Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2, and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes.

Unusual 1-3 peptidoglycan cross-links in Acetobacteraceae are made by L,D-transpeptidases with a catalytic domain distantly related to YkuD domains.

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.

The characterisation of Wickerhamomyces anomalus M15, a highly tolerant yeast for bioethanol production using seaweed derived medium.

Advanced generation biofuels have potential for replacing fossil fuels as society moves forward into a net-zero carbon future. Marine biomass is a promising source of fermentable sugars for fermentative bioethanol production; however the medium derived from seaweed hydrolysis contains various inhibitors, such as salts that affected ethanol fermentation efficiency. In this study the stress tolerance of a marine yeast, Wickerhamomyces anomalus M15 was characterised. Specific growth rate analysis results showed that Wickerhamomyces anomalus M15 could tolerate up to 600 g/L glucose, 150 g/L xylose and 250 g/L ethanol, respectively. Using simulated concentrated seaweed hydrolysates, W. anomalus M15's bioethanol production potential using macroalgae derived feedstocks was assessed, in which 5.8, 45.0, and 19.9 g/L ethanol was produced from brown (Laminaria digitata), green (Ulva linza) and red seaweed (Porphyra umbilicalis) based media. The fermentation of actual Ulva spp. hydrolysate harvested from United Kingdom shores resulted in a relatively low ethanol concentration (15.5 g/L) due to challenges that arose from concentrating the seaweed hydrolysate. However, fed-batch fermentation using simulated concentrated green seaweed hydrolysate achieved a concentration of 73 g/L ethanol in fermentations using both seawater and reverse osmosis water. Further fermentations conducted with an adaptive strain W. anomalus M15-500A showed improved bioethanol production of 92.7 g/L ethanol from 200 g/L glucose and reduced lag time from 93 h to 24 h in fermentation with an initial glucose concentration of 500 g/L. The results indicated that strains W. anomalus M15 and W. anomalus M15-500A have great potential for industrial bioethanol production using marine biomass derived feedstocks. It also suggested that if a concentrated high sugar content seaweed hydrolysate could be obtained, the bioethanol concentration could achieve 90 g/L or above, exceeding the minimum industrial production threshold.

The utilization of seawater for the hydrolysis of macroalgae and subsequent bioethanol fermentation.

A novel seawater-based pretreatment process was developed to improve the hydrolysis yield of brown (Laminaria digitata), green (Ulva linza) and red (Porphyra umbilicalis) macroalgae. Pre-treated with 5% sulphuric acid at 121 °C, 15 minutes, L. digitata, U. linza and P. umbilicalis liberated 64.63 ± 0.30%, 69.19 ± 0.11% and 63.03 ± 0.04% sugar in seawater compared with 52.82 ± 0.16%, 45.93 ± 0.37% and 48.60 ± 0.07% in reverse-osmosis water, respectively. Low hydrolysis yields (2.6-11.7%) were observed in alkali and hydrothermal pretreatment of macroalgae, although seawater led to relatively higher yields. SEM images of hydrolyzed macroalgae showed that reverse-osmosis water caused contortions in the remaining cell walls following acid and hydrothermal pre-treatments in the L. digitata and U. linza samples. Fed-batch fermentations using concentrated green seaweed hydrolysates and seawater with marine yeast Wickerhamomyces anomalus M15 produced 48.24 ± 0.01 g/L ethanol with an overall yield of 0.329 g/g available sugars. Overall, using seawater in hydrolysis of seaweed increased sugar hydrolysis yield and subsequent bioethanol production.

A Role for COX20 in Tolerance to Oxidative Stress and Programmed Cell Death in Saccharomyces cerevisiae.

