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The major pathogen Staphylococcus aureus has to cope with host‐derived oxidative stress to cause infections in humans. Here, we report that S. aureus tolerates high concentrations of hypothiocyanous acid (HOSCN), a key antimicrobial oxidant produced in the respiratory tract. We discovered that the flavoprotein disulfide reductase (FDR) MerA protects S. aureus from this oxidant by functioning as a HOSCN reductase, with its deletion sensitizing bacteria to HOSCN. Crystal structures of homodimeric MerA (2.4 Å) with a Cys43–Cys48 intramolecular disulfide, and reduced MerACys43S (1.6 Å) showed the FAD cofactor close to the active site, supporting that MerA functions as a group I FDR. MerA is controlled by the redox‐sensitive repressor HypR, which we show to be oxidized to intermolecular disulfides under HOSCN stress, resulting in its inactivation and derepression of merA transcription to promote HOSCN tolerance. Our study highlights the HOSCN tolerance of S. aureus and characterizes the structure and function of MerA as a major HOSCN defense mechanism. Crippling the capacity to respond to HOSCN may be a novel strategy for treating S. aureus infections. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae has to cope with the strong oxidant hypochlorous acid (HOCl), during host–pathogen interactions. Thus, we analyzed the global gene expression profile of S. pneumoniae D39 towards HOCl stress. In the RNA‐seq transcriptome, the NmlR, SifR, CtsR, HrcA, SczA and CopY regulons and the etrx1‐ccdA1‐msrAB2 operon were most strongly induced under HOCl stress, which participate in the oxidative, electrophile and metal stress response in S. pneumoniae. The MerR‐family regulator NmlR harbors a conserved Cys52 and controls the alcohol dehydrogenase‐encoding adhC gene under carbonyl and NO stress. We demonstrated that NmlR senses also HOCl stress to activate transcription of the nmlR‐adhC operon. HOCl‐induced transcription of adhC required Cys52 of NmlR in vivo. Using mass spectrometry, NmlR was shown to be oxidized to intersubunit disulfides or S‐glutathionylated under oxidative stress in vitro. A broccoli‐FLAP‐based assay further showed that both NmlR disulfides significantly increased transcription initiation at the nmlR promoter by RNAP in vitro, which depends on Cys52. Phenotype analyses revealed that NmlR functions in the defense against oxidative stress and promotes survival of S. pneumoniae during macrophage infections. In conclusion, NmlR was characterized as HOCl‐sensing transcriptional regulator, which activates transcription of adhC under oxidative stress by thiol switches in S. pneumoniae. [ABSTRACT FROM AUTHOR]

Targeting immune evasion tactics of pathogenic bacteria may hold the key to treating recalcitrant bacterial infections. Staphylococcus aureus produces bacillithiol (BSH), its major low‐molecular‐weight thiol, which is thought to protect this opportunistic human pathogen against the bombardment of oxidants inside neutrophil phagosomes. Here, we show that BSH was oxidized when human neutrophils phagocytosed S. aureus, but provided limited protection to the bacteria. We used mass spectrometry to measure the oxidation of BSH upon exposure of S. aureus USA300 to either a bolus of hypochlorous acid (HOCl) or a flux generated by the neutrophil enzyme myeloperoxidase. Oxidation of BSH and loss of bacterial viability were strongly correlated (r = 0.99, p < 0.001). BSH was fully oxidized after exposure of S. aureus to lethal doses of HOCl. However, there was no relationship between the initial BSH levels and the dose of HOCl required for bacterial killing. In contrast to the HOCl systems, only 50% of total BSH was oxidized when neutrophils killed the majority of phagocytosed bacteria. Oxidation of BSH was decreased upon inhibition of myeloperoxidase, implicating HOCl in phagosomal BSH oxidation. A BSH‐deficient S. aureus USA300 mutant was slightly more susceptible to treatment with either HOCl or ammonia chloramine, or to killing within neutrophil phagosomes. Collectively, our data show that myeloperoxidase‐derived oxidants react with S. aureus inside neutrophil phagosomes, leading to partial BSH oxidation, and contribute to bacterial killing. However, BSH offers only limited protection against the neutrophil's multifaceted killing mechanisms. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus has to cope with oxidative stress during infections. In this study, S. aureus was found to be resistant to 100 mM H2O2 during aerobic growth. While KatA was essential for this high aerobic H2O2 resistance, the peroxiredoxin AhpC contributed to detoxification of 0.4 mM H2O2 in the absence of KatA. In addition, the peroxiredoxins AhpC, Tpx and Bcp were found to be required for detoxification of cumene hydroperoxide (CHP). The high H2O2 tolerance of aerobic S. aureus cells was associated with priming by endogenous H2O2 levels, which was supported by an oxidative shift of the bacillithiol redox potential to −291 mV compared to −310 mV in microaerophilic cells. In contrast, S. aureus could be primed by sub-lethal doses of 100 µM H2O2 during microaerophilic growth to acquire an improved resistance towards the otherwise lethal triggering stimulus of 10 mM H2O2. This microaerophilic priming was dependent on increased KatA activity, whereas aerobic cells showed constitutive high KatA activity. Thus, KatA contributes to the high H2O2 resistance of aerobic cells and to microaerophilic H2O2 priming in order to survive the subsequent lethal triggering doses of H2O2, allowing the adaptation of S. aureus under infections to different oxygen environments. [ABSTRACT FROM AUTHOR]

