The oxidative stress response, in particular the katY gene, is temperature-regulated in Yersinia pseudotuberculosis.

Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5'-untranslated region (5'-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37°C. The trxA gene is transcribed into two mRNA isoforms, of which the most abundant short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5'-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region of katY we discovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37°C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with the upregulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37°C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature.

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx).

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

Functional metagenomics of the thioredoxin superfamily.

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22,000 thioredoxins found in the Global Ocean Sampling data set. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (ΔtrxA), isomerase (ΔdsbC), or oxidase (ΔdsbA). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified, and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

The effects of neutrophil-generated hypochlorous acid and other hypohalous acids on host and pathogens.

Neutrophils are predominant immune cells that protect the human body against infections by deploying sophisticated antimicrobial strategies including phagocytosis of bacteria and neutrophil extracellular trap (NET) formation. Here, we provide an overview of the mechanisms by which neutrophils kill exogenous pathogens before we focus on one particular weapon in their arsenal: the generation of the oxidizing hypohalous acids HOCl, HOBr and HOSCN during the so-called oxidative burst by the enzyme myeloperoxidase. We look at the effects of these hypohalous acids on biological systems in general and proteins in particular and turn our attention to bacterial strategies to survive HOCl stress. HOCl is a strong inducer of protein aggregation, which bacteria can counteract by chaperone-like holdases that bind unfolding proteins without the need for energy in the form of ATP. These chaperones are activated by HOCl through thiol oxidation (Hsp33) or N-chlorination of basic amino acid side-chains (RidA and CnoX) and contribute to bacterial survival during HOCl stress. However, neutrophil-generated hypohalous acids also affect the host system. Recent studies have shown that plasma proteins act not only as sinks for HOCl, but get actively transformed into modulators of the cellular immune response through N-chlorination. N-chlorinated serum albumin can prevent aggregation of proteins, stimulate immune cells, and act as a pro-survival factor for immune cells in the presence of cytotoxic antigens. Finally, we take a look at the emerging role of HOCl as a potential signaling molecule, particularly its role in neutrophil extracellular trap formation.

Redox regulation in host-pathogen interactions: thiol switches and beyond.

Our organism is exposed to pathogens on a daily basis. Owing to this age-old interaction, both pathogen and host evolved strategies to cope with these encounters. Here, we focus on the consequences of the direct encounter of cells of the innate immune system with bacteria. First, we will discuss the bacterial strategies to counteract powerful reactive species. Our emphasis lies on the effects of hypochlorous acid (HOCl), arguably the most powerful oxidant produced inside the phagolysosome of professional phagocytes. We will highlight individual examples of proteins in gram-negative bacteria activated by HOCl via thiol-disulfide switches, methionine sulfoxidation, and N-chlorination of basic amino acid side chains. Second, we will discuss the effects of HOCl on proteins of the host. Recent studies have shown that both host and bacteria address failing protein homeostasis by activation of chaperone-like holdases through N-chlorination. After discussing the role of individual proteins in the HOCl-defense, we will turn our attention to the examination of effects on host and pathogen on a systemic level. Recent studies using genetically encoded redox probes and redox proteomics highlight differences in redox homeostasis in host and pathogen and give first hints at potential cellular HOCl signaling beyond thiol-disulfide switch mechanisms.

Comparison of Proteomic Responses as Global Approach to Antibiotic Mechanism of Action Elucidation.

New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.

Allicin, a natural antimicrobial defence substance from garlic, inhibits DNA gyrase activity in bacteria.

Allicin (diallylthiosulfinate) is a potent antimicrobial substance, produced by garlic tissues upon wounding as a defence against pathogens and pests. Allicin is a reactive sulfur species (RSS) that oxidizes accessible cysteines in glutathione and proteins. We used a differential isotopic labelling method (OxICAT) to identify allicin targets in the bacterial proteome. We compared the proteomes of allicin-susceptible Pseudomonas fluorescens Pf0-1 and allicin-tolerant PfAR-1 after a sublethal allicin exposure. Before exposure to allicin, proteins were in a predominantly reduced state, with approximately 77% of proteins showing less than 20% cysteine oxidation. Protein oxidation increased after exposure to allicin, and only 50% of proteins from allicin-susceptible Pf0-1, but 65% from allicin-tolerant PfAR-1, remained less than 20% oxidised. DNA gyrase was identified as an allicin target. Cys433 in DNA gyrase subunit A (GyrA) was approximately 6% oxidized in untreated bacteria. After allicin treatment the degree of Cys433 oxidation increased to 55% in susceptible Pf0-1 but only to 10% in tolerant PfAR-1. Allicin inhibited E. coli DNA gyrase activity in vitro in the same concentration range as nalidixic acid. Purified PfAR-1 DNA gyrase was inhibited to greater extent by allicin in vitro than the Pf0-1 enzyme. Substituting PfAR-1 GyrA into Pf0-1 rendered the exchange mutants more susceptible to allicin than the Pf0-1 wild type. Taken together, these results suggest that GyrA was protected from oxidation in vivo in the allicin-tolerant PfAR-1 background, rather than the PfAR-1 GyrA subunit being intrinsically less susceptible to oxidation by allicin than the Pf0-1 GyrA subunit. DNA gyrase is a target for medicinally important antibiotics; thus, allicin and its analogues may have potential to be developed as gyrase inhibitors, either alone or in conjunction with other therapeutics.

