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The reducing system of SoxR consists of a putative electron transfer system encoded by the rsxABCDGE operon, RseC encoded from the unlinked rpoE-rseABC operon, and ApbE. RseC is composed of two transmembrane helices, with both the N-terminal and C-terminal domains located in the cytoplasm. The N-terminal domain has a four-cysteine motif, CX 5 CX 2 CX 5 C, in the cytoplasm, with the latter three cysteines highly conserved in RseC homologs, allowing the SoxR reducer complex to function in Escherichia coli . These three cysteines can form an oxygen-sensitive Fe-S cluster when only the N-terminal domain is expressed in a truncated form. Without the C-terminal domain, RseC shows no significant difference in interaction with the SoxR reducer complex, but its ability to complement the function of an rseC mutant is greatly reduced. Additionally, the rseC mutant exhibits weak resistance to cumene hydrogen peroxide in the stationary phase and increased sensitivity to hydrogen peroxide in the exponential phase, independent of the SoxR regulon. This suggests that the full-length sequence of RseC is essential for its function and that it may have SoxR-independent additional roles.

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., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Zur is a Fur-family metalloregulator that is widely used to control zinc homeostasis in bacteria. In Streptomyces coelicolor, Zur (ScZur) acts as both a repressor for zinc uptake (znuA) gene and an activator for zinc exporter (zitB) gene. Previous structural studies revealed three zinc ions specifically bound per ScZur monomer; a structural one to allow dimeric architecture and two regulatory ones for DNA-binding activity. In this study, we present evidence that Zur contains a fourth specific zinc-binding site with a key histidine residue (H36), widely conserved among actinobacteria, for regulatory function. Biochemical, genetic, and calorimetric data revealed that H36 is critical for hexameric binding of Zur to the zitB zurbox and further binding to its upstream region required for full activation. A comprehensive thermodynamic model demonstrated that the DNA-binding affinity of Zur to both znuA and zitB zurboxes is remarkably enhanced upon saturation of all three regulatory zinc sites. The model also predicts that the strong coupling between zinc binding and DNA binding equilibria of Zur drives a biphasic activation of the zitB gene in response to a wide concentration change of zinc. Similar mechanisms may be pertinent to other metalloproteins, expanding their response spectrum through binding multiple regulatory metals., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Vertebrate sex is typically determined genetically, but in many ectotherms sex can be determined by genes (genetic sex determination, GSD), temperature (temperature-dependent sex determination, TSD), or interactions between genes and temperature during development. TSD may involve GSD systems with either male or female heterogamety (XX/XY or ZZ/ZW) where temperature overrides chromosomal sex determination to cause a mismatch between genetic sex and phenotypic sex (sex reversal). In these temperature-sensitive lineages, phylogenetic investigations point to recurrent evolutionary shifts between genotypic and temperature-dependent sex determination. These evolutionary transitions in sex determination can occur rapidly if selection favours the reversed sex over the concordant phenotypic sex. To investigate the consequences of sex reversal on offspring phenotypes, we measured two energy-driven traits (metabolism and growth) and 6 month survival in two species of reptile with different patterns of temperature-induced sex reversal. Male sex reversal occurs in Bassiana duperreyi when chromosomal females (female XX) develop male phenotypes (maleSR XX), while female sex reversal occurs in Pogona vitticeps when chromosomal males (male ZZ) develop female phenotypes (femaleSR ZZ). We show metabolism in maleSR XX was like that of male XY; that is, reflective of phenotypic sex and lower than genotypic sex. In contrast, for Pogona vitticeps, femaleSR ZZ metabolism was intermediate between male ZZ and female ZW metabolic rate. For both species, our data indicate that differences in metabolism become more apparent as individuals become larger. Our findings provide some evidence for an energetic advantage from sex reversal in both species but do not exclude energetic processes as a constraint on the distribution of sex reversal in nature., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Turtle populations are in decline worldwide, requiring immediate conservation and management actions. For species with broad geographic ranges that cover diverse environmental contexts, region-specific information on declining species could inform more targeted management plans. I examined the ecology of a Spotted Turtle ( Clemmys guttata ) population in a temporally dynamic wetland system in the Southeastern Plains ecoregion of North Carolina. Turtles selected forested wetlands and streams while avoiding open wetlands and river habitats, but used terrestrial habitats minimally and randomly. Turtles responded to wetland drying by remaining in wetlands and maintaining modest levels of activity during short-duration drying events (< 1 mo), but moved very little during longer droughts (7 mo). Turtles had prolonged active seasons (February-October) as long as wetlands and streams were flooded, with movement rates peaking in late spring at 23.6 ± 3.7 m/d (mean ± standard error). Turtles had large home ranges (14.1 ± 4.3 ha) that often included multiple local stream networks used as movement corridors between forested wetland patches and as activity centers when wetlands dried. I suggest that conservation plans for C. guttata include large management areas that protect a network of streams and adjacent forested swamps in the Southeastern Plains and perhaps other lowland ecoregions in the Southeastern U.S. Because terrestrial habitats were not used for extended refuge during drought, certain low-impact land uses in adjacent terrestrial areas are not likely to negatively impact C. guttata populations, but terrestrial forests would still be important in maintaining overland travel corridors.