Allicin (diallyl thiosulfinate) is the major thiol-reactive organosulfur compound produced by garlic plants (Allium sativum) upon tissue damage. Allicin exerts its strong antimicrobial activity against bacteria and fungi via S -thioallylation of protein thiols and low molecular weight thiols. Here, we investigated the effect of allicin on SARS-CoV-2 infected Vero E6 and Calu-3 cells. Toxicity tests revealed that Calu-3 cells showed greater allicin tolerance, probably due to >4-fold higher GSH levels compared to the very sensitive Vero E6 cells. Exposure of infected Vero E6 and Calu-3 cells to biocompatible allicin doses led to a ∼60–70% decrease of viral RNA and infectious viral particles. Label-free quantitative proteomics was used to investigate the changes in the Calu-3 proteome after SARS-CoV-2 infection and the effect of allicin on the host-virus proteome. SARS-CoV-2 infection of Calu-3 cells caused a strong induction of the antiviral interferon-stimulated gene (ISG) signature, including several antiviral effectors, such as cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, OAS, and ISG15, pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTCL1, and ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin treatment of infected Calu-3 cells reduced the expression of IFN signaling pathways and ISG effectors and reverted several host pathways to levels of uninfected cells. Allicin further reduced the abundance of the structural viral proteins N, M, S and ORF3 in the host-virus proteome. In conclusion, our data demonstrate the antiviral and immunomodulatory activity of biocompatible doses of allicin in SARS-CoV-2-infected cell cultures. Future drug research should be directed to exploit the thiol-reactivity of allicin derivatives with increased stability and lower human cell toxicity as antiviral lead compounds. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) isolates are often resistant to multiple antibiotics and pose a major health burden due to limited treatment options. The novel AGXX® surface coating exerts strong antimicrobial activity and successfully kills multi-resistant pathogens, including MRSA. The mode of action of AGXX® particles involves the generation of reactive oxygen species (ROS), which induce an oxidative and metal stress response, increased protein thiol-oxidations, protein aggregations, and an oxidized bacillithiol (BSH) redox state in S. aureus. In this work, we report that the AGXX® particle size determines the effective dose and time-course of S. aureus USA300JE2 killing. We found that the two charges AGXX®373 and AGXX®383 differ strongly in their effective concentrations and times required for microbial killing. While 20–40 μg/ml AGXX®373 of the smaller particle size of 1.5–2.5 μm resulted in >99.9% killing after 2 h, much higher amounts of 60–80 μg/ml AGXX®383 of the larger particle size of >3.2 μm led to a >99% killing of S. aureus USA300JE2 within 3 h. Smaller AGXX® particles have a higher surface/volume ratio and therefore higher antimicrobial activity to kill at lower concentrations in a shorter time period compared to the larger particles. Thus, in future preparations of AGXX® particles, the size of the particles should be kept at a minimum for maximal antimicrobial activity. [ABSTRACT FROM AUTHOR]

Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family, whose molecular mechanisms and cellular functions are poorly understood. Here, we show that the Escherichia coli DEAH/RHA protein, HrpA, is an ATP-dependent 3 to 5' RNA helicase and that the RNA helicase activity of HrpA influences bacterial survival under antibiotics treatment. Limited proteolysis, crystal structure analysis, and functional assays showed that HrpA contains an N-terminal DEAH/RHA helicase cassette preceded by a unique N-terminal domain and followed by a large C-terminal region that modulates the helicase activity. Structures of an expanded HrpA helicase cassette in the apo and RNA-bound states in combination with cross-linking/mass spectrometry revealed ratchet-like domain movements upon RNA engagement, much more pronounced than hitherto observed in related eukaryotic DEAH/RHA enzymes. Structure-based functional analyses delineated transient interdomain contact sites that support substrate loading and unwinding, suggesting that similar conformational changes support RNA translocation. Consistently, modeling studies showed that analogous dynamic intramolecular contacts are not possible in the related but helicase-inactive RNA-dependent nucleoside-triphosphatase, HrpB. Our results indicate that HrpA may be an interesting target to interfere with bacterial tolerance toward certain antibiotics and suggest possible interfering strategies. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major human pathogen, which encounters reactive oxygen, nitrogen, chlorine, electrophile and sulfur species (ROS, RNS, RCS, RES and RSS) by the host immune system, during cellular metabolism or antibiotics treatments. To defend against redox active species and antibiotics, S. aureus is equipped with redox sensing regulators that often use thiol switches to control the expression of specific detoxification pathways. In addition, the maintenance of the redox balance is crucial for survival of S. aureus under redox stress during infections, which is accomplished by the low molecular weight (LMW) thiol bacillithiol (BSH) and the associated bacilliredoxin (Brx)/BSH/bacillithiol disulfide reductase (YpdA)/NADPH pathway. Here, we present an overview of thiol-based redox sensors, its associated enzymatic detoxification systems and BSH-related regulatory mechanisms in S. aureus, which are important for the defense under redox stress conditions. Application of the novel Brx-roGFP2 biosensor provides new insights on the impact of these systems on the BSH redox potential. These thiol switches of S. aureus function in protection against redox active desinfectants and antimicrobials, including HOCl, the AGXX® antimicrobial surface coating, allicin from garlic and the naphthoquinone lapachol. Thus, thiol switches could be novel drug targets for the development of alternative redox-based therapies to combat multi-drug resistant S. aureus isolates. [ABSTRACT FROM AUTHOR]

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections. The low molecular weight thiol bacillithiol (BSH) is an important defense mechanism of S. aureus for detoxification of ROS and HOCl stress to maintain the reduced state of the cytoplasm. Under HOCl stress, BSH forms mixed disulfides with proteins, termed as S -bacillithiolations, which are reduced by bacilliredoxins (BrxA and BrxB). The NADPH-dependent flavin disulfide reductase YpdA is phylogenetically associated with the BSH synthesis and BrxA/B enzymes and was recently suggested to function as BSSB reductase (Mikheyeva et al., 2019). Here, we investigated the role of the complete bacilliredoxin BrxAB/BSH/YpdA pathway in S. aureus COL under oxidative stress and macrophage infection conditions in vivo and in biochemical assays in vitro. Using HPLC thiol metabolomics, a strongly enhanced BSSB level and a decreased BSH/BSSB ratio were measured in the S. aureus COL Δ ypdA deletion mutant under control and NaOCl stress. Monitoring the oxidation degree (OxD) of the Brx-roGFP2 biosensor revealed that YpdA is required for regeneration of the reduced BSH redox potential (E BSH) upon recovery from oxidative stress. In addition, the Δ ypdA mutant was impaired in H2O2 detoxification as measured with the novel H2O2-specific Tpx-roGFP2 biosensor. Phenotype analyses further showed that BrxA and YpdA are required for survival under NaOCl and H2O2 stress in vitro and inside murine J-774A.1 macrophages in infection assays in vivo. Finally, NADPH-coupled electron transfer assays provide evidence for the function of YpdA in BSSB reduction, which depends on the conserved Cys14 residue. YpdA acts together with BrxA and BSH in de-bacillithiolation of S -bacillithiolated GapDH. In conclusion, our results point to a major role of the BrxA/BSH/YpdA pathway in BSH redox homeostasis in S. aureus during recovery from oxidative stress and under infections. [ABSTRACT FROM AUTHOR]

Bacteria encounter reactive oxygen species (ROS) as a consequence of the aerobic life or as an oxidative burst of activated neutrophils during infections. In addition, bacteria are exposed to other redox-active compounds, including hypochloric acid (HOCl) and reactive electrophilic species (RES) such as quinones and aldehydes. These reactive species often target the thiol groups of cysteines in proteins and lead to thiol-disulfide switches in redox-sensing regulators to activate specific detoxification pathways and to restore the redox balance. Here, we review bacterial thiol-based redox sensors that specifically sense ROS, RES and HOCl via thiol-based mechanisms and regulate gene transcription in Gram-positive model bacteria and in human pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis. We also pay particular attention to emerging widely conserved HOCl-specific redox regulators that have been recently characterized in Escherichia coli. Different mechanisms are used to sense and respond to ROS, RES and HOCl by 1-Cys-type and 2-Cys-type thiol-based redox sensors that include versatile thiol-disulfide switches (OxyR, OhrR, HypR, YodB, NemR, RclR, Spx, RsrA/RshA) or alternative Cys phosphorylations (SarZ, MgrA, SarA), thiol-S-alkylation (QsrR), His-oxidation (PerR) and methionine oxidation (HypT). In pathogenic bacteria, these redox-sensing regulators are often important virulence regulators and required for adapation to the host immune defense. [ABSTRACT FROM AUTHOR]