Industrial production of bioethanol from lignocellulosic materials (LCM's) is reliant on a microorganism being tolerant to the stresses inherent to fermentation. Previous work has highlighted the importance of a cytochrome oxidase chaperone gene (COX20) in improving yeast tolerance to acetic acid, a common inhibitory compound produced during pre-treatment of LCM's. The presence of acetic acid has been shown to induce oxidative stress and programmed cell death, so the role of COX20 in oxidative stress was determined. Analysis using flow cytometry revealed that COX20 expression was associated with reduced levels of reactive oxygen species (ROS) in hydrogen peroxide and metal-induced stress, and there was a reduction in apoptotic and necrotic cells when compared with a strain without COX20. Results on the functionality of COX20 have revealed that overexpression of COX20 induced respiratory growth in Δimp1 and Δcox18, two genes whose presence is essential for yeast respiratory growth. COX20 also has a role in protecting the yeast cell against programmed cell death.

The Development of a Sorghum Bran-Based Biorefining Process to Convert Sorghum Bran into Value Added Products.

Sorghum bran, a starch rich food processing waste, was investigated for the production of glucoamylase in submerged fungal fermentation using Aspergillus awamori. The fermentation parameters, such as cultivation time, substrate concentration, pH, temperature, nitrogen source, mineral source and the medium loading ratio were investigated. The glucoamylase activity was improved from 1.90 U/mL in an initial test, to 19.3 U/mL at 10% (w/v) substrate concentration, pH 6.0, medium loading ratio of 200 mL in 500 mL shaking flask, with the addition of 2.5 g/L yeast extract and essential minerals. Fermentation using 2 L bioreactors under the optimum conditions resulted in a glucoamylase activity of 23.5 U/mL at 72 h, while further increase in sorghum bran concentration to 12.5% (w/v) gave an improved gluco-amylase activity of 37.6 U/mL at 115 h. The crude glucoamylase solution was used for the enzymatic hydrolysis of the sorghum bran. A sorghum bran hydrolysis carried out at 200 rpm, 55 °C for 48 h at a substrate loading ratio of 80 g/L resulted in 11.7 g/L glucose, similar to the results obtained using commercial glucoamylase. Large-scale sorghum bran hydrolysis in 2 L bioreactors using crude glucoamylase solution resulted in a glucose concentration of 38.7 g/L from 200 g/L sorghum bran, corresponding to 94.1% of the theoretical hydrolysis yield.

The establishment of a marine focused biorefinery for bioethanol production using seawater and a novel marine yeast strain.

Current technologies for bioethanol production rely on the use of freshwater for preparing the fermentation media and use yeasts of a terrestrial origin. Life cycle assessment has suggested that between 1,388 to 9,812 litres of freshwater are consumed for every litre of bioethanol produced. Hence, bioethanol is considered a product with a high-water footprint. This paper investigated the use of seawater-based media and a novel marine yeast strain 'Saccharomyces cerevisiae AZ65' to reduce the water footprint of bioethanol. Results revealed that S. cerevisiae AZ65 had a significantly higher osmotic tolerance when compared with the terrestrial reference strain. Using 15-L bioreactors, S. cerevisiae AZ65 produced 93.50 g/L ethanol with a yield of 83.33% (of the theoretical yield) and a maximum productivity of 2.49 g/L/h when using seawater-YPD media. This approach was successfully applied using an industrial fermentation substrate (sugarcane molasses). S. cerevisiae AZ65 produced 52.23 g/L ethanol using molasses media prepared in seawater with a yield of 73.80% (of the theoretical yield) and a maximum productivity of 1.43 g/L/h. These results demonstrated that seawater can substitute freshwater for bioethanol production without compromising production efficiency. Results also revealed that marine yeast is a potential candidate for use in the bioethanol industry especially when using seawater or high salt based fermentation media.

The application of phenotypic microarray analysis to anti-fungal drug development.

Candida albicans metabolic activity in the presence and absence of acetylcholine was measured using phenotypic microarray analysis. Acetylcholine inhibited C. albicans biofilm formation by slowing metabolism independent of biofilm forming capabilities. Phenotypic microarray analysis can therefore be used for screening compound libraries for novel anti-fungal drugs and measuring antifungal resistance.