An individual's behavioral tendencies (i.e., personality or temperament) can influence its interactions with the environment and thus have important ecological and evolutionary consequences for animal populations. Boldness, defined as an individual's tendency to engage in risk-taking activities, is a phenotypically variable trait linked with numerous behavioral and fitness outcomes in free-ranging animals. We examined variation and repeatability of boldness and other behavioral characteristics in two wild Eastern Box Turtle ( Terrapene carolina carolina ) populations using radiotelemetry, and assessed fitness correlates of boldness over multiple years. We observed large amounts of among-individual variation and within-individual consistency (i.e., repeatability) of boldness as measured by their head emergence latency following a standardized confinement assay. Individuals were also consistent in several in-field behaviors including movement rate, home range size, and date of emergence from overwintering refuges. Individuals with shorter head emergence latencies (i.e., bolder turtles) had larger home ranges, emerged earlier from overwintering dormancy, and experienced moderately lower survival compared with shy individuals. Boldness did not affect time spent within the thermal preference range, somatic growth rates, or the frequency of mating or same-sex aggressive encounters. Boldness and its effects on in-field behaviors differed between sexes and populations, and the relationship between boldness and survival was temporally variable. Our results suggest possible intrinsic behavioral types in T. c. carolina and highlight the importance of long-term and multipopulation studies when examining ecological and evolutionary processes that shape personality phenotypes in turtles.

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.

The sex of vertebrates is typically determined genetically, but reptile sex can also be determined by developmental temperature. In some reptiles, temperature interacts with genotype to reverse sex, potentially leading to transitions from a chromosomal to a temperature-dependent sex determining system. Transitions between such systems in nature are accelerated depending on the frequency and fitness of sex-reversed individuals. The Central Bearded Dragon, Pogona vitticeps, exhibits female heterogamety (ZZ/ZW) but can have its sex reversed from ZZ male to ZZ female by high incubation temperatures. The species exhibits sex-reversal in the wild and it has been suggested that climate change and fitness of sex-reversed individuals could be increasing the frequency of reversal within the species range. Transitions to temperature-dependent sex determination require low levels of dispersal and high (>50%) rates of sex-reversal. Here, we combine genotype-by-sequencing, identification of phenotypic and chromosomal sex, exhaustive field surveys, and radio telemetry to examine levels of genetic structure, rates of sex-reversal, movement, space use, and survival of P. vitticeps in a location previously identified as a hot spot for sex-reversal. We find that the species exhibits low levels of population structure (FST ~0.001) and a modest (~17%) rate of sex-reversal, and that sex-reversed and nonsex-reversed females have similar survival and behavioural characteristics to each other. Overall, our data indicate this system is evolutionary stable, although we do not rule out the prospect of a more gradual transition in sex-determining mechanisms in the future in a more fragmented landscape and as global temperatures increase., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

A reducing system of SoxR, a regulator of redox-active molecules, was identified as rsxABCDGE gene products and RseC in Escherichia coli through genetic studies. We found that ApbE was an additional component of the reducer system. Bacterial two hybrid analysis revealed that these proteins indeed had multiple interactions among themselves. RseC and RsxB formed the core of the complex, interacting with more than five other components. RsxC, the only cytoplasmic component of the system, interacted with SoxR. It might be linked with the rest of the complex via RsxB. Membrane fractions containing the wild type complex but not the mutant complex reduced purified SoxR using NADH as an electron source. These results suggest that Rsx genes, RseC, and ApbE can form a complex using NAD(P)H to reduce SoxR., (© 2022. The Microbiological Society of Korea.)