Low molecular weight (LMW) thiols function as thiol-redox buffers to maintain the reduced state of the cytoplasm. The best studied LMW thiol is the tripeptide glutathione (GSH) present in all eukaryotes and Gram-negative bacteria. Firmicutes bacteria, including Bacillus and Staphylococcus species utilize the redox buffer bacillithiol (BSH) while Actinomycetes produce the related redox buffer mycothiol (MSH). In eukaryotes, proteins are post-translationally modified to S-glutathionylated proteins under conditions of oxidative stress. S-glutathionylation has emerged as major redox-regulatory mechanism in eukaryotes and protects active site cysteine residues against overoxidation to sulfonic acids. First studies identified S-glutathionylated proteins also in Gram-negative bacteria. Advances in mass spectrometry have further facilitated the identification of protein S-bacillithiolations and S-mycothiolation as BSH- and MSH-mixed protein disulfides formed under oxidative stress in Firmicutes and Actinomycetes, respectively. In Bacillus subtilis, protein S-bacillithiolation controls the activities of the redox-sensing OhrR repressor and the methionine synthase MetE in vivo. In Corynebacterium glutamicum, protein S-mycothiolation was more widespread and affected the functions of the maltodextrin phosphorylase MalP and thiol peroxidase (Tpx). In addition, novel bacilliredoxins (Brx) and mycoredoxins (Mrx1) were shown to function similar to glutaredoxins in the reduction of BSH- and MSH-mixed protein disulfides. Here we review the current knowledge about the functions of the bacterial thiol-redox buffers glutathione, bacillithiol, and mycothiol and the role of protein S-thiolation in redox regulation and thiol protection in model and pathogenic bacteria. [ABSTRACT FROM AUTHOR]

The respiratory pathogen Streptococcus pneumoniae has evolved efficient mechanisms to resist oxidative stress conditions and to displace other bacteria in the nasopharynx. Here we characterize at physiological, functional and structural levels two novel surface-exposed thioredoxin-family lipoproteins, Etrx1 and Etrx2. The impact of both Etrx proteins and their redox partner methionine sulfoxide reductase SpMsrAB2 on pneumococcal pathogenesis was assessed in mouse virulence studies and phagocytosis assays. The results demonstrate that loss of function of either both Etrx proteins or SpMsrAB2 dramatically attenuated pneumococcal virulence in the acute mouse pneumonia model and that Etrx proteins compensate each other. The deficiency of Etrx proteins or SpMsrAB2 further enhanced bacterial uptake by macrophages, and accelerated pneumococcal killing by H2O2 or free methionine sulfoxides (MetSO). Moreover, the absence of both Etrx redox pathways provokes an accumulation of oxidized SpMsrAB2 in vivo. Taken together our results reveal insights into the role of two extracellular electron pathways required for reduction of SpMsrAB2 and surface-exposed MetSO. Identification of this system and its target proteins paves the way for the design of novel antimicrobials. [ABSTRACT FROM AUTHOR]

Mycobacteria produce millimolar concentrations of mycothiol ( MSH) as their major low molecular weight thiol redox buffer. MSH-deficient mutants display increased sensitivity towards reactive oxygen, nitrogen and electrophilic species as well as alkylating agents and antibiotics. MSH is maintained in its reduced thiol state by the NADPH-dependent mycothiol disulphide reductase ( Mtr). However, the redoxin that uses the MSH/ Mtr/ NADPH pathway for reduction of MSH-mixed protein disulphides, formed during oxidative stress, has long remained unknown. In this issue, Van Laer et al. report that MSH provides the reducing power for mycoredoxin-1 ( Mrx1) in reduction of synthetic MSH-mixed disulphides. The reduced (dithiol) and oxidized (disulphide) solution structures of Mrx1 have been solved by nuclear magnetic resonance ( NMR) spectroscopy. NMR time course experiments have also demonstrated the transient S-mycothiolation of the active site Cys14 of oxidized Mrx1 during reduction by the MSH/ Mtr/ NADPH electron pathway. The paper opens a new era of research to identify S-mycothiolated Mrx1 substrates and the function of MSH in redox regulation and virulence in Mycobacterium tuberculosis. [ABSTRACT FROM AUTHOR]