A New Isolation and Evaluation Method for Marine-Derived Yeast spp. with Potential Applications in Industrial Biotechnology.

Yeasts that are present in marine environments have evolved to survive hostile environments that are characterized by high exogenous salt content, high concentrations of inhibitory compounds, and low soluble carbon and nitrogen levels. Therefore, yeasts isolated from marine environments could have interesting characteristics for industrial applications. However, the application of marine yeast in research or industry is currently very limited owing to the lack of a suitable isolation method. Current methods for isolation suffer from fungal interference and/or low number of yeast isolates. In this paper, an efficient and non-laborious isolation method has been developed and successfully isolated large numbers of yeasts without bacterial or fungal growth. The new method includes a three-cycle enrichment step followed by an isolation step and a confirmation step. Using this method, 116 marine yeast strains were isolated from 14 marine samples collected in the UK, Egypt, and the USA. These strains were further evaluated for the utilization of fermentable sugars (glucose, xylose, mannitol, and galactose) using a phenotypic microarray assay. Seventeen strains with higher sugar utilization capacity than the reference terrestrial yeast Saccharomyces cerevisiae NCYC 2592 were selected for identification by sequencing of the ITS and D1/D2 domains. These strains belonged to six species: S. cerevisiae, Candida tropicalis, Candida viswanathii, Wickerhamomyces anomalus, Candida glabrata, and Pichia kudriavzevii. The ability of these strains for improved sugar utilization using seawater-based media was confirmed and, therefore, they could potentially be utilized in fermentations using marine biomass in seawater media, particularly for the production of bioethanol and other biochemical products.

Expression of Mitochondrial Cytochrome C Oxidase Chaperone Gene (COX20) Improves Tolerance to Weak Acid and Oxidative Stress during Yeast Fermentation.

INTRODUCTION: Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid. RESULTS: A Δcox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a Δcox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress. CONCLUSION: This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress.

Screening of Non- Saccharomyces cerevisiae Strains for Tolerance to Formic Acid in Bioethanol Fermentation.

Formic acid is one of the major inhibitory compounds present in hydrolysates derived from lignocellulosic materials, the presence of which can significantly hamper the efficiency of converting available sugars into bioethanol. This study investigated the potential for screening formic acid tolerance in non-Saccharomyces cerevisiae yeast strains, which could be used for the development of advanced generation bioethanol processes. Spot plate and phenotypic microarray methods were used to screen the formic acid tolerance of 7 non-Saccharomyces cerevisiae yeasts. S. kudriavzeii IFO1802 and S. arboricolus 2.3319 displayed a higher formic acid tolerance when compared to other strains in the study. Strain S. arboricolus 2.3319 was selected for further investigation due to its genetic variability among the Saccharomyces species as related to Saccharomyces cerevisiae and availability of two sibling strains: S. arboricolus 2.3317 and 2.3318 in the lab. The tolerance of S. arboricolus strains (2.3317, 2.3318 and 2.3319) to formic acid was further investigated by lab-scale fermentation analysis, and compared with S. cerevisiae NCYC2592. S. arboricolus 2.3319 demonstrated improved formic acid tolerance and a similar bioethanol synthesis capacity to S. cerevisiae NCYC2592, while S. arboricolus 2.3317 and 2.3318 exhibited an overall inferior performance. Metabolite analysis indicated that S. arboricolus strain 2.3319 accumulated comparatively high concentrations of glycerol and glycogen, which may have contributed to its ability to tolerate high levels of formic acid.

Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model.

Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

The genetic basis of variation in clean lineages of Saccharomyces cerevisiae in response to stresses encountered during bioethanol fermentations.