Antimicrobial properties of ternary eutectic aluminum alloys.

Several Escherichia coli deletion mutants of the Keio collection were selected for analysis to better understand which genes may play a key role in copper or silver homeostasis. Each of the selected E. coli mutants had a deletion of a single gene predicted to encode proteins for homologous recombination or contained functions directly linked to copper or silver transport or transformation. The survival of these strains on pure copper surfaces, stainless steel, and alloys of aluminum, copper and/or silver was investigated. When exposed to pure copper surfaces, E. coli ΔcueO was the most sensitive, whereas E. coli ΔcopA was the most resistant amongst the different strains tested. However, we observed a different trend in sensitivities in E. coli strains upon exposure to alloys of the system Al-Ag-Cu. While minor antimicrobial effects were detected after exposure of E. coli ΔcopA and E. coli ΔrecA to Al-Ag alloys, no effect was detected after exposure to Al-Cu alloys. The release of copper ions and cell-associated copper ion concentrations were determined for E. coli ΔcopA and the wild-type E. coli after exposure to pure copper surfaces. Altogether, compared to binary alloys, ternary eutectic alloys (Al-Ag-Cu) had the highest antimicrobial effect and thus, warrant further investigation.

In silico approach to designing rational metagenomic libraries for functional studies.

BACKGROUND: With the development of Next Generation Sequencing technologies, the number of predicted proteins from entire (meta-) genomes has risen exponentially. While for some of these sequences protein functions can be inferred from homology, an experimental characterization is still a requirement for the determination of protein function. However, functional characterization of proteins cannot keep pace with our capabilities to generate more and more sequence data. RESULTS: Here, we present an approach to reduce the number of proteins from entire (meta-) genomes to a reasonably small number for further experimental characterization without loss of important information. About 6.1 million predicted proteins from the Global Ocean Sampling Expedition Metagenome project were distributed into classes based either on homology to existing hidden markov models (HMMs) of known families, or de novo by assessment of pairwise similarity. 5.1 million of these proteins could be classified in this way, yielding 18,437 families. For 4,129 protein families, which did not match existing HMMs from databases, we could create novel HMMs. For each family, we then selected a representative protein, which showed the closest homology to all other proteins in this family. We then selected representatives of four families based on their homology to known and well-characterized lipases. From these four synthesized genes, we could obtain the novel esterase/lipase GOS54, validating our approach. CONCLUSIONS: Using an in silico approach, we were able improve the success rate of functional screening and make entire (meta-) genomes amenable for biochemical characterization.

Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines.

Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.

Proteome analysis reveals differential expression of proteins involved in triacylglycerol accumulation by Rhodococcus jostii RHA1 after addition of methyl viologen.

Rhodococcus jostii RHA1 is able to degrade toxic compounds and accumulate high amounts of triacylglycerols (TAG) upon nitrogen starvation. These NADPH-dependent processes are essential for the adaptation of rhodococci to fluctuating environmental conditions. In this study, we used an MS-based, label-free and quantitative proteomic approach to better understand the integral response of R. jostii RHA1 to the presence of methyl viologen (MV) in relation to the synthesis and accumulation of TAG. The addition of MV promoted a decrease of TAG accumulation in comparison to cells cultivated under nitrogen-limiting conditions in the absence of this pro-oxidant. Proteomic analyses revealed that the abundance of key proteins of fatty acid biosynthesis, the Kennedy pathway, glyceroneogenesis and methylmalonyl-CoA pathway, among others, decreased in the presence of MV. In contrast, some proteins involved in lipolysis and ß-oxidation of fatty acids were upregulated. Some metabolic pathways linked to the synthesis of NADPH remained activated during oxidative stress as well as under nitrogen starvation conditions. Additionally, exposure to MV resulted in the activation of complete antioxidant machinery comprising superoxide dismutases, catalases, mycothiol biosynthesis, mycothione reductase and alkyl hydroperoxide reductases, among others. Our study suggests that oxidative stress response affects TAG accumulation under nitrogen-limiting conditions through programmed molecular mechanisms when both stresses occur simultaneously.

