Jung-Hye Roe

RsrA, an anti‐sigma factor regulated by redox change

SigR (σR) is a sigma factor responsible for inducing the thioredoxin system in response to oxidative stress in the antibiotic‐producing, Gram‐positive bacterium Streptomyces coelicolor A3(2). Here we identify a redox‐sensitive, σR‐specific anti‐sigma factor, RsrA, which binds σR and inhibits σR‐directed transcription in vitro only under reducing conditions. Exposure to H2O2 or to the thiol‐specific oxidant diamide caused the dissociation of the σR–RsrA complex, thereby allowing σR‐dependent transcription. This correlated with intramolecular disulfide bond formation in RsrA. Thioredoxin was able to reduce oxidized RsrA, suggesting that σR, RsrA and the thioredoxin system comprise a novel feedback homeostasis loop that senses and responds to changes in the intracellular thiol–disulfide redox balance.

Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation

Glutathione (GSH) is an abundant cellular thiol which has been implicated in many cellular processes including protection against xenobiotics, carcinogens and free radicals. Utilization of GSH in both enzymic and non‐enzymic defence mechanisms results in its conversion to the oxidized form (GSSG), and it must be recycled to GSH to maintain the high intracellular ratio of GSH to GSSG. Glutathione reductase (GLR) is a flavoenzyme, which catalyses reduction of GSSG to GSH using the reducing power of NADPH. We show that yeast mutants deleted for GLR1, encoding glutathione reductase, lack GLR activity and accumulate increased levels of GSSG. In addition, the glr1 mutant strain was unaffected in the inducible adaptive response to hydrogen peroxide, but showed increased sensitivity to oxidants including both peroxides and superoxide, indicating a requirement for GLR in protection against oxidative stress. Furthermore, GLR1 expression was elevated two to threefold in the presence of oxidants, and regulation was dependent upon the yAP‐1 transcriptional activator protein. Thus, GLR1 is one of a growing number of genes involved in the protection of yeast cells against oxidative stress and regulated by yAP‐1.

σR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2)

We have identified an RNA polymerase sigma factor, σR, that is part of a system that senses and responds to thiol oxidation in the Gram‐positive, antibiotic‐producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding σR caused sensitivity to the thiol‐specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of σR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5–6 bp. trxBp1 is transiently induced at least 50‐fold in response to diamide treatment in a sigR‐dependent manner. Purified σR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for σR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR‐null mutant, and purified σR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70‐fold) following treatment with diamide. We propose a model in which σR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, σR activity is switched off, resulting in down‐regulation of trxBA and sigR. We present evidence that the σR system also functions in the actinomycete pathogen Mycobacterium tuberculosis.

IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins.

In Escherichia coli, Fe‐S clusters are assembled by gene products encoded from the isc and suf operons. Both the iscRSUA and sufABCDSE operons are induced highly by oxidants, reflecting an increased need for providing and maintaining Fe‐S clusters under oxidative stress conditions. Three cis‐acting oxidant‐responsive elements (ORE‐I, II, III) in the upstream of the sufA promoter serve as the binding sites for OxyR, IHF and an uncharacterized factor respectively. Using DNA affinity fractionation, we isolated an ORE‐III‐binding factor that positively regulates the suf operon in response to various oxidants. MALDI‐TOF mass analysis identified it with IscR, known to serve as a repressor of the iscRSUA gene expression under anaerobic condition as a [2Fe‐2S]‐bound form. The iscR null mutation abolished ORE‐III‐binding activity in cell extracts, and caused a significant decrease in the oxidant induction of sufA in vivo. OxyR and IscR contributed almost equally to activate the sufA operon in response to oxidants. Purified IscR that lacked Fe‐S cluster bound to the ORE‐III site and activated transcription from the sufA promoter in vitro. Mutations in Fe‐S‐binding sites of IscR enabled sufA activation in vivo and in vitro. These results support a model that IscR in its demetallated form directly activates sufA transcription, while it de‐represses isc operon, under oxidative stress condition.

A reducing system of the superoxide sensor SoxR in Escherichia coli

The soxRS regulon functions in protecting Escherichia coli cells against superoxide and nitric oxide. When SoxR is activated by oxidation of its [2Fe–2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression. How the oxidized SoxR returns to and is maintained in its reduced state has been under question. To identity genes that constitute the SoxR‐reducing system, we screened an E.coli mutant library carrying a chromosomal soxSp::lacZ fusion, for constitutive mutants. Mutations mapped to two loci: the rsxABCDGE operon (named for reducer of SoxR) that is highly homologous to the rnfABCDGE operon in Rhodobacter capsulatus involved in transferring electrons to nitrogenase, and the rseC gene in the rpoE–rseABC operon. In‐frame deletion of each open reading frame in the rsxABCDGE operon produced a similar constitutive phenotype. The double mutation of rsx and rseC suggested that rsxABCDGE and rseC gene products act together in the same pathway in reducing SoxR. Electron paramagnetic resonance analysis of SoxR and measurement of re‐reduction kinetics support the proposal that rsx and rseC gene products constitute a reducing system for SoxR.

Mutational analysis of RsrA, a zinc‐binding anti‐sigma factor with a thiol–disulphide redox switch

In the Gram‐positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called σR in response to changes in the cytoplasmic thiol–disulphide status, and the activity of σR is controlled post‐translationally by an anti‐sigma factor, RsrA. In vitro, the anti‐sigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol–disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and σR‐dependent transcription, confirming that RsrA is a negative regulator of σR and a key sensor of thiol–disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti‐sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo. We also show that RsrA is a metalloprotein, containing near‐stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol–disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti‐sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free σR.