Summary In eukaryotes, lysine acetylation is a well-established post-translational modification that has been implicated in virtually all aspects of eukaryotic physiology. Although homologues of the enzymes that catalyse protein acetylation are widely conserved and distributed among bacterial species, not much is known about the impact of protein acetylation on bacterial physiology. Here, we present evidence that the Gcn5-like acetyltransferase YfiQ and the sirtuin deacetylase CobB play crucial roles in the transcription regulation of the periplasmic stress-responsive promoter cpxP when cells of Escherichia coli grow in the presence of glucose, an environment that induces protein acetylation. Under this growth condition, several acetylation sites were detected on three of the RNA polymerase subunits: β, β′ and α. We focused on acetylations of the carboxy-terminal domain (CTD) of α because of its relative small size and its limited acetylation. We determined that K298 of α is acetylated in a glucose and YfiQ-dependent manner and that K298 is specifically required for glucose-induced cpxP transcription. Because the αCTD aids in promoter recognition by RNA polymerase, we propose its acetylation may influence bacterial physiology through effects on gene expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described ''minimal Tat translocase'' consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate. [ABSTRACT FROM AUTHOR]

The acoABCL and acuABC operons of Bacillus licheniformis DSM13 are strongly induced at the transcriptional level during glucose starvation conditions. Primer extension analyses of this study indicate that the acoABCL operon is controlled by a sigmaL-dependent promoter and the acuABC operon by a sigmaA-dependent promoter. Transcription at the acoA promoter is repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The acuA promoter shows a similar induction pattern, but its activity is independent from the presence of acetoin. It is demonstrated that the acoABCL operon is mainly responsible for acetoin and 2,3-butanediol degradation in B. licheniformis. [ABSTRACT FROM AUTHOR]

Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and Δ tatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The Δ tatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild-type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild-type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence. [ABSTRACT FROM AUTHOR]

Bacillithiol (BSH), the α-anomeric glycoside of L-cysteinyl-D-glucosamine with 1-malic acid, is a major low-molecular-weight thiol in Bacillus subtills and related bacteria. Here, we identify genes required for BSH biosynthesis and provide evidence that the synthetic pathway has similarities to that established for the related thiol (mycothiol) in the Actinobacteria. Consistent with a key role for BSH in detoxification of electroph lIes, the BshA glycosyltransferase and BshBl deacetylase are encoded in an operon with methylglyoxal synthase. BshBl is partially redundant in function with BshB2, a deacetylase of the LmbE family. Phylogenomic profiling identif led a conserved unknown function protein (COG4365) as a candidate cysteine-adding enzyme (BshC) that cooccurs in genomes also encoding BshA, BshB1, and BshB2. Additional evolutionarily linked proteins include a thioredoxin reductase homolog and two thiol:disulfide oxidoreductases of the DUF1094 (CxC motif) family. Mutants lacking BshA, BshC, or both BshBl and BshB2 are devoid of BSH. BSH is at least partially redundant in function with other low-molecular-weight thiols: redox proteomics indicates that protein thiols are largely reduced even in the absence of BSH. At the transcriptional level, the induction of genes controlled by two thiol-based regulators (OhrR, Spx) occurs normally. However, BSH null cells are significantly altered in acid and salt resistance, sporulation, and resistance to electrophiles and thiol reactive compounds. Moreover, cells lacking BSH are highly sensitive to fosfomycin, an epoxidecontaining antibiotic detoxified by FosB, a prototype for bacillithiol-Stransferase enzymes. [ABSTRACT FROM AUTHOR]

