Disulfide Stress Research Articles

Genome-wide identification of Bacillus subtilis Zur-binding sites associated with a Zur box expands its known regulatory network.

BackgroundThe Bacillus subtilis Zur transcription factor recognizes a specific DNA motif, the Zur box, to repress expression of genes in response to zinc availability. Although several Zur-regulated genes are well characterized, a genome-wide mapping of Zur-binding sites is needed to define further the set of genes directly regulated by this protein.ResultsUsing chromatin immunoprecipitation coupled with hybridization to DNA tiling arrays (ChIP-on-chip), we reported the identification of 80 inter- and intragenic chromosomal sites bound by Zur. Seven Zur-binding regions constitute the Zur primary regulon while 35 newly identified targets were associated with a predicted Zur box. Using transcriptional fusions an intragenic Zur box was showed to promote a full Zur-mediated repression when placed within a promoter region. In addition, intragenic Zur boxes appeared to mediate a transcriptional cis-repressive effect (4- to 9-fold) but the function of Zur at these sites remains unclear. Zur binding to intragenic Zur boxes could prime an intricate mechanisms of regulation of the transcription elongation, possibly with other transcriptional factors. However, the disruption of zinc homeostasis in Δzur cells likely affects many cellular processes masking direct Zur-dependent effects. Finally, most Zur-binding sites were located near or within genes responsive to disulfide stress. These findings expand the potential Zur regulon and reveal unknown interconnections between zinc and redox homeostasis regulatory networks.ConclusionsOur findings considerably expand the potential Zur regulon, and reveal a new level of complexity in Zur binding to its targets via a Zur box motif and via a yet unknown mechanism that remains to be characterized.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0345-4) contains supplementary material, which is available to authorized users.

Disulfide stress and its targets in acute pancreatitis.

Under physiological conditions, the balance between ROS production and removal properly maintains the intracellular redox-sensitive signaling as well as the appropriate status of protein thiols and disulfides. However, inflammation among other factors can modify this balance causing a rapid increase in intracellular ROS levels and hence thiol oxidation, eventually leading to oxidative stress. In the case of acute pancreatitis, both redox signaling and oxidative stress seem to contribute to the progression of the severe form of the disease. In this review we will focus on the reversible oxidation of protein cysteines during the course of acute pancreatitis. We describe disulfide stress in an acute inflammatory process, which is characterized by thiol oxidation in proteins, particularly protein cysteinylation, without significant changes in the glutathione redox status.

Regulated protein aggregation: a mechanism to control the activity of the ClpXP adaptor protein YjbH.

Bacteria use stress response pathways to activate diverse target genes to react to a variety of stresses. The Bacillus subtilis Spx protein is a global transcriptional regulator that controls expression of more than 140 genes and operons in response to thiol-specific oxidative stress. Under nonstress conditions the concentration of Spx is kept low by proteolysis catalyzed by the ClpXP complex. Spx protein levels increase in response to disulfide stress and decrease when the cells cope with the stress. The cytosolic adaptor protein YjbH is required to target Spx for efficient proteolysis by ClpXP. We demonstrate that YjbH aggregates in response to disulfide stress, that is, the YjbH protein is soluble under nonstressed conditions and destabilized during stress leading to aggregation. Stress conditions (heat and ethanol) that cause severe perturbations in protein stability/folding also induced aggregation of YjbH and led to induction of Spx. By heterologous expression of a less aggregation prone YjbH homolog Spx induction was abolished. Thus we show that moderation of YjbH solubility is an important mechanism of signal transduction and represents a new mechanism of controlling the activity of adaptor proteins.

Bile salts act as effective protein-unfolding agents and instigators of disulfide stress in vivo

Commensal and pathogenic bacteria must deal with many different stress conditions to survive in and colonize the human gastrointestinal tract. One major challenge that bacteria encounter in the gut is the high concentration of bile salts, which not only aid in food absorption but also act as effective physiological antimicrobials. The mechanism by which bile salts limit bacterial growth is still largely unknown. Here, we show that bile salts cause widespread protein unfolding and aggregation, affecting many essential proteins. Simultaneously, the bacterial cytosol becomes highly oxidizing, indicative of disulfide stress. Strains defective in reducing oxidative thiol modifications, restoring redox homeostasis, or preventing irreversible protein aggregation under disulfide stress conditions are sensitive to bile salt treatment. Surprisingly, cholate and deoxycholate, two of the most abundant and very closely related physiological bile salts, vary substantially in their destabilizing effects on proteins in vitro and cause protein unfolding of different subsets of proteins in vivo. Our results provide a potential mechanistic explanation for the antimicrobial effects of bile salts, help explain the beneficial effects of bile salt mixtures, and suggest that we have identified a physiological source of protein-unfolding disulfide stress conditions in bacteria.

