated that the outer membrane channel tunnel protein TolC but none of the respective seven resistance nodulation cell division (RND) proteins CusA, AcrB, AcrD, AcrF, MdtF (YhiV), or the twin RND MdtBC (YegNO) was essential for enterobactin export across the outer membrane. Mutant E. coli strains with additional deletion of tolC or the major facilitator entS were growth deficient in iron-depleted medium. Strains with deletion of tolC or entS, but not with deletion of genes encoding RND transporters, excreted very little enterobactin into the growth medium. Enterobactin excretion in E. coli is thus probably a two-step process involving the major facilitator EntS and the outer membrane channel tunnel protein TolC. Quantitative reverse transcription-PCR analysis of gene-specific transcripts showed no significant changes in tolC expression upon iron depletion. However, iron starvation led to increased expression of the RND gene mdtF and a decrease in acrD.Enterobactin (33), also known as enterochelin (30), is the catecholate-type siderophore of Escherichia coli and of several other bacteria. Enterobactin, a cyclic triester of 2,3-dihydroxybenzoylserine (DHBS), is one of the most effective ferric iron chelating compounds known (1, 36). While the molecular processes involved in ferric enterobactin uptake by TonB-energized outer membrane receptor proteins such as FepA (recently reviewed in reference 15) were studied exhaustively over the last decades, a mechanism for enterobactin efflux across the cytoplasmic membrane was discovered only recently. EntS (the ybdA gene product) (5), a member of the vast major facilitator superfamily (MFS) of membrane-bound transporters, was shown to be necessary for effective enterobactin export in E. coli (9). Cells with entS deleted excreted very little enterobactin into the surrounding medium, but degradation products of enterobactin were released into supernatants. Since those degradation products are themselves efficient siderophores and were still exported, strains lacking entS suffered no iron depletion (9).Because enterobactin is too big to diffuse freely through the porins of the outer membrane, transport from the periplasm to the outside has to be accomplished by another still unknown transport system. Previously, we and others (18,20,26,27) could demonstrate that transport systems of the resistance nodulation cell division (RND) type (38) may transport their substrates from the periplasm (or from the cytoplasmic membrane in the case of hydrophobic substances) rather than from the cytoplasm to the outside. At least for copper and cobalt, we provided evidence that efflux is probably a two-step process involving a transp...

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Glutathione-dependent detoxification is a key pathway that allows plants to efficiently remove toxic compounds like heavy metals or electrophilic xenobiotics. Under persistent exposure to toxins plants need to respond to continuous demand with efficient synthesis of glutathione (GSH) and ideally fast and efficient removal of potentially toxic glutathione S-conjugates. With the aim of studying the respective degradation pathway in Physcomitrella patens we initially characterized fluorescence labeling of protonema cultures with GSH-specific xenobiotic monochlorobimane (MCB). Incubation of protonema with 200 μM MCB for 24 h resulted in a steady increase of total bimane label, which was not confined to glutathione S-bimane (GS-B), but predominantly present in γ-glutamylcysteine S-bimane (γ-EC-B) and cysteine S-bimane (Cys-B). Pulse-chase experiments identified γ-EC-B and Cys-B as degradation products of GS-B, suggesting initial cleavage of the C-terminal glycine, followed by cleavage of the γ-glutamyl bond. The amount of GS-B formed, increased linearly at 90 nmol GSH g fw⁻¹ h⁻¹ for 24 h and after ∼1.5 h already surpassed the amount of GSH present in control protonema. This demand-driven biosynthesis of GSH depends on sufficient supply of sulfate in the incubation medium.

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5-dimethylphenylcarbamate)-coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid-liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors.