Non-Mitochondrial Aconitase-2 Mediates the Transcription of Nuclear-Encoded Electron Transport Chain Genes in Fission Yeast.

Aconitase-2 (Aco2) is present in the mitochondria, cytosol, and nucleus of fission yeast. To explore its function beyond the well-known role in the mitochondrial tricarboxylic acid (TCA) cycle, we conducted genome-wide profiling using the aco2ΔNLS mutant, which lacks a nuclear localization signal (NLS). The RNA sequencing (RNA-seq) data showed a general downregulation of electron transport chain (ETC) genes in the aco2ΔNLS mutant, except for those in the complex II, leading to a growth defect in respiratory-prone media. Complementation analysis with non-catalytic Aco2 [aco2ΔNLS + aco2(3CS)], where three cysteines were substituted with serine, restored normal growth and typical ETC gene expression. This suggests that Aco2's catalytic activity is not essential for its role in ETC gene regulation. Our mRNA decay assay indicated that the decrease in ETC gene expression was due to transcriptional regulation rather than changes in mRNA stability. Additionally, we investigated the Php complex's role in ETC gene regulation and found that ETC genes, except those within complex II, were downregulated in php3Δ and php5Δ strains, similar to the aco2ΔNLS mutant. These findings highlight a novel role for nuclear aconitase in ETC gene regulation and suggest a potential connection between the Php complex and Aco2.

Iron-Induced Respiration Promotes Antibiotic Resistance in Actinomycete Bacteria.

The bacterial response to antibiotics eliciting resistance is one of the key challenges in global health. Despite many attempts to understand intrinsic antibiotic resistance, many of the underlying mechanisms still remain elusive. In this study, we found that iron supplementation promoted antibiotic resistance in Streptomyces coelicolor. Iron-promoted resistance occurred specifically against bactericidal antibiotics, irrespective of the primary target of antibiotics. Transcriptome profiling revealed that some genes in the central metabolism and respiration were upregulated under iron-replete conditions. Iron supported the growth of S. coelicolor even under anaerobic conditions. In the presence of potassium cyanide, which reduces aerobic respiration of cells, iron still promoted respiration and antibiotic resistance. This suggests the involvement of a KCN-insensitive type of respiration in the iron effect. This phenomenon was also observed in another actinobacterium, Mycobacterium smegmatis. Taken together, these findings provide insight into a bacterial resistance strategy that mitigates the activity of bactericidal antibiotics whose efficacy accompanies oxidative damage by switching the respiration mode. IMPORTANCE A widely investigated mode of antibiotic resistance occurs via mutations and/or by horizontal acquisition of resistance genes. In addition to this acquired resistance, most bacteria exhibit intrinsic resistance as an inducible and adaptive response to different classes of antibiotics. Increasing attention has been paid recently to intrinsic resistance mechanisms because this may provide novel therapeutic targets that help rejuvenate the efficacy of the current antibiotic regimen. In this study, we demonstrate that iron promotes the intrinsic resistance of aerobic actinomycetes Streptomyces coelicolor and Mycobacterium smegmatis against bactericidal antibiotics. A surprising role of iron to increase respiration, especially in a mode of using less oxygen, appears a fitting strategy to cope with bactericidal antibiotics known to kill bacteria through oxidative damage. This provides new insights into developing antimicrobial treatments based on the availability of iron and oxygen.

Activity modulation of anti-sigma factor via cysteine alkylation in Actinobacteria.

σR (SigR) is an alternative sigma factor that enables gene expression in Streptomyces coelicolor to cope with thiol oxidation and antibiotic stresses. Its activity is repressed by a zinc-containing anti-sigma (ZAS) factor RsrA that senses thiol oxidants and electrophiles. Inactivation of RsrA by disulfide formation has been well studied. Here we investigated another pathway of RsrA inactivation by electrophiles. Mass spectrometry revealed alkylation of RsrA in vivo by N-ethylmaleimide (NEM) at C61 and C62 located in the C-terminal loop. Substitution mutation (C61S/C62S) in RsrA decreased the induction of σR target genes by electrophiles and made cells more sensitive to electrophiles. In contrast to stable protein of oxidized RsrA, alkylated RsrA is subjected to degradation partly mediated by ClpP proteases. RsrA2, a redox-sensitive homolog of RsrA in S. coelicolor lacking cysteine in the terminal loop, did not respond to electrophiles. However, redox-sensitive RsrA homologs in other Actinobacteria also harboring terminal loop cysteines all responded to electrophiles. These results indicate that the activity of RsrA can be modulated via cysteine alkylation, apart from disulfide formation of zinc-coordinating cysteines. This pathway expands the spectrum of signals that the σR -RsrA system can sense and reveals another intricate regulatory layer for optimal survival of Actinobacteria.