FRET-based system for probing protein-protein interactions between σR and RsrA from Streptomyces coelicolor in response to the redox environment.

Protein-protein interactions between sigma factor σR and its corresponding zinc-binding anti-sigma (ZAS) protein RsrA trigger the thioredoxin system for maintaining cellular redox homeostasis in S. coelicolor. RsrA bound to zinc associates with σR, inhibiting its transcriptional activity in a reducing environment. During disulfide stress it forms intramolecular disulfide bonds, leading to zinc release and dissociation from σR, which initiates transcription to produce reductase and thioredoxin. We designed a fluorescence resonance energy transfer (FRET) based system for monitoring protein-protein interactions between σR and RsrA to further understand how this redox switch regulates the thioredoxin system in S. coelicolor in response to its redox environment, especially various reactive oxygen species (ROS) derived from different metabolic pathways, and clarify the different response mechanisms between Zn-RsrA and apo-RsrA. By the use of the FRET approach described here, we showed that zinc protected thiols in RsrA and causes the σR-RsrA complex to form a more compact structure. This system was also utilized to detect changes in redox status induced by ROS and diamide in real time in E. coli cells.

Redox Regulation in Bacillus subtilis: The Bacilliredoxins BrxA(YphP) and BrxB(YqiW) Function in De-Bacillithiolation of S-Bacillithiolated OhrR and MetE

In bacillithiol (BSH)-utilizing organisms, protein S-bacillithiolation functions as a redox switch in response to oxidative stress and protects critical Cys residues against overoxidation. In Bacillus subtilis, both the redox-sensing repressor OhrR and the methionine synthase MetE are redox controlled by S-bacillithiolation in vivo. Here, we identify pathways of protein de-bacillithiolation and test the hypothesis that YphP(BrxA) and YqiW(BrxB) act as bacilliredoxins (Brx) to remove BSH from OhrR and MetE mixed disulfides. We present evidence that the BrxA and BrxB paralogs have de-bacillithiolation activity. This Brx activity results from attack of the amino-terminal Cys residue in a CGC motif on protein BSH-mixed disulfides. B. subtilis OhrR DNA-binding activity is eliminated by S-thiolation on its sole Cys residue. Both the BrxA and BrxB bacilliredoxins mediate de-bacillithiolation of OhrR accompanied by the transfer of BSH to the amino-terminal cysteine of their CGC active site motif. In vitro studies demonstrate that BrxB can restore DNA-binding activity to OhrR which is S-bacillithiolated, but not to OhrR that is S-cysteinylated. MetE is most strongly S-bacillithiolated at Cys719 in vitro and can be efficiently de-bacillithiolated by both BrxA and BrxB. We demonstrate that BrxA and BrxB function in the reduction of BSH mixed protein disulfides with two natural substrates (MetE, OhrR). These results provide biochemical evidence for a new class of bacterial redox-regulatory proteins, the bacilliredoxins, which function analogously to glutaredoxins. Bacilliredoxins function in concert with other thiol-disulfide oxidoreductases to maintain redox homeostasis in response to disulfide stress conditions.

Disulfide stress: a novel type of oxidative stress in acute pancreatitis

Glutathione oxidation and protein glutathionylation are considered hallmarks of oxidative stress in cells because they reflect thiol redox status in proteins. Our aims were to analyze the redox status of thiols and to identify mixed disulfides and targets of redox signaling in pancreas in experimental acute pancreatitis as a model of acute inflammation associated with glutathione depletion. Glutathione depletion in pancreas in acute pancreatitis is not associated with any increase in oxidized glutathione levels or protein glutathionylation. Cystine and homocystine levels as well as protein cysteinylation and γ-glutamyl cysteinylation markedly rose in pancreas after induction of pancreatitis. Protein cysteinylation was undetectable in pancreas under basal conditions. Targets of disulfide stress were identified by Western blotting, diagonal electrophoresis, and proteomic methods. Cysteinylated albumin was detected. Redox-sensitive PP2A and tyrosine protein phosphatase activities diminished in pancreatitis and this loss was abrogated by N-acetylcysteine. According to our findings, disulfide stress may be considered a specific type of oxidative stress in acute inflammation associated with protein cysteinylation and γ-glutamylcysteinylation and oxidation of the pair cysteine/cystine, but without glutathione oxidation or changes in protein glutathionylation. Two types of targets of disulfide stress were identified: redox buffers, such as ribonuclease inhibitor or albumin, and redox-signaling thiols, which include thioredoxin 1, APE1/Ref1, Keap1, tyrosine and serine/threonine phosphatases, and protein disulfide isomerase. These targets exhibit great relevance in DNA repair, cell proliferation, apoptosis, endoplasmic reticulum stress, and inflammatory response. Disulfide stress would be a specific mechanism of redox signaling independent of glutathione redox status involved in inflammation.

Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress

Protein S-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in Firmicutes bacteria. Here we used transcriptomics to analyze the NaOCl stress response in the mycothiol (MSH)-producing Corynebacterium glutamicum. We further applied thiol-redox proteomics and mass spectrometry (MS) to identify protein S-mycothiolation. Transcriptomics revealed the strong upregulation of the disulfide stress σ(H) regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH. We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol- and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives.

Transcriptomic and phenotypic analysis of paralogous spx gene function in Bacillus anthracis Sterne

Spx of Bacillus subtilis is a redox-sensitive protein, which, under disulfide stress, interacts with RNA polymerase to activate genes required for maintaining thiol homeostasis. Spx orthologs are highly conserved among low %GC Gram-positive bacteria, and often exist in multiple paralogous forms. In this study, we used B. anthracis Sterne, which harbors two paralogous spx genes, spxA1 and spxA2, to examine the phenotypes of spx null mutations and to identify the genes regulated by each Spx paralog. Cells devoid of spxA1 were sensitive to diamide and hydrogen peroxide, while the spxA1 spoxA2 double mutant was hypersensitive to the thiol-specific oxidant, diamide. Bacillus anthracis Sterne strains expressing spxA1DD or spxA2DD alleles encoding protease-resistant products were used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses in order to uncover genes under SpxA1, SpxA2, or SpxA1/SpxA2 control. Comparison of transcriptomes identified many genes that were upregulated when either SpxA1DD or SpxA2DD was produced, but several genes were uncovered whose transcript levels increased in only one of the two SpxADD-expression strains, suggesting that each Spx paralog governs a unique regulon. Among genes that were upregulated were those encoding orthologs of proteins that are specifically involved in maintaining intracellular thiol homeostasis or alleviating oxidative stress. Some of these genes have important roles in B. anthracis pathogenesis, and a large number of upregulated hypothetical genes have no homology outside of the B. cereus/thuringiensis group. Microarray and RT-qPCR analyses also unveiled a regulatory link that exists between the two spx paralogous genes. The data indicate that spxA1 and spxA2 are transcriptional regulators involved in relieving disulfide stress but also control a set of genes whose products function in other cellular processes.Bacillus anthracis harbors two paralogs of the global transcriptional regulator of stress response, SpxA. SpxA1 and SpxA2 contribute to disulfide stress tolerance, but only SpxA1 functions in resistance to peroxide. Transcriptome analysis uncovered potential SpxA1 and SpxA2 regulon members, which include genes activated by both paralogs. However, paralog-specific gene activation was also observed. Genes encoding glutamate racemase, CoA disulfide reductase, and products functioning in bacillithiol biosynthesis, are among the genes activated by the SpxA paralogs.

Nonnative Disulfide Bond Formation Activates the σ 32 -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.