σ R (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 σ R 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 σ R -RsrA system can sense and reveals another intricate regulatory layer for optimal survival of Actinobacteria., (© 2021 John Wiley & Sons Ltd.)

WhiB7/WblC is a transcriptional factor of actinomycetes conferring intrinsic resistance to multiple translation-inhibitory antibiotics. It positively autoregulates its own transcription in response to the same antibiotics. The presence of a uORF and a potential Rho-independent transcription terminator in the 5' leader region has suggested a possibility that the whiB7/wblC gene is regulated via a uORF-mediated transcription attenuation. However, experimental evidence for the molecular mechanism to explain how antibiotic stress suppresses the attenuator, if any, and induces transcription of the whiB7/wblC gene has been lacking. Here we report that the 5' leader sequences of the whiB7/wblC genes in sub-clades of actinomycetes include conserved antiterminator RNA structures. We confirmed that the putative antiterminator in the whiB7/wblC leader sequences of both Streptomyces and Mycobacterium indeed suppresses Rho-independent transcription terminator and facilitates transcription readthrough, which is required for WhiB7/WblC-mediated antibiotic resistance. The antibiotic-mediated suppression of the attenuator can be recapitulated by amino acid starvation, indicating that translational inhibition of uORF by multiple signals is a key to induce whiB7/wblC expression. Our findings of a mechanism leading to intrinsic antibiotic resistance could provide an alternative to treat drug-resistant mycobacteria., (© 2021 John Wiley & Sons Ltd.)

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., (© 2021. The Microbiological Society of Korea.)

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., (© 2021. The Microbiological Society of Korea.)

Prescribed fire is an essential management practice in pyrogenic ecosystems, but fire can also be a significant disturbance and source of mortality for both target and non-target species. Seasonal periods of animal inactivity may provide opportunities to design burn plans that minimize negative impacts to species of conservation concern, but few studies have rigorously examined this possibility. Using radiotelemetry, we studied overwintering behavior and interactions with fire in a forest-dwelling terrestrial turtle, the Eastern Box Turtle ( Terrapene carolina carolina ), over an eight-year period at two sites that use prescribed fire in forest management. Turtles at both sites selected predominantly hardwood forests and mesic habitats and avoided upland pine forests. Turtles buried deepest (2.9 - 3.2 cm) below the soil-litter interface in late February and then moved gradually shallower until emergence in early April. Emergence timing varied over a 58-day period, but was consistent within individuals from year to year. Turtles also maintained fidelity to refuge locations, but those overwintering in burned areas selected sites over twice as far from refuges used in previous years compared to those in unburned areas. The areas and habitats selected by turtles during winter served as refugia from fire, and those whose refuges did burn remained buffered from lethal temperatures even at shallow burial depths. The only fire-related injury or mortality occurred during seasons of surface activity. Timing burning and other forest management practices during periods of winter dormancy may thus minimize threats to turtle populations, but modifications to prescribed fire regimes must also be balanced with other management objectives.

