The role of glutathione in periplasmic redox homeostasis and oxidative protein folding inEscherichia coli

Abstract

The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo redox potential imposed onto protein thiol disulfide pairs in the periplasm remains unknown. Here, we used genetically encoded redox probes (roGFP2 and roGFP-iL), targeted to the periplasm, to directly probe the thiol redox homeostasis in this compartment. These probes contain two cysteine residues, that are virtually completely reduced in the cytoplasm, but once exported into the periplasm, can form a disulfide bond, a process that can be monitored by fluorescence spectroscopy. Even in the absence of DsbA, roGFP2, exported to the periplasm, was fully oxidized, suggesting the presence of an alternative system for the introduction of disulfide bonds into exported proteins. However, the absence of DsbA shifted the periplasmic thiol-redox potential from -228 mV to a more reducing -243 mV and the capacity to re-oxidize periplasmic roGFP2 after a reductive pulse was significantly decreased. Re-oxidation in a DsbA strain could be fully restored by exogenous oxidized glutathione (GSSG), while reduced GSH accelerated re-oxidation of roGFP2 in the WT. In line, a strain devoid of endogenous glutathione showed a more reducing periplasm, and was significantly worse in oxidatively folding PhoA, a native periplasmic protein and substrate of the oxidative folding machinery. PhoA oxidative folding could be enhanced by the addition of exogenous GSSG in the WT and fully restored in a delta dsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.

Comprehensive elucidation of glutathione import inEscherichia coli

“…For the construction of E. coli gshA double mutants, the gshA KEIO mutant without kanamycin cassette generated previously (Knoke et al, 2023) was used as acceptor for P1 transduction as described previously (Thomason et al, 2007). The KEIO gsiB (JW5113-1), gsiC (JW0815-2), gsiD (JW0816-1), oppA (JW1235-1) and oppC (JW1237-3) mutants were used as donors for P1 transduction.…”

“…The Grx1-roGFP2 oxidation state was determined as described before, with minor changes (Degrossoli et al, 2018;Knoke et al, 2023;Xie et al, 2020). Briefly, for determination of the Grx1-roGFP2 oxidation state in different E. coli mutant cells (Table 1), cells were transformed with Grx1-roGFP2 coding plasmids, and after expression of the probes for 16 h at 20 °C, as mentioned above, cells were harvested, washed twice in HEPES (40 mM, pH 7.…”

“…Briefly, for determination of the Grx1-roGFP2 oxidation state in different E. coli mutant cells (Table 1), cells were transformed with Grx1-roGFP2 coding plasmids, and after expression of the probes for 16 h at 20 °C, as mentioned above, cells were harvested, washed twice in HEPES (40 mM, pH 7. Subsequently, the probe's oxidation state was calculated as described before (Knoke et al, 2023;Xie et al, 2020) from the ratios of the fluorescence excitation intensities (405/488 nm).…”

“…Although we showed that neither glutathione reductase, nor EDTA prevented Grx1-roGFP2 reduction by exogenous reduced glutathione in a DgshA DoppC strain, we can, however, not exclude that Gsi might be specific for oxidized glutathione. GSH in solution forms GSSG over time (Stevens et al, 1983) and our efforts with the addition of EDTA or glutathione reductase might have fully inhibited this oxidation, but before entering the cytosol, GSH also needs to pass the periplasm, a highly oxidative environment (Knoke et al, 2023;Manta et al, 2019). In this compartment GSH might get oxidized, especially, when direct GSH import is abolished, and the resulting GSSG could then be transported by Gsi.…”