Disulfide stress-induced aluminium toxicity: molecular insights through genome-wide screening of Saccharomyces cerevisiae

Formation of non-native disulfide bonds within or between proteins can lead to protein misfolding and disruption to cellular metabolism. Such a process is defined as disulfide stress. A marked effect of disulfide stress in cells is the elevated accumulation of the intracellular aluminium ion (Al(3+)) accompanied by increased cytotoxicity. To gain an in-depth understanding of the underlying molecular mechanism for disulfide stress-induced aluminium toxicity, the complete set of Saccharomyces cerevisiae deletion mutants (5047) was screened in this study simultaneously with a combination of the two stressors, diamide and Al(3+). The combined treatment of a benign concentration of diamide (0.8 mM) with a sublethal concentration of aluminium sulfate (0.4 mM) revealed 494 sensitive deletion mutants, distinct from those found when either of the single stressors (0.8 mM diamide or 0.4 mM aluminium sulfate) was used. Hierarchical clustering and functional analyses of the 494 mutants sensitive to the dual stressors indicated a significant enrichment in the genes involved in cell wall homeostasis, signaling cascades, secretory transport machinery and detoxification. The results highlight the process of maintaining cell wall integrity as the central response to the combined exposure of diamide and Al(3+), which is mediated by the signaling pathways and transcription activation via Rlm1p and Swi6p for biosynthesis of the essential cell wall components such as glucan and chitin. Sensitivity of mutants associated with endoplasmic reticulum (ER), vesicle and vacuole functions demonstrates that secretory machinery is essential for surviving the stress conditions, probably due to their roles in transporting polysaccharides to the cell wall and detoxification of accumulated Al(3+). Finally, the phenotype of 100 previously uncharacterized genes against the dual stressors will contribute to their eventual functional annotation.

Disulfide stress as a novel type of oxidative stress in acute inflammation

Disulfide stress as a novel type of oxidative stress in acute inflammation

Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis

The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx–RNA polymerase (Spx–RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx–RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx–RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended −35 box in which nucleotide composition at the −43/−44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.

MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa

MexT is a global LysR transcriptional regulator known to modulate antibiotic resistance and virulence in Pseudomonas aeruginosa. In this study, a novel role for MexT in mediating intrinsic disulfide stress resistance was demonstrated, representing the first identified phenotype associated with inactivation of this regulator in wild-type cells. Disruption of mexT resulted in increased susceptibility to the disulfide stress elicitor diamide [diazenedicarboxylic acid bis(N,N,-di-methylamide)]. This compound is known to elicit a specific stress response via depletion of reduced glutathione and alteration of the cellular redox environment, implicating MexT in redox control. In support of this, MexT-regulated targets, including the MexEF-OprN multidrug efflux system, were induced by subinhibitory concentrations of diamide. A mexF insertion mutant also exhibited increased diamide susceptibility, implicating the MexEF-OprN efflux system in MexT-associated disulfide stress resistance. Purified MexT protein was observed to form an oligomeric complex in the presence of oxidized glutathione, with a calculated redox potential of -189 mV. This value far exceeds the thiol-disulfide redox potential of the bacterial cytoplasm, ensuring that MexT remains reduced under normal physiological conditions. MexT is activated by mutational disruption of the predicted quinone oxidoreductase encoded by mexS. Alterations in the cellular redox state were observed in a mexS mutant (PA14nfxC), supporting a model whereby the perception of MexS-associated redox signals by MexT leads to the induction of the MexEF-OprN efflux system, which, in turn, may mediate disulfide stress resistance via efflux of electrophilic compounds.

P38MAPK and ERK1/2 dictate cell death/survival response to different pro-oxidant stimuli via p53 and Nrf2 in neuroblastoma cells SH-SY5Y