Bacteria that encounter antibiotics can efficiently change their physiology to develop resistance. This intrinsic antibiotic resistance is mediated by multiple pathways, including a regulatory system(s) that activates specific genes. In some Streptomyces and Mycobacterium spp., the WblC/WhiB7 transcription factor is required for intrinsic resistance to translation-targeting antibiotics. Wide conservation of WblC/WhiB7 within Actinobacteria indicates a critical role of WblC/WhiB7 in developing resistance to such antibiotics. Here, we identified 312 WblC target genes in Streptomyces coelicolor , a model antibiotic-producing bacterium, using a combined analysis of RNA sequencing and chromatin immunoprecipitation sequencing. Interestingly, WblC controls many genes involved in translation, in addition to previously identified antibiotic resistance genes. Moreover, WblC promotes translation rate during antibiotic stress by altering the ribosome-associated protein composition. Our genome-wide analyses highlight a previously unappreciated antibiotic resistance mechanism that modifies ribosome composition and maintains the translation rate in the presence of sub-MIC levels of antibiotics. IMPORTANCE The emergence of antibiotic-resistant bacteria is one of the top threats in human health. Therefore, we need to understand how bacteria acquire resistance to antibiotics and continue growth even in the presence of antibiotics. Streptomyces coelicolor , an antibiotic-producing soil bacterium, intrinsically develops resistance to translation-targeting antibiotics. Intrinsic resistance is controlled by the WblC/WhiB7 transcription factor that is highly conserved within Actinobacteria , including Mycobacterium tuberculosis Here, identification of the WblC/WhiB7 regulon revealed that WblC/WhiB7 controls ribosome maintenance genes and promotes translation in the presence of antibiotics by altering the composition of ribosome-associated proteins. Also, the WblC-mediated ribosomal alteration is indeed required for resistance to translation-targeting antibiotics. This suggests that inactivation of the WblC/WhiB7 regulon could be a potential target to treat antibiotic-resistant mycobacteria., (Copyright © 2020 Lee et al.)

Protein lysine acetylation, one of the most abundant post-translational modifications in eukaryotes, occurs in prokaryotes as well. Despite the evidence of lysine acetylation in bacterial RNA polymerases (RNAPs), its function remains unknown. We found that the housekeeping sigma factor (HrdB) was acetylated throughout the growth of an actinobacterium, Streptomyces venezuelae, and the acetylated HrdB was enriched in the RNAP holoenzyme complex. The lysine (K259) located between 1.2 and 2 regions of the sigma factor, was determined to be the acetylated residue of HrdB in vivo by LC-MS/MS analyses. Specifically, the label-free quantitative analysis revealed that the K259 residues of all the HrdB subunits were acetylated in the RNAP holoenzyme. Using mutations that mimic or block acetylation (K259Q and K259R), we found that K259 acetylation enhances the interaction of HrdB with the RNAP core enzyme as well as the binding activity of the RNAP holoenzyme to target promoters in vivo. Taken together, these findings provide a novel insight into an additional layer of modulation of bacterial RNAP activity., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2019 Federation of European Biochemical Societies.)

Animal movements and use of space are in part determined by interactions between individual attributes such as sex and body size and extrinsic environmental factors such as the seasonal availability, quality and spatial configuration of resource patches in the landscape. Fire is a common and widespread disturbance process that has the potential to affect animal movements through modifications to the environment. Using radiotelemetry, we examined the contribution of these factors to variation in movements and home range over a 5-year period in a forest-dwelling terrestrial turtle, Terrapene carolina , at fire-maintained and unburned habitats in the southeastern United States. Female turtles had annual home-range sizes twice as large as males and moved longer distances per day during the nesting season (June and July), but males exhibited greater spatial fidelity from year to year. Turtles at the unburned site had home-range sizes twice as large as those at the fire-maintained site, and home-range size also decreased with increasing frequency and extent of fire, but this latter effect was strongest in females. Home-range behavior was highly repeatable within individuals of both sexes over time. This is the first evidence that fire influences the spatial ecology and movements of turtles, most likely through fire's impact on the spatial configuration, availability and quality of critical resources. That individuals behaved consistently through time, but differently from one another according to both intrinsic individual attributes and extrinsic environmental factors provides strong evidence of consistent inter- and intra-population variation in space use and movement behaviors in T. carolina . Such intra-specific behavioral variation suggests applying caution when extrapolating results to other sites across the geographic range of a species for use in conservation and management.