Regulation of iron homeostasis by peroxide-sensitive CatR, a Fur-family regulator in Streptomyces coelicolor.

CatR, a peroxide-sensing transcriptional repressor of Fur family, can de-repress the transcription of the catA gene encoding catalase upon peroxide stress in Streptomyces coelicolor. Since CatR-regulated genes other than catA and its own gene catR have not been identified in detail, the understanding of the role of CatR regulon is very limited. In this study, we performed transcriptomic analysis to identify genes influenced by both ΔcatR mutation and hydrogen peroxide treatment. Through ChIP-qPCR and other analyses, a new consensus sequence was found in CatR-responsive promoter region of catR gene and catA operon for direct regulation. In addition, vtlA (SCO2027) and SCO4983 were identified as new members of the CatR regulon. Expression levels of iron uptake genes were reduced by hydrogen peroxide and a DmdR1 binding sequence was identified in promoters of these genes. The increase in free iron by hydrogen peroxide was thought to suppress the iron import system by DmdR1. A putative exporter protein VtlA regulated by CatR appeared to reduce intracellular iron to prevent oxidative stress. The name vtlA (VIT1-like transporter) was proposed for iron homeostasis related gene SCO2027.

Non-mitochondrial aconitase regulates the expression of iron-uptake genes by controlling the RNA turnover process in fission yeast.

Aconitase, a highly conserved protein across all domains of life, functions in converting citrate to isocitrate in the tricarboxylic acid cycle. Cytosolic aconitase is also known to act as an iron regulatory protein in mammals, binding to the RNA hairpin structures known as iron-responsive elements within the untranslated regions of specific RNAs. Aconitase-2 (Aco2) in fission yeast is a fusion protein consisting of an aconitase and a mitochondrial ribosomal protein, bL21, residing not only in mitochondria but also in cytosol and the nucleus. To investigate the role of Aco2 in the nucleus and cytoplasm of fission yeast, we analyzed the transcriptome of aco2ΔN mutant that is deleted of nuclear localization signal (NLS). RNA sequencing revealed that the aco2ΔN mutation caused increase in mRNAs encoding iron uptake transporters, such as Str1, Str3, and Shu1. The half-lives of mRNAs for these genes were found to be significantly longer in the aco2ΔN mutant than the wild-type strain, suggesting the role of Aco2 in mRNA turnover. The three conserved cysteines required for the catalytic activity of aconitase were not necessary for this role. The UV cross-linking RNA immunoprecipitation analysis revealed that Aco2 directly bound to the mRNAs of iron uptake transporters. Aco2-mediated degradation of iron-uptake mRNAs appears to utilize exoribonuclease pathway that involves Rrp6 as evidenced by genetic interactions. These results reveal a novel role of non-mitochondrial aconitase protein in the mRNA turnover in fission yeast to fine-tune iron homeostasis, independent of regulation by transcriptional repressor Fep1.

The role of Rsv1 in the transcriptional regulation of genes involved in sugar metabolism for long-term survival.

Glucose limitation is a major stress condition that cells must respond to by altering their metabolism to ensure survival. Rsv1 is a zinc finger protein previously shown to be required for survival during stationary phase. In this study, we present a novel mechanism regulated by Rsv1 in the fission yeast Schizosaccharomyces pombe that is involved in altering glucose metabolic flux. We found that rsv1 gene expression is induced by Rst2 and Atf1, two transcription factors regulated by the cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) cascade, respectively. The downstream target genes of Rsv1 were identified by genome-wide ChIP sequencing of Rsv1-bound DNA sites and RNA sequencing analysis of Rsv1-dependent transcripts that were differentially expressed under glucose starvation. Rsv1 directly regulated the expression of at least 21 genes that mostly encode transporters and proteins related to sugar metabolism. Among these, gcd1, which encodes glucose dehydrogenase in the gluconate shunt for the pentose phosphate pathway, was most remarkably repressed by Rsv1. The defect in survival of Δrsv1 mutant under glucose starvation condition was mitigated by additional deletion of a gcd1, idn1, or a gene for a putative lactonase (SPCC16c4.10), suggesting the critical importance of downregulating the gluconate shunt and pentose phosphate pathway for long-term survival. These results show an intricate response to glucose starvation: increasing the synthesis of a transcription factor via two signal transduction pathways, which sheds light on the importance of remodeling a metabolic circuit to secure glucose for cell survival.