Redox changes are often reported as causative of neoplastic transformation and chemoresistance, but are also exploited as clinical tools to selectively kill tumor cells. We previously demonstrated that gastrointestinal-derived tumor histotypes are resistant to ROS-based treatments by means of the redox activation of Nrf2, but highly sensitive to disulfide stressors triggering apoptosis via the redox induction of Trx1/p38MAPK/p53 signaling pathway.Here, we provide evidence that neuroblastoma SH-SY5Y has a complete opposite behavior, being sensitive to H2O2, but resistant to the glutathione (GSH)-oxidizing molecule diamide. Consistent with these observations, the apoptotic pathway activated upon H2O2 treatment relies upon Trx1 oxidation, and is mediated by the p38MAPK/p53 signaling axis. Pre-treatment with different antioxidants, pharmacological inhibitor of p38MAPK, or small interfering RNA against p53 rescue cell viability. On the contrary, cell survival to diamide relies upon redox activation of Nrf2, in a way independent on Keap1 oxidation, but responsive to ERK1/2 activation. Chemical inhibition of GSH neo-synthesis or ERK1/2 phosphorylation, as well as overexpression of the dominant-negative form of Nrf2 sensitizes cells to diamide toxicity. In the searching for the molecular determinant(s) unifying these phenomena, we found that SH-SY5Y cells show high GSH levels, but exhibit very low GPx activity. This feature allows to efficiently buffer disulfide stress, but leaves them being vulnerable to H2O2-mediated insult. The increase of GPx activity by means of selenium supplementation or GPx1 ectopic expression completely reverses death phenotype, indicating that the response of tumor cells to diverse oxidative stimuli deeply involves the entire GSH redox system.

Roles of thioredoxin and thioredoxin reductase in the resistance to oxidative stress in Lactobacillus casei

The Lactobacillus casei strain Shirota used in this study has in the genome four putative thioredoxin genes designated trxA1, trxA2, trxA3 and trxA4, and one putative thioredoxin reductase gene designated trxB. To elucidate the roles of the thioredoxins and the thioredoxin reductase against oxidative stress in L. casei, we constructed gene disruption mutants, in which each of the genes trxA1, trxA2 and trxB, or both trxA1 and trxA2 were disrupted, and we characterized their growth and response to oxidative stresses. In aerobic conditions, the trxA1 (MS108) and the trxA2 (MS109) mutants had moderate growth defects, and the trxA1 trxA2 double mutant (MS110) had a severe growth defect, which was characterized by elongation of doubling time and a lower final turbidity level. Furthermore, the trxB mutant (MS111), which is defective in thioredoxin reductase, lost the ability to grow under aerobic conditions, although it grew partially under anaerobic conditions. The growth of these mutants, however, could be substantially restored by the addition of dithiothreitol or reduced glutathione. In addition, MS110 and MS111 were more sensitive to hydrogen peroxide and disulfide stress than the wild-type. In particular, the stress sensitivity of MS111 was significantly increased. On the other hand, transcription of all these genes was only weakly affected by these oxidative stresses. Taken together, these results suggest that the thioredoxin-thioredoxin reductase system is the major thiol/disulfide redox system and is essential to allow the facultative anaerobe L. casei to grow under aerobic conditions.

Transcriptome-Wide Identification of Preferentially Expressed Genes in the Hypothalamus and Pituitary Gland

To identify preferentially expressed genes in the central endocrine organs of the hypothalamus and pituitary gland, we generated transcriptome-wide mRNA profiles of the hypothalamus, pituitary gland, and parietal cortex in male mice (12–15 weeks old) using serial analysis of gene expression (SAGE). Total counts of SAGE tags for the hypothalamus, pituitary gland, and parietal cortex were 165824, 126688, and 161045 tags, respectively. This represented 59244, 45151, and 55131 distinct tags, respectively. Comparison of these mRNA profiles revealed that 22 mRNA species, including three potential novel transcripts, were preferentially expressed in the hypothalamus. In addition to well-known hypothalamic transcripts, such as hypocretin, several genes involved in hormone function, intracellular transduction, metabolism, protein transport, steroidogenesis, extracellular matrix, and brain disease were identified as preferentially expressed hypothalamic transcripts. In the pituitary gland, 106 mRNA species, including 60 potential novel transcripts, were preferentially expressed. In addition to well-known pituitary genes, such as growth hormone and thyroid stimulating hormone beta, a number of genes classified to function in transport, amino acid metabolism, intracellular transduction, cell adhesion, disulfide bond formation, stress response, transcription, protein synthesis, and turnover, cell differentiation, the cell cycle, and in the cytoskeleton and extracellular matrix were also preferentially expressed. In conclusion, the current study identified not only well-known hypothalamic and pituitary transcripts but also a number of new candidates likely to be involved in endocrine homeostatic systems regulated by the hypothalamus and pituitary gland.