Label-free and redox proteomic analyses of the triacylglycerol-accumulating Rhodococcus jostii RHA1.

The bacterium Rhodococcus jostii RHA1 synthesizes large amounts of triacylglycerols (TAGs) under conditions of nitrogen starvation. To better understand the molecular mechanisms behind this process, we performed proteomic studies in this oleaginous bacterium. Upon nitrogen starvation, we observed a re-routing of the carbon flux towards the formation of TAGs. Under these conditions, the cellular lipid content made up more than half of the cell's dry weight. On the proteome level, this coincided with a shift towards non-glycolytic carbohydrate-metabolizing pathways. These pathways (Entner-Doudoroff and pentose-phosphate shunt) contribute NADPH and precursors of glycerol 3-phosphate and acetyl-CoA to lipogenesis. The expression of proteins involved in the degradation of branched-chain amino acids and the methylmalonyl-CoA pathway probably provided propionyl-CoA for the biosynthesis of odd-numbered fatty acids, which make up almost 30 % of RHA1 fatty acid composition. Additionally, lipolytic and glycerol-degrading enzymes increased in abundance, suggesting a dynamic cycling of cellular lipids. Conversely, abundance of proteins involved in consuming intermediates of lipogenesis decreased. Furthermore, we identified another level of lipogenesis regulation through redox-mediated thiol modification in R. jostii. Enzymes affected included acetyl-CoA carboxylase and a ß-ketoacyl-[acyl-carrier protein] synthase II (FabF). An integrative metabolic model for the oleaginous RHA1 strain is proposed on the basis of our results.

Incidence and physiological relevance of protein thiol switches.

A few small-molecule oxidants, most notably hydrogen peroxide, can act as messengers in signal transduction. They trigger so-called 'thiol switches', cysteine residues that are reversibly oxidized to transiently change the functional properties of their host proteins. The proteome-wide identification of functionally relevant 'thiol switches' is of significant interest. Unfortunately, prediction of redox-active cysteine residues on the basis of surface accessibility and other computational parameters appears to be of limited use. Proteomic thiol labeling approaches remain the most reliable strategy to discover new thiol switches in a hypothesis-free manner. We discuss if and how genomic knock-in strategies can help establish the physiological relevance of a 'thiol switch' on the organismal level. We conclude that surprisingly few attempts have been made to thoroughly verify the physiological relevance of thiol-based redox switches in mammalian model organisms.

Redox proteomics uncovers peroxynitrite-sensitive proteins that help Escherichia coli to overcome nitrosative stress.

Peroxynitrite is a highly reactive chemical species with antibacterial properties that are synthesized in immune cells. In a proteomic approach, we identified specific target proteins of peroxynitrite-induced modifications in Escherichia coli. Although peroxynitrite caused a fairly indiscriminate nitration of tyrosine residues, reversible modifications of protein thiols were highly specific. We used a quantitative redox proteomic method based on isotope-coded affinity tag chemistry and identified four proteins consistently thiol-modified in cells treated with peroxynitrite as follows: AsnB, FrmA, MaeB, and RidA. All four were required for peroxynitrite stress tolerance in vivo. Three of the identified proteins were modified at highly conserved cysteines, and MaeB and FrmA are known to be directly involved in the oxidative and nitrosative stress response in E. coli. In in vitro studies, we could show that the activity of RidA, a recently discovered enamine/imine deaminase, is regulated in a specific manner by the modification of its single conserved cysteine. Mutation of this cysteine 107 to serine generated a constitutively active protein that was not susceptible to peroxynitrite.

The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis.

The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.

About the dangers, costs and benefits of living an aerobic lifestyle.