SigR, a hub of multilayered regulation of redox and antibiotic stress responses.

Signal-specific activation of alternative sigma factors redirects RNA polymerase to induce transcription of distinct sets of genes conferring protection against the damage the signal and the related stresses incur. In Streptomyces coelicolor, σR (SigR), a member of ECF12 subfamily of Group IV sigma factors, responds to thiol-perturbing signals such as oxidants and electrophiles, as well as to translation-blocking antibiotics. Oxidants and electrophiles interact with and inactivate the zinc-containing anti-sigma factor, RsrA, via disulfide bond formation or alkylation of reactive cysteines, subsequently releasing σR for target gene induction. Translation-blocking antibiotics induce the synthesis of σR , via the WhiB-like transcription factor, WblC/WhiB7. Signal transduction via RsrA produces a dramatic transient response that involves positive feedback to produce more SigR as an unstable isoform σ R ' and negative feedbacks to degrade σ R ' , and reduce oxidized RsrA that subsequently sequester σR and σ R ' . Antibiotic stress brings about a prolonged response by increasing stable σR levels. The third negative feedback, which occurs via IF3, lowers the translation efficiency of the sigRp1 transcript that utilizes a non-canonical start codon. σR is a global regulator that directly activates > 100 transcription units in S. coelicolor, including genes for thiol homeostasis, protein quality control, sulfur metabolism, ribosome modulation and DNA repair. Close homologues in Actinobacteria, such as σH in Mycobacteria and Corynebacteria, show high conservation of the signal transduction pathways and target genes, thus reflecting the robustness of this type of regulation in response to redox and antibiotic stresses.

Simultaneous Activation of Iron- and Thiol-Based Sensor-Regulator Systems by Redox-Active Compounds.

Bacteria in natural habitats are exposed to myriad redox-active compounds (RACs), which include producers of reactive oxygen species (ROS) and reactive electrophile species (RES) that alkylate or oxidize thiols. RACs can induce oxidative stress in cells and activate response pathways by modulating the activity of sensitive regulators. However, the effect of a certain compound on the cell has been investigated primarily with respect to a specific regulatory pathway. Since a single compound can exert multiple chemical effects in the cell, its effect can be better understood by time-course monitoring of multiple sensitive regulatory pathways that the compound induces. We investigated the effect of representative RACs by monitoring the activity of three sensor-regulators in the model actinobacterium Streptomyces coelicolor; SoxR that senses reactive compounds directly through oxidation of its [2Fe-2S] cluster, CatR/PerR that senses peroxides through bound iron, and an anti-sigma factor RsrA that senses RES via disulfide formation. The time course and magnitude of induction of their target transcripts were monitored to predict the chemical activities of each compound in S. coelicolor. Phenazine methosulfate (PMS) was found to be an effective RAC that directly activated SoxR and an effective ROS-producer that induced CatR/PerR with little thiol-perturbing activity. p-Benzoquinone was an effective RAC that directly activated SoxR, with slower ROS-producing activity, and an effective RES that induced the RsrA-SigR system. Plumbagin was an effective RAC that activated SoxR, an effective ROS-producer, and a less agile but effective RES. Diamide was an RES that effectively formed disulfides and a weak RAC that activated SoxR. Monobromobimane was a moderately effective RES and a slow producer of ROS. Interestingly, benzoquinone induced the SigR system by forming adducts on cysteine thiols in RsrA, revealing a new pathway to modulate RsrA activity. Overall, this study showed that multiple chemical activities of a reactive compound can be conveniently monitored in vivo by examining the temporal response of multiple sensitive regulators in the cell to reveal novel activities of the chemicals.

