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 ...

The higher affinity of Cd 2؉ for sulfur compounds than for nitrogen and oxygen led to the theoretical consideration that cadmium toxicity should result mainly from the binding of Cd 2؉ to sulfide, thiol groups, and sulfur-rich complex compounds rather than from Cd 2؉ replacement of transition-metal cations from nitrogenor oxygen-rich biological compounds. This hypothesis was tested by using Escherichia coli for a global transcriptome analysis of cells synthesizing glutathione (GSH; wild type), ␥-glutamylcysteine (⌬gshB mutant), or neither of the two cellular thiols (⌬gshA mutant). The resulting data, some of which were validated by quantitative reverse transcription-PCR, were sorted using the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology system, which groups genes hierarchically with respect to the cellular functions of their respective products. The main difference among the three strains concerned tryptophan biosynthesis, which was up-regulated in wild-type cells upon cadmium shock and strongly up-regulated in ⌬gshA cells but repressed in ⌬gshB cells containing ␥-glutamylcysteine instead of GSH. Overall, however, all three E. coli strains responded to cadmium shock similarly, with the up-regulation of genes involved in protein, disulfide bond, and oxidative damage repair; cysteine and iron-sulfur cluster biosynthesis; the production of proteins containing sensitive iron-sulfur clusters; the storage of iron; and the detoxification of Cd 2؉ by efflux. General energy conservation pathways and iron uptake were down-regulated. These findings indicated that the toxic action of Cd 2؉ indeed results from the binding of the metal cation to sulfur, lending support to the hypothesis tested.With the exception of that of copper, the affinities of the borderline metals of the first transition group for the ligands oxygen and sulfur increase in the same manner as those of the metals of the second group, in order from Mn 2ϩ to Zn 2ϩ . In contrast, Cd 2ϩ (and Pb 2ϩ ) has a much higher affinity for sulfur than for oxygen (40). Thus, Cd 2ϩ toxicity should be the result mainly of the affinity of Cd 2ϩ for sulfur. If in a physiological complex, the first-shell ligands around a transition-metal cation are mainly nitrogen or oxygen, Cd 2ϩ should not be able to replace that particular cation. Cadmium should replace the cations, however, if the first shell is composed of sulfur atoms predominantly. Theoretically, cadmium toxicity should be the result of the binding of Cd 2ϩ to sulfide, generated during the biosynthesis of cysteine and of iron-sulfur centers (FeS centers); binding to thiol groups, e.g., of proteins; and the replacement of other transition-metal cations from such sulfur-rich complex compounds.Cellular thiols may interfere with this mode of action of cadmium toxicity. The main cellular thiol in cyanobacteria and proteobacteria is glutathione (GSH), which is absent in many other prokaryotes that contain other thiol compounds like mycothiol and ergothioneine (14, 39). GSH (L-␥-glutamyl-Lcysteine-glycine) is esse...

We have used analytical ultracentrifugation to explore the oligomeric states of AcrB and CusA in micellar solution of detergent. These two proteins belong to the resistance, nodulation and cell division (RND) family of efflux proteins that are involved in multiple drug and heavy metal resistance. Only the structure of AcrB has been determined so far. Although functional RND proteins should assemble as trimers as AcrB does, both AcrB and CusA form a mixture of quaternary structures (from monomer to heavy oligomer) in detergent solution. The distribution of the oligomeric states was studied as a function of different parameters: nature and concentration of the detergent, ionic strength, pH, protein concentration. This pseudo-heterogeneity does not hamper the crystallization of AcrB as a homotrimer.