The era in which ROS (reactive oxygen species) were simply the 'bad boys of biology' is clearly over. High levels of ROS are still rightfully considered to be toxic to many cellular processes and, as such, contribute to disease conditions and cell death. However, the high toxicity of ROS is also extremely beneficial, particularly as it is used to kill invading micro-organisms during mammalian host defence. Moreover, a transient, often more localized, increase in ROS levels appears to play a major role in signal transduction processes and positively affects cell growth, development and differentiation. At the heart of all these processes are redox-regulated proteins, which use oxidation-sensitive cysteine residues to control their function and by extension the function of the pathways that they are part of. Our work has contributed to changing the view about ROS through: (i) our characterization of Hsp33 (heat-shock protein 33), one of the first redox-regulated proteins identified, whose function is specifically activated by ROS, (ii) the development of quantitative tools that reveal extensive redox-sensitive processes in bacteria and eukaryotes, and (iii) the discovery of a link between early exposure to oxidants and aging. Our future research programme aims to generate an integrated and system-wide view of the beneficial and deleterious effects of ROS with the central goal to develop more effective antioxidant strategies and more powerful antimicrobial agents.

Nonnative disulfide bond formation activates the σ32-dependent heat shock response in Escherichia coli.

Formation of nonnative disulfide bonds in the cytoplasm, so-called disulfide stress, is an integral component of oxidative stress. Quantification of the extent of disulfide bond formation in the cytoplasm of Escherichia coli revealed that disulfide stress is associated with oxidative stress caused by hydrogen peroxide, paraquat, and cadmium. To separate the impact of disulfide bond formation from unrelated effects of these oxidative stressors in subsequent experiments, we worked with two complementary approaches. We triggered disulfide stress either chemically by diamide treatment of cells or genetically in a mutant strain lacking the major disulfide-reducing systems TrxB and Gor. Studying the proteomic response of E. coli exposed to disulfide stress, we found that intracellular disulfide bond formation is a particularly strong inducer of the heat shock response. Real-time quantitative PCR experiments showed that disulfide stress induces the heat shock response in E. coli σ(32) dependently. However, unlike heat shock treatment, which induces these genes transiently, transcripts of σ(32)-dependent genes accumulated over time in disulfide stress-treated cells. Analyzing the stability of σ(32), we found that this constant induction can be attributed to an increase of the half-life of σ(32) upon disulfide stress. This is concomitant with aggregation of E. coli proteins treated with diamide. We conclude that oxidative stress triggers the heat shock response in E. coli σ(32) dependently. The component of oxidative stress responsible for the induction of heat shock genes is disulfide stress. Nonnative disulfide bond formation in the cytoplasm causes protein unfolding. This stabilizes σ(32) by preventing its DnaK- and FtsH-dependent degradation.

Extracting iron and manganese from bacteria with ionophores - a mechanism against competitors characterized by increased potency in environments low in micronutrients.

To maintain their metal ion homeostasis, bacteria critically depend on membrane integrity and controlled ion translocation. Terrestrial Streptomyces species undermine the function of the cytoplasmic membrane as diffusion barrier for metal cations in competitors using ionophores. Although the properties of the divalent cation ionophores calcimycin and ionomycin have been characterized to some extent in vitro, their effects on bacterial ion homeostasis, the factors leading to bacterial cell death, and their ecological role are poorly understood. To gain insight into their antibacterial mechanism, we determined the metal ion composition of the soil bacterium Bacillus subtilis after treatment with calcimycin and ionomycin. Within 15 min the cells lost approximately half of their cellular iron and manganese content whereas calcium levels increased. The proteomic response of B. subtilis provided evidence that disturbance of metal cation homeostasis is accompanied by intracellular oxidative stress, which was confirmed with a ROS-specific fluorescent probe. B. subtilis showed enhanced sensitivity to the ionophores in medium lacking iron or manganese. Furthermore, in the presence of ionophores bacteria were sensitive to high calcium levels. These findings suggest that divalent cation ionophores are particularly effective against competing microorganisms in soils rich in available calcium and low in available iron and manganese.

Global approaches for protein thiol redox state detection.

Due to its nucleophilicity, the thiol group of cysteine is chemically very versatile. Hence, cysteine often has important functions in a protein, be it as the active site or, in extracellular proteins, as part of a structural disulfide. Within the cytosol, cysteines are typically reduced. But the nucleophilicity of its thiol group makes it also particularly prone to post-translational oxidative modifications. These modifications often lead to an alteration of the function of the affected protein and are reversible in vivo, e.g. by the thioredoxin and glutaredoxin system. The in vivo-reversible nature of these modifications and their genesis in the presence of localized high oxidant levels led to the paradigm of thiol-based redox regulation, the adaptation, and modulation of the cellular metabolism in response to oxidative stimuli by thiol oxidation in regulative proteins. Consequently, the proteomic study of these oxidative posttranslational modifications of cysteine plays an indispensable role in redox biology.

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

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 "steady state" 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 almost 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 steady state 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 ΔdsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.