The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines.

Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1.

Essential function of Aco2, a fusion protein of aconitase and mitochondrial ribosomal protein bL21, in mitochondrial translation in fission yeast.

A possible interaction between aconitase and a mitochondrial ribosomal protein was suggested in a genome-wide interactome study. In fission yeast Schizosaccharomyces pombe, the aco2(+) gene encodes a fusion protein between aconitase and a putative mitochondrial ribosomal protein bL21 (Mrpl49). Two types of aco2(+) transcripts are generated via alternative poly (A) site selection, producing both a single aconitase domain protein and the fusion form. The bL21-fused Aco2 protein resides in mitochondria as well as in the cytosol and the nucleus. The viability defect of aco2 mutation is complemented not by the aconitase domain but by the bL21 domain, which enables mitochondrial translation.

Genome-scale analysis reveals a role for NdgR in the thiol oxidative stress response in Streptomyces coelicolor.

BACKGROUND: NdgR is an IclR-type transcription factor that regulates leucine biosynthesis and other metabolic pathways in Streptomyces coelicolor. Recent study revealed that NdgR is one of the regulatory targets of SigR, an oxidative stress response sigma factor, suggesting that the NdgR plays an important physiological role in response to environmental stresses. Although the regulatory functions of NdgR were partly characterized, determination of its regulon is required for better understanding of the transcriptional regulatory network related with the oxidative stress response. RESULTS: We determined genome-wide binding loci of NdgR by using chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and explored its physiological roles. The ChIP-seq profiles revealed 19 direct binding loci with a 15-bp imperfect palindromic motif, including 34 genes in their transcription units. Most genes in branched-chain amino acid and cysteine biosynthesis pathways were involved in the NdgR regulon. We proved that ndgR is induced by SigR under the thiol oxidation, and that an ndgR mutant strain is sensitive to the thiol oxidizing agent, diamide. Through the expression test of NdgR and the target genes for NdgR under diamide treatment, regulatory motifs were suggested. Interestingly, NdgR constitutes two regulatory motifs, coherent and incoherent feed-forward loops (FFL), in order to control its regulon under the diamide treatment. Using the regulatory motifs, NdgR regulates cysteine biosynthesis in response to thiol oxidative stress, enabling cells to maintain sulfur assimilation with homeostasis under stress conditions. CONCLUSIONS: Our analysis revealed that NdgR is a global transcriptional regulator involved in the regulation of branched-chain amino acids biosynthesis and sulphur assimilation. The identification of the NdgR regulon broadens our knowledge regarding complex regulatory networks governing amino acid biosynthesis in the context of stress responses in S. coelicolor.

Regulation of a nickel-cobalt efflux system and nickel homeostasis in a soil actinobacterium Streptomyces coelicolor.

In Streptomyces coelicolor, a soil actinobacterium capable of morphological differentiation and complex secondary metabolism, nickel deficiency is sensed by Nur, a Ni-specific Fur family regulator that controls nickel uptake systems (NikABCDE and NikMNOQ) and both Fe-containing and Ni-containing superoxide dismutases (SodF and SodN). On the other hand, the nickel efflux system and its regulator have not been elucidated. In this study, we demonstrate that an ArsR/SmtB family metalloregulator NmtR, a close homologue of NmtR from Mycobacterium tuberculosis, controls a putative efflux pump of P1-type ATPase (NmtA) in S. coelicolor. NmtR binds to the nmtA promoter region to repress its transcription, and is dissociated in the presence of Ni(ii) and Co(ii). Disruption of the nmtA gene makes cells more sensitive to nickel and cobalt, consistent with its predicted role in encoding a Ni-Co-efflux pump. Growth of S. coelicolor in complex YEME medium is only marginally inhibited by up to 0.5 mM Ni(ii), with significant growth retardation at 1 mM. Nur-regulated sodF and nikA genes are repressed at less than 0.1 µM added NiSO4 whereas NmtR-regulated nmtA transcription is induced at 0.5 mM or more Ni(ii). This reveals the extreme sensitivity of S. coelicolor to nickel deficiency as well as tolerance to surplus nickel. How this organism and possibly other actinomycetes have evolved to develop such a highly Ni-tolerant physiology and how the highly sensitive regulator Nur and the obtuse regulator NmtR achieve their characteristic Ni-sensitivity are interesting questions to solve in the future.

Alternative zinc-binding sites explain the redox sensitivity of zinc-containing anti-sigma factors.

Certain bacterial zinc-containing anti-sigma (ZAS) factors respond sensitively to thiol-induced oxidative stress by undergoing conformational changes, which in turn reduce binding affinities for their cognate sigma factors. This redox sensitivity provides a mechanism for coping with oxidative stress by activating the transcription of antioxidant genes. Not all ZAS proteins are redox-sensitive, but the mechanism of redox sensitivity is not fully understood. Here we propose that alternative zinc-binding sites determine redox sensitivity. To support this proposal, we performed protein modeling and zinc docking on redox-sensitive and redox-insensitive ZAS proteins complexed with their cognate sigma factors. At least one strong alternative zinc-binding pocket was detected for all known redox-sensitive ZAS factors in actinomycetes, while no strong alternative zinc-binding pocket was identified in redox-insensitive ZAS factors, except for one controversial case. This hypothesis of alternative zinc-binding sites can also explain residue-specific contributions to the redox sensitivity of RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor, for which alanine mutagenesis experiments are available. Our results suggest a mechanistic model for redox sensitivity as follows: zinc ion can probabilistically occupy multiple sites in redox-sensitive ZAS proteins, increasing the susceptibility of zinc-coordinating cysteine residues to oxidation. This picture of probabilistic zinc occupation agrees with a previous structure and energy analysis on zinc finger proteins, and thus it may be more widely applicable to other classes of reactive zinc-binding proteins.

Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response.

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K(33)FEHH(37)FEEC(41)SPC(44)LEK(47) that encompass the conserved HX(3)CX(2)C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information.

Multi-domain CGFS-type glutaredoxin Grx4 regulates iron homeostasis via direct interaction with a repressor Fep1 in fission yeast.

The fission yeast Schizosaccharomyces pombe contains two CGFS-type monothiol glutaredoxins, Grx4 and Grx5, which are localized primarily in the nucleus and mitochondria, respectively. We observed involvement of Grx4 in regulating iron-responsive gene expression, which is modulated by a repressor Fep1. Lack of Grx4 caused defects not only in growth but also in the expression of both iron-uptake and iron-utilizing genes regardless of iron availability. In order to unravel how Grx4 is involved in Fep1-mediated regulation, interaction between them was investigated. Co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) revealed that Grx4 physically interacts with Fep1 in vivo. BiFC revealed localized nuclear dots produced by interaction of Grx4 with Fep1. Mutation of cysteine-172 in the CGFS motif to serine (C172S) produced effects similarly observed under Grx4 depletion, such as the loss of iron-dependent gene regulation and the absence of nuclear dots in BiFC analysis. These results suggest that the ability of Grx4 to bind iron, most likely Fe-S cofactor, could be critical in interacting with and modulating the activity of Fep1.

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast.

Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Deltagrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1(+) gene encoding a putative A-type Fe-S scaffold, in addition to mas5(+) and hsc1(+) genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2(+), but not isu1(+) and ssc1(+), complemented the growth phenotype of Deltagrx5 mutant as isa1(+) did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.

Positive and negative feedback regulatory loops of thiol-oxidative stress response mediated by an unstable isoform of sigmaR in actinomycetes.

Alternate sigma factors provide an effective way of diversifying bacterial gene expression in response to environmental changes. In Streptomyces coelicolor where more than 65 sigma factors are predicted, sigma(R) is the major regulator for response to thiol-oxidative stresses. sigma(R) becomes available when its bound anti-sigma factor RsrA is oxidized at sensitive cysteine thiols to form disulphide bonds. sigma(R) regulon includes genes for itself and multiple thiol-reducing systems, which constitute positive and negative feedback loops respectively. We found that the positive amplification loop involves an isoform of sigma(R) (sigma(R')) with an N-terminal extension of 55 amino acids, produced from an upstream start codon. A major difference between constitutive sigma(R) and inducible sigma(R') is that the latter is markedly unstable (t(1/2) approximately 10 min) compared with the former (> 70 min). The rapid turnover of sigma(R') is partly due to induced ClpP1/P2 proteases from the sigma(R) regulon. This represents a novel way of elaborating positive and negative feedback loops in a control circuit. Similar phenomenon may occur in other actinomycetes that harbour multiple start codons in the sigR homologous gene. We observed that sigH gene, the sigR orthologue in Mycobacterium smegmatis, produces an unstable larger isoform of sigma(H) upon induction by thiol-oxidative stress.