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of monomeric sugars into bioethanol. Industrial bioethanol fermentations are intrinsically stressful environments for yeast and the adaptive protective response varies between strain backgrounds. With the aim of identifying quantitative trait loci (QTL's) that regulate phenotypic variation, linkage analysis on six F1 crosses from four highly divergent clean lineages of S. cerevisiae was performed. Segregants from each cross were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Tolerance levels within populations of F1 segregants to stress conditions differed and displayed transgressive variation. Linkage analysis resulted in the identification of QTL's for tolerance to weak acid and osmotic stress. We tested candidate genes within loci identified by QTL using reciprocal hemizygosity analysis to ascertain their contribution to the observed phenotypic variation; this approach validated a gene (COX20) for weak acid stress and a gene (RCK2) for osmotic stress. Hemizygous transformants with a sensitive phenotype carried a COX20 allele from a weak acid sensitive parent with an alteration in its protein coding compared with other S. cerevisiae strains. RCK2 alleles reveal peptide differences between parental strains and the importance of these changes is currently being ascertained.

The kinetics of inhibitor production resulting from hydrothermal deconstruction of wheat straw studied using a pressurised microwave reactor.

BACKGROUND: The use of a microwave synthesis reactor has allowed kinetic data for the hydrothermal reactions of straw biomass to be established from short times, avoiding corrections required for slow heating in conventional reactors, or two-step heating. Access to realistic kinetic data is important for predictions of optimal reaction conditions for the pretreatment of biomass for bioethanol processes, which is required to minimise production of inhibitory compounds and to maximise sugar and ethanol yields. RESULTS: The gravimetric loss through solubilisation of straw provided a global measure of the extent of hydrothermal deconstruction. The kinetic profiles of furan and lignin-derived inhibitors were determined in the hydrothermal hydrolysates by UV analysis, with concentrations of formic and acetic acid determined by HPLC. Kinetic analyses were either carried out by direct fitting to simple first order equations or by numerical integration of sequential reactions. CONCLUSIONS: A classical Arrhenius activation energy of 148 kJmol-1 has been determined for primary solubilisation, which is higher than the activation energy associated with historical measures of reaction severity. The gravimetric loss is primarily due to depolymerisation of the hemicellulose component of straw, but a minor proportion of lignin is solubilised at the same rate and hence may be associated with the more hydrophilic lignin-hemicellulose interface. Acetic acid is liberated primarily from hydrolysis of pendant acetate groups on hemicellulose, although this occurs at a rate that is too slow to provide catalytic enhancement to the primary solubilisation reactions. However, the increase in protons may enhance secondary reactions leading to the production of furans and formic acid. The work has suggested that formic acid may be formed under these hydrothermal conditions via direct reaction of sugar end groups rather than furan breakdown. However, furan degradation is found to be significant, which may limit ultimate quantities generated in hydrolysate liquors.

Tolerance of pentose utilising yeast to hydrogen peroxide-induced oxidative stress.

BACKGROUND: Bioethanol fermentations follow traditional beverage fermentations where the yeast is exposed to adverse conditions such as oxidative stress. Lignocellulosic bioethanol fermentations involve the conversion of pentose and hexose sugars into ethanol. Environmental stress conditions such as osmotic stress and ethanol stress may affect the fermentation performance; however, oxidative stress as a consequence of metabolic output can also occur. However, the effect of oxidative stress on yeast with pentose utilising capabilities has yet to be investigated. RESULTS: Assaying for the effect of hydrogen peroxide-induced oxidative stress on Candida, Pichia and Scheffersomyces spp. has demonstrated that these yeast tolerate hydrogen peroxide-induced oxidative stress in a manner consistent with that demonstrated by Saccharomyces cerevisiae. Pichia guillermondii appears to be more tolerant to hydrogen peroxide-induced oxidative stress when compared to Candida shehatae, Candida succiphila or Scheffersomyces stipitis. CONCLUSIONS: Sensitivity to hydrogen peroxide-induced oxidative stress increased in the presence of minimal media; however, addition of amino acids and nucleobases was observed to increase tolerance. In particular adenine increased tolerance and methionine reduced tolerance to hydrogen peroxide-induced oxidative stress.