Quinones and α,β-unsaturated carbonyls are naturally occurring electrophiles that target cysteine residues via thiol-(S)-alkylation. We analysed the global expression profile of Bacillus subtilis to the toxic carbonyls methylglyoxal (MG) and formaldehyde (FA). Both carbonyl compounds cause a stress response characteristic for thiol-reactive electrophiles as revealed by the induction of the Spx, CtsR, CymR, PerR, ArsR, CzrA, CsoR and SigmaD regulons. MG and FA triggered also a SOS response which indicates DNA damage. Protection against FA is mediated by both the hxlAB operon, encoding the ribulose monophosphate pathway for FA fixation, and a thiol-dependent formaldehyde dehydrogenase (AdhA) and DJ-1/PfpI-family cysteine proteinase (YraA). The adhA–yraA operon and the yraC gene, encoding a γ-carboxymuconolactone decarboxylase, are positively regulated by the MerR-family regulator, YraB(AdhR). AdhR binds specifically to its target promoters which contain a 7-4-7 inverted repeat (CTTAAAG-N4-CTTTAAG) between the −35 and −10 elements. Activation of adhA–yraA transcription by AdhR requires the conserved Cys52 residue in vivo. We speculate that AdhR is redox-regulated via thiol-(S)-alkylation by aldehydes and that AdhA and YraA are specifically involved in reduction of aldehydes and degradation or repair of damaged thiol-containing proteins respectively. [ABSTRACT FROM AUTHOR]

The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can secrete numerous proteins into the surrounding medium enabling them to use high-molecular-weight substances, which are abundant in soils, as nutrient sources. The availability of complete genome sequences allows for the prediction of the proteins containing signals for secretion into the extracellular milieu and also of the proteins which form the secretion machinery needed for protein translocation through the cytoplasmic membrane. To confirm the predicted subcellular localization of proteins, proteomics is the best choice. The extracellular proteomes of B. subtilis and B. licheniformis have been analyzed under different growth conditions allowing comparisons of the extracellular proteomes and conclusions regarding similarities and differences of the protein secretion mechanisms between the two species. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Quinones are highly toxic naturally occurring thiol-reactive compounds. We have previously described novel pathways for quinone detoxification in the Gram-positive bacterium Bacillus subtilis. In this study, we have investigated the extent of irreversible and reversible thiol modifications caused in vivo by electrophilic quinones. Exposure to toxic benzoquinone (BQ) concentrations leads to depletion of numerous Cys-rich cytoplasmic proteins in the proteome of B. subtilis. Mass spectrometry and immunoblot analyses demonstrated that these BQ-depleted proteins represent irreversibly damaged BQ aggregates that escape the two-dimensional gel separation. This enabled us to quantify the depletion of thiol-containing proteins which are the in vivo targets for thiol-(S)-alkylation by toxic quinone compounds. Metabolomic approaches confirmed that protein depletion is accompanied by depletion of the low-molecular-weight (LMW) thiol cysteine. Finally, no increased formation of disulphide bonds was detected in the thiol-redox proteome in response to sublethal quinone concentrations. The glyceraldehyde-3-phosphate dehydrogenase (GapA) was identified as the only new target for reversible thiol modifications after exposure to toxic quinones. Together our data show that the thiol-(S)-alkylation reaction with protein and non-protein thiols is the in vivo mechanism for thiol depletion and quinone toxicity in B. subtilis and most likely also in other bacteria. [ABSTRACT FROM AUTHOR]

Regulation of bacterial iron homeostasis is often controlled by the iron-sensing ferric uptake repressor (Fur). The Bacillus subtilis Fur protein acts as an iron-dependent repressor for siderophore biosynthesis and iron transport proteins. Here, we demonstrate that Fur also coordinates an iron-sparing response that acts to repress the expression of iron-rich proteins when iron is limiting. When Fur is inactive, numerous iron-containing proteins are down-regulated, including succinate dehydrogenase, aconitase, cytochromes, and biosynthetic enzymes for heme, cysteine, and branched chain amino acids. As a result, a fur mutant grows slowly in a variety of nutrient conditions. Depending on the growth medium, rapid growth can be restored by mutations in one or more of the molecular effectors of the iron-sparing response. These effectors include the products of three Fur-regulated operons that encode a small RNA (FsrA) and three small, basic proteins (FbpA, FbpB, and FbpC). Extensive complementarity between FsrA and the leader region of the succinate dehydrogenase operon is consistent with an RNA-mediated translational repression mechanism for this target. Thus, iron deprivation in B. subtilis activates pathways to remodel the proteome to preserve iron for the most critical cellular functions. [ABSTRACT FROM AUTHOR]