The YjbH Adaptor Protein Enhances Proteolysis of the Transcriptional Regulator Spx in Staphylococcus aureus

Spx is a global regulator that is widespread among the low-G+C-content gram-positive bacteria. Spx has been extensively studied in Bacillus subtilis, where it acts as an activator and a repressor of transcription in response to disulfide stress. Under nonstress conditions, Spx is rapidly degraded by the ClpXP protease. This degradation is enhanced by the YjbH adaptor protein. Upon disulfide stress, the amount of Spx rapidly increases due to a decrease in degradation. In the opportunistic pathogen Staphylococcus aureus, Spx is a global regulator influencing growth, biofilm formation, and general stress protection, and cells lacking the spx gene exhibit poor growth also under nonstress conditions. To investigate the mechanism by which the activity of Spx is regulated, we identified a homolog in S. aureus of the B. subtilis yjbH gene. The gene encodes a protein that shows approximately 30% sequence identity to YjbH of B. subtilis. Heterologous expression of S. aureus yjbH in a B. subtilis yjbH mutant restored Spx to wild-type levels both under nonstress conditions and under conditions of disulfide stress. From these studies, we conclude that the two YjbH homologues have a conserved physiological function. Accordingly, inactivation of yjbH in S. aureus increased the level of Spx protein and transcription of the Spx-regulated gene trxB. Notably, the yjbH mutant exhibited reduced growth and increased pigmentation, and both phenotypes were reversed by complementation of the yjbH gene.

New Role of the Disulfide Stress Effector YjbH in β-Lactam Susceptibility of Staphylococcus aureus

Staphylococcus aureus is exposed to multiple antimicrobial compounds, including oxidative burst products and antibiotics. The various mechanisms and regulatory pathways governing susceptibility or resistance are complex and only superficially understood. Bacillus subtilis recently has been shown to control disulfide stress responses by the thioredoxin-related YjbH protein, which binds to the transcriptional regulator Spx and controls its degradation via the proteasome-like ClpXP protease. We show that the S. aureus YjbH homolog has a role in susceptibility to the disulfide stress-inducing agent diamide that is similar to that in B. subtilis, and we demonstrate that the four cysteine residues in YjbH are required for this activity. In addition, the inactivation of YjbH led to moderate resistance to oxacillin and other β-lactam antibiotics, and this phenotypic change was associated with higher penicillin-binding protein 4 levels and increased peptidoglycan cross-linking. Of note, the impact of YjbH on β-lactam susceptibility still was observed when the four cysteines of YjbH were mutated, indicating that the roles of YjbH in disulfide stress and β-lactam resistance rely on different types of interactions. These data suggest that the ClpXP adaptor YjbH has more target proteins than previously thought, and that oxidative burst and β-lactam resistance mechanisms of S. aureus are closely linked.

Phenotype Enhancement Screen of a Regulatory spx Mutant Unveils a Role for the ytpQ Gene in the Control of Iron Homeostasis

Spx is a global regulator of genes that are induced by disulfide stress in Bacillus subtilis. The regulon that it governs is comprised of over 120 genes based on microarray analysis, although it is not known how many of these are under direct Spx control. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products that are conserved in low %GC Gram-positive bacteria. Using a gene-disruption library of B. subtilis genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as poor growth in minimal medium and sensitivity to methyglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an spx mutant background to determine which mutant SRG allele would enhance the spx mutant phenotype. One of the SRGs, ytpQ was the site of a mutation that, when combined with an spx null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methyglyoxal. The ytpQ mutant showed elevated oxidative protein damage when exposed to methylglyoxal, and reduced growth rate in liquid culture. Proteomic and transcriptomic data indicated that the ytpQ mutation caused the derepression of the Fur and PerR regulons of B. subtilis. Our study suggests that the ytpQ gene, encoding a conserved DUF1444 protein, functions directly or indirectly in iron homeostasis. The ytpQ mutant phenotype mimics that of a fur mutation, suggesting a condition of low cellular iron. In vitro transcription analysis indicated that Spx stimulates transcription from the ytpPQR operon within which the ytpQ gene resides. The work uncovers a link between Spx and control of iron homeostasis.