Structural basis for the specialization of Nur, a nickel-specific Fur homolog, in metal sensing and DNA recognition.

Nur, a member of the Fur family, is a nickel-responsive transcription factor that controls nickel homeostasis and anti-oxidative response in Streptomyces coelicolor. Here we report the 2.4-A resolution crystal structure of Nur. It contains a unique nickel-specific metal site in addition to a nonspecific common metal site. The identification of the 6-5-6 motif of the Nur recognition box and a Nur/DNA complex model reveals that Nur mainly interacts with terminal bases of the palindrome on complex formation. This contrasts with more distributed contacts between Fur and the n-1-n type of the Fur-binding motif. The disparity between Nur and Fur in the conformation of the S1-S2 sheet in the DNA-binding domain can explain their different DNA-recognition patterns. Furthermore, the fact that the specificity of Nur in metal sensing and DNA recognition is conferred by the specific metal site suggests that its introduction drives the evolution of Nur orthologs in the Fur family.

Thiol-independent action of mitochondrial thioredoxin to support the urea cycle of arginine biosynthesis in Schizosaccharomyces pombe.

Thioredoxins usually perform a role as a thiol-disulfide oxidoreductase using their active-site cysteines. The fission yeast Schizosaccharomyces pombe contains two thioredoxins: Trx1 for general stress protection and Trx2 for mitochondrial functions. The Deltatrx2 mutant grows as well as the wild type on complex media containing glucose. However, on nonfermentable carbon source such as glycerol, the mutant did not grow, indicating a defect in mitochondrial function. The mutant also exhibited auxotrophy for arginine and cysteine on minimal medium. In order to find the reason for the unexpected arginine auxotrophy, we searched for multicopy suppressors and found that the arg3(+) gene encoding ornithine carbamoyltransferase (OCTase) in the urea cycle of the arginine biosynthetic pathway rescued the arginine auxotrophy. The levels of arg3(+) transcript, Arg3 protein, and OCTase activity were all decreased in Deltatrx2. Through immunocoprecipitation, we observed a direct interaction between Trx2 and Arg3 in cell extracts. The mutant forms of Trx2 lacking either one or both of the active site cysteines through substitution to serines also rescued the arginine auxotrophy and restored the decreased OCTase activity. They also rescued the growth defect of Deltatrx2 on glycerol medium. This contrasts with the thiol-dependent action of overproduced Trx2 in complementing glutathione reductase. Therefore, Trx2 serves multiple functions in mitochondria, protecting mitochondrial components against thiol-oxidative damage as a thiol-disulfide oxidoreductase, and supporting urea cycle and respiration in mitochondria in a manner independent of active site thiols.

The role and regulation of Trxl, a cytosolic thioredoxin in Schizosaccharomyces pombe.

The genome of fission yeast Schizosaccharomyces pombe harbors two genes for thioredoxins, trx1(+) and trx2(+), which encode cytosolic and mitochondrial thioredoxins, respectively. The Deltatrx1 mutant was found sensitive to diverse external stressors such as various oxidants, heat, and salt, whereas Deltatrx2 mutant was not sensitive except to paraquat, a superoxide generator. Both Deltatrx1 and Deltatrx2 mutants were more resistant to diamide, a thiol-specific oxidant, than the wild type. The trx1(+) gene expression was induced by H(2)O(2) and menadione, being mediated through a stress-responsive transcription factor Papl. In Deltatrx1 cells, the basal expression of Pap1-regulated genes were elevated, suggesting a role for Trxl as a reducer for oxidized (activated) Papl. The Deltatrx1 mutant exhibited cysteine auxotrophy, which can be overcome by adding sulfite. This suggests that Trxl serves as a primary electron donor for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase and thus is an essential protein for sulfur assimilation in S. pombe. These results suggest that, in contrast to Trx2 whose role is more confined to mitochondrial functions, Trxl plays a major role in protecting S. pombe against various stressful conditions and enables proper sulfur metabolism.