Search Results (11)

Although bacterial thioredoxin reductase-like ferredoxin/flavodoxin NAD(P)⁺ oxidoreductases (FNRs) are similar in terms of primary sequences and structures, they participate in diverse biological processes by catalyzing a range of different redox reactions. Many of the reactions are critical for the growth, survival of, and infection by pathogens, and insight into the structural basis for substrate preference, specificity, and reaction kinetics is crucial for the detailed understanding of these redox pathways. Bacillus cereus (Bc) encodes three FNR paralogs, two of which have assigned distinct biological functions in bacillithiol disulfide reduction and flavodoxin (Fld) reduction. Bc FNR2, the endogenous reductase of the Fld-like protein NrdI, belongs to a distinct phylogenetic cluster of homologous oxidoreductases containing a conserved His residue stacking the FAD cofactor. In this study, we have assigned a function to FNR1, in which the His residue is replaced by a conserved Val, in the reduction of the heme-degrading monooxygenase IsdG, ultimately facilitating the release of iron in an important iron acquisition pathway. The Bc IsdG structure was solved, and IsdG-FNR1 interactions were proposed through protein–protein docking. Mutational studies and bioinformatics analyses confirmed the importance of the conserved FAD-stacking residues on the respective reaction rates, proposing a division of FNRs into four functionally unique sequence similarity clusters likely related to the nature of this residue. Full article

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. Full article

Deinococcus species possess remarkable tolerance to extreme environmental conditions that generate oxidative damage to macromolecules. Among enzymes fulfilling key functions in metabolism regulation and stress responses, thiol reductases (TRs) harbour catalytic cysteines modulating the redox status of Cys and Met in partner proteins. We present here a detailed description of Deinococcus TRs regarding gene occurrence, sequence features, and physiological functions that remain poorly characterised in this genus. Two NADPH-dependent thiol-based systems are present in Deinococcus. One involves thioredoxins, disulfide reductases providing electrons to protein partners involved notably in peroxide scavenging or in preserving protein redox status. The other is based on bacillithiol, a low-molecular-weight redox molecule, and bacilliredoxin, which together protect Cys residues against overoxidation. Deinococcus species possess various types of thiol peroxidases whose electron supply depends either on NADPH via thioredoxins or on NADH via lipoylated proteins. Recent data gained on deletion mutants confirmed the importance of TRs in Deinococcus tolerance to oxidative treatments, but additional investigations are needed to delineate the redox network in which they operate, and their precise physiological roles. The large palette of Deinococcus TR representatives very likely constitutes an asset for the maintenance of redox homeostasis in harsh stress conditions. Full article

The transposition of insertion sequence elements was evaluated among different Deinococcus geothermalis lineages, including the wild-type, a cystine importer-disrupted mutant, a complemented strain, and a cystine importer-overexpressed strain. Cellular growth reached early exponential growth at OD₆₀₀ 2.0 and late exponential growth at OD₆₀₀ 4.0. Exposing the cells to hydrogen peroxide (80–100 mM) resulted in the transposition of insertion sequences (ISs) in genes associated with the carotenoid biosynthesis pathway. Particularly, ISDge7 (an IS5 family member) and ISDge5 (an IS701 family member) from the cystine importer-disrupted mutant were transposed into phytoene desaturase (dgeo_0524) via replicative transposition. Further, the cystine importer-overexpressed strain Δdgeo_1985R showed transposition of both ISDge2 and ISDge5 elements. In contrast, IS transposition was not detected in the complementary strain. Interestingly, a cystine importer-overexpressing strain exhibited streptomycin resistance, indicating that point mutation occurred in the rpsL (dgeo_1873) gene encoding ribosomal protein S12. qRT-PCR analyses were then conducted to evaluate the expression of oxidative stress response genes, IS elements, and low-molecular-weight thiol compounds such as mycothiol and bacillithiol. Nevertheless, the mechanisms that trigger IS transposition in redox imbalance conditions remain unclear. Here, we report that the active transposition of different IS elements was affected by intracellular redox imbalances caused by cystine importer deficiencies or overexpression. Full article