In Schizosaccharomyces pombe, there are two aconitases, Aco1 and Aco2, involved in the Krebs cycle in mitochondria. Interestingly, Aco2 is localized to nucleus as well. Here, we investigated the nuclear role of Aco2 by deleting its nuclear localization signal. The aco2ΔNLS mutation suppressed the gene-silencing defects of RNAi mutants at the centromere, where heterochromatin formation depends on RNAi pathway. In Δago1, the aco2ΔNLS mutation restored heterochromatin through elevating Chp1 binding. Aco2 physically interacted with Chp1 via the N-terminal chromodomain that binds to methylated histone H3K9. In the sub-telomeric region, where heterochromatin forms independent of RNAi pathway, the single aco2ΔNLS mutation caused extra gene silencing via elevating Chp1 binding, without increasing histone methylation. The anti-silencing effect did not require the catalytic function of aconitase. Taken together, Aco2 functions as an epigenetic regulator of gene expression, through associating with chromodomain of Chp1 to maintain heterochromatin., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Genomic analysis of the clavulanic acid (CA)-high-producing Streptomyces clavuligerus strains, OL13 and OR, developed through random mutagenesis revealed a frameshift mutation in the cas1 gene-encoding clavaminate synthase 1. Overexpression of the intact cas1 in S. clavuligerus OR enhanced the CA titer by approximately 25%, producing ~ 4.95 g/L of CA, over the OR strain in the flask culture. Moreover, overexpression of the pathway-specific positive regulatory genes, ccaR and claR, in the OR strain improved CA yield by approximately 43% (~ 5.66 g/L) in the flask. However, co-expression of the intact cas1 with ccaR-claR did not further improve CA production. In the 7 L fermenter culture, maximum CA production by the OR strain expressing the wild-type cas1 and ccaR-claR reached approximately 5.52 g/L and 6.01 g/L, respectively, demonstrating that reverse engineering or simple rational metabolic engineering is an efficient method for further improvement of industrial strains.

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, σ R (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 σ R for target gene induction. Translation-blocking antibiotics induce the synthesis of σ R , 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 σ R and σ R ' . Antibiotic stress brings about a prolonged response by increasing stable σ R levels. The third negative feedback, which occurs via IF3, lowers the translation efficiency of the sigRp1 transcript that utilizes a non-canonical start codon. σ R 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 σ H 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., (© 2019 John Wiley & Sons Ltd.)

Streptomycetes naturally produce a variety of secondary metabolites, in the process of physiological differentiation. Streptomyces venezuelae differentiates into spores in liquid media, serving as a good model system for differentiation and a host for exogenous gene expression. Here, we report the growth and differentiation properties of S. venezuelae ATCC-15439 in liquid medium, which produces pikromycin, along with genome-wide gene expression profile. Comparison of growth properties on two media (SPA, MYM) revealed that the stationary phase cell viability rapidly decreased in SPA. Submerged spores showed partial resistance to lysozyme and heat, similar to what has been observed for better-characterized S. venezuelae ATCC10712, a chloramphenicol producer. TEM revealed that the differentiated cells in the submerged culture showed larger cell size, thinner cell wall than the aerial spores. We analyzed transcriptome profiles of cells grown in liquid MYM at various growth phases. During transition and/or stationary phases, many differentiationrelated genes were well expressed as judged by RNA level, except some genes forming hydrophobic coats in aerial mycelium. Since submerged spores showed thin cell wall and partial resistance to stresses, we examined cellular expression of MreB protein, an actin-like protein known to be required for spore wall synthesis in Streptomycetes. In contrast to aerial spores where MreB was localized in septa and spore cell wall, submerged spores showed no detectable signal. Therefore, even though the mreB transcripts are abundant in liquid medium, its protein level and/or its interaction with spore wall synthetic complex appear impaired, causing thinner- walled and less sturdy spores in liquid culture.

Prescribed fire is commonly used as a tool to meet a range of forest management goals. Owing to their limited movement abilities, terrestrial turtles are likely to be at high risk of injury and mortality, and to experience other fitness consequences with population-level implications from fire. Using radiotelemetry, we studied the responses of Eastern Box Turtles, Terrapene carolina carolina , to prescribed fire management in a sandhills Longleaf Pine forest system over a five-year period and compared our results to a nearby population in an unburned coastal plain location. Individual variation in turtle survival was strongly dependent on how frequently and extensively the areas were burned, with annual survival rates of 94.5% in unburned areas decreasing to 45.9 % in the most extensively burned areas. Turtles at the fire-maintained sandhills site had annual survival rates 4.9 % less than at the unburned coastal plain site, and females had annual survival rates 6.8 % less than males. Survival varied seasonally, with greatest mortality rates in winter and spring, especially among females. Growth rates and body condition did not differ between sites, nor did they vary according to fire extent and frequency at the fire-maintained site. Although mortality was greater and spatially variable at the fire-maintained site, annual survival rates across the site (86 - 90 % for females and males, respectively) were comparable to other stable populations of T. carolina . The lesser than expected mortality rate at the fire-maintained site was likely the result of turtles' strong selection of mesic hardwood forests near bottomlands and streams - habitats that may serve as refugia from fire. In areas where T. carolina conservation is a priority, land managers should integrate maintenance of fire refuge habitats into burn planning to minimize unintended negative impacts to this imperiled species.