Induction of the sufA operon encoding Fe‐S assembly proteins by superoxide generators and hydrogen peroxide: involvement of OxyR, IHF and an unidentified oxidant‐responsive factor

A promoter (sufAp), inducible by various oxidants, directs transcription of the sufABCDSE operon encoding an alternative Fe‐S cluster assembly system in Escherichia coli. Superoxide generators and H2O2 induced expression of sufA–lacZ even in ΔsoxRS and ΔoxyR mutants, suggesting participation of an additional regulator(s) in oxidant induction of the sufA operon. Through deletion and linker scanning mutagenesis, we found three cis‐acting oxidant‐responsive elements (OREs). ORE‐I lies between −236 and −197 nucleotides from the transcription start site, overlapping extensively with the OxyR binding site reported previously. ORE‐II (−156 to −127) was found to be the site of IHF action. ORE‐III (−56 to −35) had no predictable binding sites for known regulators. Gel mobility shift assays with a 50 bp DNA probe containing ORE‐III revealed the presence of an ORE‐III‐specific factor that binds only when cells are treated with oxidants. S1 mapping analysis revealed that phenazine methosulphate (PMS) and H2O2 induced sufA expression by more than 40‐fold. In a ΔoxyR mutant, sufA was still induced more than 10‐fold. Fur, a ferric uptake regulator that negatively regulates this operon in response to iron availability, did not mediate the oxidant induction. Deletion of the suf operon caused cells to be more sensitive to superoxide‐generating agents without affecting sensitivity to H2O2. From these results, we propose that the oxidant induction of the sufA operon is mediated through OxyR, IHF, plus an unidentified oxidant‐responsive factor, and that the suf gene products are needed to defend cells against oxidative stress caused by superoxide generators.

The Escherichia coli glucose transporter enzyme IICB(Glc) recruits the global repressor Mlc.

In addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes. Exposure of cells to glucose can result in repression or induction of gene expression. While the mechanism for carbon catabolite repression by glucose was well documented, that for glucose induction was not clearly understood in Escherichia coli. Recently, glucose induction of several E.coli genes has been shown to be mediated by the global repressor Mlc. Here, we elucidate a general mechanism for glucose induction of gene expression in E.coli, revealing a novel type of regulatory circuit for gene expression mediated by the phosphorylation state‐dependent interaction of a membrane‐bound protein with a repressor. The dephospho‐form of enzyme IICBGlc, but not its phospho‐form, interacts directly with Mlc and induces transcription of Mlc‐regulated genes by displacing Mlc from its target sequences. Therefore, the glucose induction of Mlc‐regulated genes is caused by dephosphorylation of the membrane‐bound transporter enzyme IICBGlc, which directly recruits Mlc to derepress its regulon.

A master regulator σB governs osmotic and oxidative response as well as differentiation via a network of sigma factors in Streptomyces coelicolor

The differentiating bacterium Streptomyces coelicolor harbours some 66 sigma factors, which support its complex life cycle. σB, a functional homologue of σS from Escherichia coli, controls both osmoprotection and differentiation in S. coelicolor A3(2). Microarray analysis revealed σB‐dependent induction of more than 280 genes by 0.2 M KCl. These genes encode several sigma factors, oxidative defence proteins, chaperones, systems to provide osmolytes, cysteine, mycothiol, and gas vesicle. σB controlled induction of itself and its two paralogues (σL and σM) in a hierarchical order of σB→σL→σM, as revealed by S1 mapping and Western blot analyses. The phenotype of each sigma mutant suggested a sequential action in morphological differentiation; σB in forming aerial mycelium, σL in forming spores and σM for efficient sporulation. σB was also responsible for the increase in cysteine and mycothiol, the major thiol buffer in actinomycetes, upon osmotic shock, revealing an overlap between protections against osmotic and oxidative stresses. Proteins in sigB mutant were more oxidized (carbonylated) than the wild type. These results support a hypothesis that σB serves as a master regulator that triggers other related sigma factors in a cascade, and thus regulates differentiation and osmotic and oxidative response in S. coelicolor.

Nur, a nickel-responsive regulator of the Fur family, regulates superoxide dismutases and nickel transport in Streptomyces coelicolor

Nickel serves as a cofactor for various microbial enzymes including superoxide dismutase (SOD) found in Streptomyces spp. In Streptomyces coelicolor, nickel represses and induces production of Fe‐containing and Ni‐containing SODs, respectively, primarily at the transcriptional level. We identified the nickel‐responsive regulator (Nur), a Fur (ferric‐uptake regulator) homologue, which binds to the promoter region of the sodF gene encoding FeSOD in the presence of nickel. Disruption of the nur gene caused constitutive expression of FeSOD and no induction of NiSOD in the presence of nickel. The intracellular level of nickel was higher in a Δnur mutant than in the wild type, suggesting that Nur also regulates nickel uptake in S. coelicolor. A putative nickel‐transporter gene cluster (nikABCDE) was identified in the genome database. Its transcription was negatively regulated by Nur in the presence of nickel. Purified Nur protein bound to the nikA promoter region in a nickel‐dependent way. These results support the action of Nur as a regulator of nickel homeostasis and antioxidative response in S. coelicolor, and add a novel nickel‐responsive member to the list of versatile metal‐specific regulators of the Fur family.