Deinococcus radiodurans is a robust bacterium with extraordinary resistance to ionizing radiation and reactive oxygen species (ROS). D. radiodurans produces an antioxidant thiol compound called bacillithiol (BSH), but BSH-related enzymes have not been investigated. The D. radiodurans mutant lacking bshA (dr_1555), the first gene of the BSH biosynthetic pathway, was devoid of BSH and sensitive to hydrogen peroxide (H₂O₂) compared to the wild-type D. radiodurans strain. Three bacilliredoxin (Brx) proteins, BrxA, B, and C, have been identified in BSH-producing bacteria, such as Bacillus. D. radiodurans possesses DR_1832, a putative homolog of BrxC. However, because DR_1832 contains a novel signature motif (TCHKT) and a C-terminal region similar to the colicin-like immunity domain, we named it AbxC (atypical BrxC). The deletion of abxC also sensitized cells to H₂O₂. AbxC exhibited peroxidase activity in vitro, which was linked to nicotinamide adenine dinucleotide phosphate (NADPH) oxidation via the BSH disulfide reductase DR_2623 (DrBdr). AbxC proteins were present mainly as dimers after exposure to H₂O₂ in vitro, and the oxidized dimers were resolved to monomers by the reaction coupled with BSH as an electron donor, in which DrBdr transported reducing equivalents from NADPH to AbxC through BSH recycling. We identified 25 D. radiodurans proteins that potentially interact with AbxC using AbxC-affinity chromatography. Most of them are associated with cellular metabolisms, such as glycolysis and amino acid biosynthesis, and stress response. Interestingly, AbxC could bind to the proposed peroxide-sensing transcription regulator, DrOxyR. These results suggest that AbxC may be involved in the H₂O₂ signaling mechanism mediated by DrOxyR. Full article

The volatile organic sulfur compound allicin (diallyl thiosulfinate) is produced as a defense substance when garlic (Allium sativum) tissues are damaged, for example by the activities of pathogens or pests. Allicin gives crushed garlic its characteristic odor, is membrane permeable and readily taken up by exposed cells. It is a reactive thiol-trapping sulfur compound that S-thioallylates accessible cysteine residues in proteins and low molecular weight thiols including the cellular redox buffer glutathione (GSH) in eukaryotes and Gram-negative bacteria, as well as bacillithiol (BSH) in Gram-positive firmicutes. Allicin shows dose-dependent antimicrobial activity. At higher doses in eukaryotes allicin can induce apoptosis or necrosis, whereas lower, biocompatible amounts can modulate the activity of redox-sensitive proteins and affect cellular signaling. This review summarizes our current knowledge of how bacterial and eukaryotic cells are specifically affected by, and respond to, allicin. Full article

Low molecular weight (LMW) thiols have many functions in bacteria and eukarya, ranging from redox homeostasis to acting as cofactors in numerous reactions, including detoxification of xenobiotic compounds. The LMW thiol, glutathione (GSH), is found in eukaryotes and many species of bacteria. Analogues of GSH include the structurally different LMW thiols: bacillithiol, mycothiol, ergothioneine, and coenzyme A. Many advances have been made in understanding the diverse and multiple functions of GSH and GSH analogues in bacteria but much less is known about distribution and functions of GSH and its analogues in archaea, which constitute the third domain of life, occupying many niches, including those in extreme environments. Archaea are able to use many energy sources and have many unique metabolic reactions and as a result are major contributors to geochemical cycles. As LMW thiols are major players in cells, this review explores the distribution of thiols and their biochemistry in archaea. Full article

Garlic plants (Allium sativum L.) produce antimicrobial compounds, such as diallyl thiosulfinate (allicin) and diallyl polysulfanes. Here, we investigated the transcriptome and protein S-thioallylomes under allicin and diallyl tetrasulfane (DAS4) exposure in the Gram-positive bacterium Bacillus subtilis. Allicin and DAS4 caused a similar thiol-specific oxidative stress response, protein and DNA damage as revealed by the induction of the OhrR, PerR, Spx, YodB, CatR, HypR, AdhR, HxlR, LexA, CymR, CtsR, and HrcA regulons in the transcriptome. At the proteome level, we identified, in total, 108 S-thioallylated proteins under allicin and/or DAS4 stress. The S-thioallylome includes enzymes involved in the biosynthesis of surfactin (SrfAA, SrfAB), amino acids (SerA, MetE, YxjG, YitJ, CysJ, GlnA, YwaA), nucleotides (PurB, PurC, PyrAB, GuaB), translation factors (EF-Tu, EF-Ts, EF-G), antioxidant enzymes (AhpC, MsrB), as well as redox-sensitive MarR/OhrR and DUF24-family regulators (OhrR, HypR, YodB, CatR). Growth phenotype analysis revealed that the low molecular weight thiol bacillithiol, as well as the OhrR, Spx, and HypR regulons, confer protection against allicin and DAS4 stress. Altogether, we show here that allicin and DAS4 cause a strong oxidative, disulfide and sulfur stress response in the transcriptome and widespread S-thioallylation of redox-sensitive proteins in B. subtilis. The results further reveal that allicin and polysulfanes have similar modes of actions and thiol-reactivities and modify a similar set of redox-sensitive proteins by S-thioallylation. Full article

