Disulfide bond formation by exported glutaredoxin indicates glutathione's presence in the E. coli periplasm

Abstract

Organisms have evolved elaborate systems that ensure the homeostasis of the thiol redox environment in their intracellular compartments. In Escherichia coli, the cytoplasm is kept under reducing conditions by the thioredoxins with the help of thioredoxin reductase and the glutaredoxins with the small molecule glutathione and glutathione reductase. As a result, disulfide bonds are constantly resolved in this compartment. In contrast to the cytoplasm, the periplasm of E. coli is maintained in an oxidized state by DsbA, which is recycled by DsbB. Thioredoxin 1, when exported to the periplasm turns from a disulfide bond reductase to an oxidase that, like DsbA, is dependent on DsbB. In this study we set out to investigate whether a subclass of the thioredoxin superfamily, the glutaredoxins, can become disulfide bond-formation catalysts when they are exported to the periplasm. We find that glutaredoxins can promote disulfide bond formation in the periplasm. However, contrary to the behavior of thioredoxin 1 in this environment, the glutaredoxins do so independently of DsbB. Furthermore, we show that glutaredoxin 3 requires the glutathione biosynthesis pathway for its function and can oxidize substrates with only a single active-site cysteine. Our data provides in vivo evidence suggesting that oxidized glutathione is present in the E. coli periplasm in biologically significant concentrations.

Fig. 1.

Redox state of various DsbA substrates. LivK-c-Myc (A), YodA-c-Myc (B), or RcsF-c-Myc (C) were expressed from plasmids (pMER154, pMER155, and pMER156) and detected by Western blot using anti-c-Myc antibody. Strains used were HK295 (wt) and HK453 (ΔdsbA ΔdsbC ΔdsbD) harboring empty vector (pMER79) or plasmids encoding TrxA_P, GrxC_P, or NrdC_P (pMER90, pMER94, and pMER96). Cultures were grown and samples were prepared as described in materials and methods. Where indicated, samples were treated with 100 mM DTT (DTT) before AMS alkylation. The mobility of DTT-treated proteins indicates the position of the reduced form. As a loading control, the same sample volumes were subjected to an immunoblot detecting SecB.

Fig. 2.

Restoration of AP activity by GrxC_P or TrxA_p. Wild-type (MER360), ΔdsbA (MER390), or ΔdsbA ΔdsbB (MER392) strains expressing GrxC_P or TrxA_p from plasmids (pTrc99a-ssTorA-GrxC or pTrc99a-ssTorA-TrxA) were grown in M63 minimal medium at 30 °C, aliquots were taken at early log-phase and AP activity measured as described in materials and methods.

Fig. 3.

Influence of glutathione on GrxC_P-dependent restoration of AP activity. A ΔdsbA ΔdsbB strain (MER392) harboring an empty vector (pMER79) or the same strain expressing GrxC_P from plasmid pTrc99a-ssTorA-GrxC or isogenic strains with a deletion in gshA (MER396, MER382) were grown to log-phase at 30 °C in M63 minimal medium containing 0 μM or 1 μM oxidized glutathione (GSSG). AP activity is expressed as % of wild-type activity.

Fig. 4.

Restoration of AP activity by active site mutants of GrxC_P or TrxA_P. AP activity of strains lacking dsbA (MER390) or dsbA and dsbB (MER392) expressing GrxC_P or TrxA_P active site variants from mutated plasmids derived from pTrc99a-ssTorA-GrxC or pTrc99a-ssTorA-TrxA, respectively. TrxA_P[AA] or GrxC_P[AA] have both active-site cysteines mutated to alanines, GrxC_P[CA] has a Cys-14→Ala mutation, GrxC_P[AC] has a Cys-11→Ala mutation, and GrxC_P[CA]C65Y has Cys-14→Ala and Cys-65→Tyr mutations. TrxA_P[CA] has a Cys-35→Ala mutation and TrxA_P[AC] has a Cys-32→Ala mutation. Strains were grown as in previous experiments.

Fig. 5.

Model for disulfide bond formation by a monothiol glutaredoxin, adopted from (19). A glutathionylated glutaredoxin is attacked by a substrate cysteine, producing reduced glutaredoxin and glutathionylated substrate. In a second step, a second cysteine within the substrate attacks the glutathione-substrate disulfide, resulting in oxidized substrate.