Phenotypic characterisation of Saccharomyces spp. yeast for tolerance to stresses encountered during fermentation of lignocellulosic residues to produce bioethanol.

BACKGROUND: During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs. RESULTS: The aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses. CONCLUSIONS: Our results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations.

Phenotype microarray technology and its application in industrial biotechnology.

Phenotype microarray (PM) technology provides an insight into the metabolic profiling of microbial cells within 96-well plate system. The PM assay allows for cells to be assessed for utilisation of nutrients or sensitivity to toxic compounds. The assay utilises a redox sensitive tetrazolium dye which becomes irreversibly reduced upon detection of cellular metabolic output, detection is synchronous with a colour change from colourless to purple. Output from PM technology can be measured visually or quantified by reader the absorbance in each well. PM technology has highlighted differences in growth requirements, nutrient utilisation, sensitivity to toxins, and genetic diversity in bacteria, fungi and mammalian cells.

Oxidation of the yeast mitochondrial thioredoxin promotes cell death.

AIMS: Yeast, like other eukaryotes, contains a complete mitochondrial thioredoxin system comprising a thioredoxin (Trx3) and a thioredoxin reductase (Trr2). Mitochondria are a main source of reactive oxygen species (ROS) in eukaryotic organisms, and this study investigates the role of Trx3 in regulating cell death during oxidative stress conditions. RESULTS: We have previously shown that the redox state of mitochondrial Trx3 is buffered by the glutathione redox couple such that oxidized mitochondrial Trx3 only accumulates in mutants simultaneously lacking Trr2 and a glutathione reductase (Glr1). We show here that the redox state of mitochondrial Trx3 is important for yeast growth and its oxidation in a glr1 trr2 mutant induces programmed cell death. Apoptosis is dependent on the Yca1 metacaspase, since loss of YCA1 abrogates cell death induced by oxidized Trx3. Our data also indicate a role for a mitochondrial 1-cysteine (Cys) peroxiredoxin (Prx1) in the oxidation of Trx3, since Trx3 does not become oxidized in glr1 trr2 mutants or in a wild-type strain exposed to hydrogen peroxide in the absence of PRX1. INNOVATION: This study provides evidence that the redox state of a mitochondrial thioredoxin regulates yeast apoptosis in response to oxidative stress conditions. Moreover, the results identify a signaling pathway, where the thioredoxin system functions in both antioxidant defense and in controlling cell death. CONCLUSIONS: Mitochondrial Prx1 functions as a redox signaling molecule that oxidizes Trx3 and promotes apoptosis. This would mean that under conditions where Prx1 cannot detoxify mitochondrial ROS, it induces cell death to remove the affected cells.

Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae.

BACKGROUND: Protein-SH groups are amongst the most easily oxidized residues in proteins, but irreversible oxidation can be prevented by protein glutathionylation, in which protein-SH groups form mixed disulphides with glutathione. Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms. Glutaredoxins have been proposed to be the predominant deglutathionylase enzymes in many plant and mammalian species, whereas, thioredoxins have generally been thought to be relatively inefficient in deglutathionylation. RESULTS: We show here that the levels of glutathionylated proteins in yeast are regulated in parallel with the growth cycle, and are maximal during stationary phase growth. This increase in glutathionylation is not a response to increased reactive oxygen species generated from the shift to respiratory metabolism, but appears to be a general response to starvation conditions. Our data indicate that glutathionylation levels are constitutively high in all growth phases in thioredoxin mutants and are unaffected in glutaredoxin mutants. We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins. Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth. CONCLUSIONS: There is increasing evidence that the thioredoxin and glutathione redox systems have overlapping functions and these present data indicate that the thioredoxin system plays a key role in regulating the modification of proteins by the glutathione system.

The thioredoxin-thioredoxin reductase system can function in vivo as an alternative system to reduce oxidized glutathione in Saccharomyces cerevisiae.

Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione E(h)'. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular E(h)'. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.