The glyoxalase system consists of two enzymes, glyoxalase I (Glo1) and glyoxalase II (Glo2), and converts a hemithioacetal substrate formed between a cytotoxic alpha-ketoaldehyde, such as methylglyoxal (MG), and an intracellular thiol, such as glutathione, to a non-toxic alpha-hydroxy acid, such as d-lactate, and the regenerated thiol. Two classes of Glo1 have been identified. The first is a Zn²⁺-activated class and is exemplified by the Homo sapiens Glo1. The second class is a Ni²⁺-activated enzyme and is exemplified by the Escherichia coli Glo1. Glutathione is the intracellular thiol employed by Glo1 from both these sources. However, many organisms employ other intracellular thiols. These include trypanothione, bacillithiol, and mycothiol. The trypanothione-dependent Glo1 from Leishmania major has been shown to be Ni²⁺-activated. Genetic studies on Bacillus subtilis and Corynebacterium glutamicum focused on MG resistance have indicated the likely existence of Glo1 enzymes employing bacillithiol or mycothiol respectively, although no protein characterizations have been reported. The current investigation provides a preliminary characterization of an isolated mycothiol-dependent Glo1 from Streptomyces coelicolor. The enzyme has been determined to display a Ni²⁺-activation profile and indicates that Ni²⁺-activated Glo1 are indeed widespread in nature regardless of the intracellular thiol employed by an organism. Full article

Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW) thiols. Bacillithiol was recently identified as a major low-molecular-weight thiol in Bacillus subtilis and related Firmicutes. Four genes (bshA, bshB1, bshB2, and bshC) are involved in bacillithiol biosynthesis. The bshA and bshB1 genes are part of a seven-gene operon (ypjD), which includes the essential gene cca, encoding CCA-tRNA nucleotidyltransferase. The inclusion of cca in the operon containing bacillithiol biosynthetic genes suggests that the integrity of the 3′ terminus of tRNAs may also be important in oxidative stress. The addition of the 3′ terminal CCA sequence by CCA-tRNA nucleotidyltransferase to give rise to a mature tRNA and functional molecules ready for aminoacylation plays an essential role during translation and expression of the genetic code. Any defects in these processes, such as the accumulation of shorter and defective tRNAs under oxidative stress, might exert a deleterious effect on cells. This review summarizes the physiological link between tRNA^Cys regulation and oxidative stress in Bacillus. Full article

The LmbE-like superfamily is comprised of a series of enzymes that use a single catalytic metal ion to catalyze the hydrolysis of various substrates. These substrates are often key metabolites for eukaryotes and prokaryotes, which makes the LmbE-like enzymes important targets for drug development. Herein we review the structure and function of the LmbE-like proteins identified to date. While this is the newest superfamily of metallohydrolases, a growing number of functionally interesting proteins from this superfamily have been characterized. Available crystal structures of LmbE-like proteins reveal a Rossmann fold similar to lactate dehydrogenase, which represented a novel fold for (zinc) metallohydrolases at the time the initial structure was solved. The structural diversity of the N-acetylglucosamine containing substrates affords functional diversity for the LmbE-like enzyme superfamily. The majority of enzymes identified to date are metal-dependent deacetylases that catalyze the hydrolysis of a N-acetylglucosamine moiety on substrate using a combination of amino acid side chains and a single bound metal ion, predominantly zinc. The catalytic zinc is coordinated to proteins via His₂-Asp-solvent binding site. Additionally, studies indicate that protein dynamics play important roles in regulating access to the active site and facilitating catalysis for at least two members of this protein superfamily. Full article