Recently, we showed that the MarR-type repressor YkvE (MhqR) regulates multiple dioxygenases/glyoxalases, oxidoreductases and the azoreductase encoding yvaB ( azoR2) gene in response to thiol-specific stress conditions, such as diamide, catechol and 2-methylhydroquinone (MHQ). Here we report on the regulation of the yocJ ( azoR1) gene encoding another azoreductase by the novel DUF24/MarR-type repressor, YodB after exposure to thiol-reactive compounds. DNA binding activity of YodB is directly inhibited by thiol-reactive compounds in vitro. Mass spectrometry identified YodB-Cys-S-adducts that are formed upon exposure of YodB to MHQ and catechol in vitro. This confirms that catechol and MHQ are auto-oxidized to toxic ortho- and para-benzoquinones which act like diamide as thiol-reactive electrophiles. Mutational analyses further showed that the conserved Cys6 residue of YodB is required for optimal repression in vivo and in vitro while substitution of all three Cys residues of YodB affects induction of azoR1 transcription. Finally, phenotype analyses revealed that both azoreductases, AzoR1 and AzoR2 confer resistance to catechol, MHQ, 1,4-benzoquinone and diamide. Thus, both azoreductases that are controlled by different regulatory mechanisms have common functions in quinone and azo-compound reduction to protect cells against the thiol reactivity of electrophiles. [ABSTRACT FROM AUTHOR]

Proteomic and transcriptomics signatures are powerful tools for visualizing global changes in gene expression in bacterial cells after exposure to stress, starvation or toxic compounds. Based on the global expression profile and the dissection into specific regulons, this knowledge can be used to predict the mode of action for novel antimicrobial compounds. This review summarizes our recent progress of proteomic signatures in the model bacterium for low-GC Gram-positive bacteria Bacillus subtilis in response to the antimicrobial compounds phenol, catechol, salicylic acid, 2-methylhydroquinone (2-MHQ) and 6-brom-2-vinyl-chroman-4-on (chromanon). Catechol, 2-MHQ and diamide displayed a common mode of action, as revealed by the induction of the thiol-specific oxidative stress response. In addition, multiple dioxygenases/glyoxalases, azoreductases and nitroreductases were induced by thiol-reactive compounds that are regulated by two novel thiol-specific regulators, YodB and MhqR (YkvE), both of which contribute to electrophile resistance in B. subtilis. These novel thiol-stress-responsive mechanisms are highly conserved among Gram-positive bacteria and are thought to have evolved to detoxify quinone-like electrophiles. [ABSTRACT FROM AUTHOR]

Catechol and 2-methylhydroquinone (2-MHQ) cause the induction of the thiol-specific stress response and four dioxygenases/glyoxalases in Bacillus subtilis. Using transcription factor arrays, the MarR-type regulator YkvE was identified as a repressor of the dioxygenase/glyoxalase-encoding mhqE gene. Transcriptional and proteome analyses of the Δ ykvE mutant revealed the upregulation of ykcA ( mhqA), ydfNOP ( mhqNOP), yodED ( mhqED) and yvaB ( azoR2) encoding multiple dioxygenases/glyoxalases, oxidoreductases and an azoreductase. Primer extension experiments identified σA-type promoter sequences upstream of mhqA, mhqNOP, mhqED and azoR2 from which transcription is elevated after thiol stress. DNase I footprinting analysis showed that YkvE protects a primary imperfect inverted repeat with the consensus sequence of tATCTcgaAtTCgAGATaaaa in the azoR2, mhqE and mhqN promoter regions. Analysis of mhqE-promoter– bgaB fusions confirmed the significance of YkvE binding to this operator in vivo. Adjacent secondary repeats were protected by YkvE in the azoR2 and mhqN promoter regions consistent with multiple DNA–protein binding complexes . DNA-binding activity of YkvE was not directly affected by thiol-reactive compounds in vitro. Mutational analyses showed that MhqA, MhqO and AzoR2 confer resistance to 2-MHQ. Moreover, the Δ ykvE mutant displayed a 2-MHQ and catechol resistant phenotype. YkvE was renamed as MhqR controlling a 2-MHQ and catechol-resistance regulon of B. subtilis. [ABSTRACT FROM AUTHOR]

Disulphide bond formation catalysed by thiol-disulphide oxidoreductases (TDORs) is a universally conserved mechanism for stabilizing extracytoplasmic proteins. In Escherichia coli, disulphide bond formation requires a concerted action of distinct TDORs in thiol oxidation and subsequent quinone reduction. TDOR function in other bacteria has remained largely unexplored. Here we focus on TDORs of low-GC Gram-positive bacteria, in particular DsbA of Staphylococcus aureus and BdbA-D of Bacillus subtilis. Phylogenetic analyses reveal that the homologues DsbA and BdbD cluster in distinct groups typical for Staphylococcus and Bacillus species respectively. To compare the function of these TDORs, DsbA was produced in various bdb mutants of B. subtilis. Next, we assessed the ability of DsbA to sustain different TDOR-dependent processes, including heterologous secretion of E. coli PhoA, competence development and bacteriocin (sublancin 168) production. The results show that DsbA can function in all three processes. While BdbD needs a quinone oxidoreductase for activity, DsbA activity appears to depend on redox-active medium components. Unexpectedly, both quinone oxidoreductases of B. subtilis are sufficient to sustain production of sublancin. Moreover, DsbA can functionally replace these quinone oxidoreductases in sublancin production. Taken together, our unprecedented findings imply that TDOR systems of low-GC Gram-positive bacteria have a modular composition. [ABSTRACT FROM AUTHOR]