Habitat selection, where observed use of a resource is disproportionate to availability, is an important behavior allowing individuals to position themselves spatially relative to critical resources in heterogeneous environments. For species that experience variable environments across broad geographic ranges, we expect resource selection templates to vary among populations accordingly. Using radiotelemetry, we examined habitat selection for populations of Eastern Box Turtles, Terrapene carolina , in fire-maintained forests of the sandhills compared to nearby unburned coastal plain forests in south-central North Carolina. Turtles at the fire-maintained sandhills site preferred bottomland habitats and areas near steams, whereas turtles in the unburned coastal plain environment preferred uplands and used streams randomly. In addition, turtles in the fire-maintained sandhills avoided Longleaf Pine and more strongly preferred hardwood and non-Longleaf Pine forests compared to turtles at the unburned coastal plain site. Body size, but not sex, was also an important source of variation in habitat selection within populations, with smaller turtles more strongly preferring areas near water. Selection of habitat structural components in the immediate area of locations did not differ between sites, sexes, or body sizes. These results highlight the variety of resource selection templates in T. carolina , underscoring a potential need for population- or region-specific conservation and management strategies.

Fire can influence the microclimate of forest habitats by removing understory vegetation and surface debris. Temperature is often higher in recently burned forests owing to increased light penetration through the open understory. Because physiological processes are sensitive to temperature in ectotherms, we expected fire-maintained forests to improve the suitability of the thermal environment for turtles, and for turtles to seasonally associate with the most thermally-optimal habitats. Using a laboratory thermal gradient, we determined the thermal preference range (T set ) of eastern box turtles, Terrapene carolina, to be 27-31°C. Physical models simulating the body temperatures experienced by turtles in the field revealed that surface environments in a fire-maintained longleaf pine forest were 3°C warmer than adjacent unburned mixed hardwood/pine forests, but the fire-maintained forest was never of superior thermal quality owing to wider T e fluctuations above T set and exposure to extreme and potentially lethal temperatures. Radiotracked turtles using fire-managed longleaf pine forests maintained shell temperatures (T s ) approximately 2°C above those at a nearby unburned forest, but we observed only moderate seasonal changes in habitat use which were inconsistent with thermoregulatory behavior. We conclude that turtles were not responding strongly to the thermal heterogeneity generated by fire in our system, and that other aspects of the environment are likely more important in shaping habitat associations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In most bacteria, zinc depletion is sensed by Zur, whereas the surplus is sensed by different regulators to achieve zinc homeostasis. Here we present evidence that zinc-bound Zur not only represses genes for zinc acquisition but also induces the zitB gene encoding a zinc exporter in Streptomyces coelicolor, a model actinobacteria. Zinc-dependent gene regulation by Zur occurs in two phases. At sub-femtomolar zinc concentrations (phase I), dimeric Zur binds to the Zur-box motif immediately upstream of the zitB promoter, resulting in low zitB expression. At the same time, Zur represses genes for zinc uptake. At micromolar zinc concentrations (phase II), oligomeric Zur binding with footprint expansion upward from the Zur box results in high zitB induction. Our findings reveal a mode of zinc-dependent gene activation that uses a single metalloregulator to control genes for both uptake and export over a wide range of zinc concentrations.