The global gene expression profile of Bacillus subtilis in response to ammonium and tryptophan starvation was analyzed using transcriptomics and proteomics which gained novel insights into these starvation responses. The results demonstrate that both starvation conditions induce specific, overlapping and general starvation responses. The TnrA regulon, the glutamine synthetase (glnA) as well as the σL -dependent bkd and roc operons were most strongly and specifically induced after ammonium starvation. These are involved in the uptake and utilization of ammonium and alternative nitrogen sources such as amino acids, γ-aminobutyrate, nitrate/nitrite, uric acid/urea and oligopeptides. In addition, several carbon catabolite-controlled genes (e.g.acsA , citB ), the α-acetolactate synthase/-decarboxylase alsSD operon and several aminotransferase genes were specifically induced after ammonium starvation. The induction of σF - and σE -dependent sporulation proteins at later time points in ammonium-starved cells was accompanied by an increased sporulation frequency. The specific response to tryptophan starvation includes the TRAP-regulated tryptophan biosynthesis genes, some RelA-dependent genes (e.g. adeC, ald ) as well as spo0E . Furthermore, we recognized overlapping responses between ammonium and tryptophan starvation (e.g. dat, maeN) as well as the common induction of the CodY and σH general starvation regulons and the RelA-dependent stringent response. Many genes encoding proteins of so far unknown functions could be assigned to specifically or commonly induced genes. [ABSTRACT FROM AUTHOR]

Aromatic organic compounds that are present in the environment can have toxic effects or provide carbon sources for bacteria. We report here the global response of Bacillus subtilis 168 to phenol and catechol using proteome and transcriptome analyses. Phenol induced the HrcA, σB and CtsR heat-shock regulons as well as the Spx disulfide stress regulon. Catechol caused the activation of the HrcA and CtsR heat-shock regulons and a thiol-specific oxidative stress response involving the Spx, PerR and FurR regulons but no induction of the σB regulon. The most surprising result was that several catabolite-controlled genes are derepressed by catechol, even if glucose is taken up under these conditions. This derepression of the carbon catabolite control was dependent on the glucose concentration in the medium, as glucose excess increased the derepression of the CcpA-dependent lichenin utilization licBCAH operon and the ribose metabolism rbsRKDACB operon by catechol. Growth and viability experiments with catechol as sole carbon source suggested that B. subtilis is not able to utilize catechol as a carbon-energy source. In addition, the microarray results revealed the very strong induction of the yfiDE operon by catechol of which the yfiE gene shares similarities to glyoxalases/bleomycin resistance proteins/extradiol dioxygenases. Using recombinant His6-YfiEBs we demonstrate that YfiE shows catechol-2,3-dioxygenase activity in the presence of catechol as the metabolite 2-hydroxymuconic semialdehyde was measured. Furthermore, both genes of the yfiDE operon are essential for the growth and viability of B. subtilis in the presence of catechol. Thus, our studies revealed that the catechol-2,3-dioxygenase YfiE is the key enzyme of a meta cleavage pathway in B. subtilis involved in the catabolism of catechol. [ABSTRACT FROM AUTHOR]

Activity of the Tat machinery for protein transport across the inner membrane ofEscherichia coliand the chloroplast thylakoidal membrane requires the presence of three membrane proteins: TatA, TatB and TatC. Here, we show that the Tat machinery of the Gram-positive bacteriumBacillus subtilisis very different because it contains at least two minimal Tat translocases, each composed of one specific TatA and one specific TatC component. A third, TatB-like component is apparently not required. This implies that TatA proteins ofB. subtilisperform the functions of both TatA and TatB ofE. coliand thylakoids. Notably, the twoB. subtilistranslocases named TatAdCd and TatAyCy both function as individual, substrate-specific translocases for the twin-arginine preproteins PhoD and YwbN, respectively. Importantly, these minimal TatAC translocases ofB. subtilisare representative for the Tat machinery of the vast majority of Gram-positive bacteria,Streptomycetesbeing the only known exception with TatABC translocases. [ABSTRACT FROM AUTHOR]