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.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Antibiotic-producing streptomycetes are rich sources of resistance mechanisms against endogenous and exogenous antibiotics. An ECF sigma factor σ(R) (SigR) is known to govern the thiol-oxidative stress response in Streptomyces coelicolor. Amplification of this response is achieved by producing an unstable isoform of σ(R) called σ(R'). In this work, we present evidence that antibiotics induce the SigR regulon via a redox-independent pathway, leading to antibiotic resistance. The translation-inhibiting antibiotics enhanced the synthesis of stable σ(R), eliciting a prolonged response. WblC/WhiB7, a WhiB-like DNA-binding protein, is responsible for inducing sigRp1 transcripts encoding the stable σ(R). The amount of WblC protein and its binding to the sigRp1 promoter in vivo increased upon antibiotic treatment. A similar phenomenon appears to exist in Mycobacterium tuberculosis as well. These findings reveal a novel antibiotic-induced resistance mechanism conserved among actinomycetes, and also give an explicit example of overlap in cellular damage and defense mechanisms between thiol-oxidative and anti- translational stresses.

Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of commercial, medical and veterinary interest. However, these products are often not detectable under laboratory culture conditions. To harness their full biosynthetic potential, it is important to develop a detailed understanding of the regulatory networks that orchestrate their metabolism. Here we integrate nucleotide resolution genome-scale measurements of the transcriptome and translatome of Streptomyces coelicolor, the model antibiotic-producing actinomycete. Our systematic study determines 3,570 transcription start sites and identifies 230 small RNAs and a considerable proportion (∼21%) of leaderless mRNAs; this enables deduction of genome-wide promoter architecture. Ribosome profiling reveals that the translation efficiency of secondary metabolic genes is negatively correlated with transcription and that several key antibiotic regulatory genes are translationally induced at transition growth phase. These findings might facilitate the design of new approaches to antibiotic discovery and development.

Streptomyces venezuelae ATCC 15439, which produces 12- and 14-membered ring macrolide antibiotics, is a platform strain for heterologous expression of secondary metabolites. Its 9.05-Mb genome sequence revealed an abundance of genes involved in the biosynthesis of secondary metabolites and their precursors, which should be useful for the production of bioactive compounds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The development of rapid and efficient genome sequencing methods has enabled us to study the evolutionary background of bacterial genetic information. Here, we present comparative genomic analysis of 17 Streptomyces species, for which the genome has been completely sequenced, using the pan-genome approach. The analysis revealed that 34,592 ortholog clusters constituted the pan-genome of these Streptomyces species, including 2,018 in the core genome, 11,743 in the dispensable genome, and 20,831 in the unique genome. The core genome was converged to a smaller number of genes than reported previously, with 3,096 gene families. Functional enrichment analysis showed that genes involved in transcription were most abundant in the Streptomyces pan-genome. Finally, we investigated core genes for the sigma factors, mycothiol biosynthesis pathway, and secondary metabolism pathways; our data showed that many genes involved in stress response and morphological differentiation were commonly expressed in Streptomyces species. Elucidation of the core genome offers a basis for understanding the functional evolution of Streptomyces species and provides insights into target selection for the construction of industrial strains.

SoxR is a [2Fe-2S]-containing sensor-regulator, which is activated through oxidation by redox-active compounds (RACs). SoxRs show differential sensitivity to RACs, partly due to different redox potentials, such that Escherichia coli (Ec) SoxR with lower potential respond to broader range of RACs than Streptomyces coelicolor (Sc) SoxR. In S. coelicolor, the RACs that do not activate ScSoxR did not inhibit growth, suggesting that ScSoxR is tuned to respond to growth-inhibitory RACs. Based on sequence comparison and mutation studies, two critical amino acids around the [2Fe-2S] binding site were proposed as key determinants of sensitivity. ScSoxR-like mutation (R127L/P131V) in EcSoxR changed its sensitivity profile as ScSoxR, whereas EcSoxR-like mutation (L126R/V130P) in ScSoxR caused relaxed response. In accordance, the redox potentials of EcSoxR(R) (127) (L) (/) (P) (131) (V) and ScSoxR(L126R/V130P) were estimated to be -192 ± 8 mV and -273 ± 10 mV, respectively, approaching that of ScSoxR (-185 mV) and EcSoxR (-290 mV). Molecular dynamics simulations revealed that the R127L and P131V substitutions in EcSoxR caused more electropositive environment around [2Fe-2S], making it harder to get oxidized. This reveals a mechanism to modulate redox-potential in [Fe-S]-containing sensors by point mutations and to evolve a sensor with differential sensitivity to achieve optimal cellular physiology., (© 2015 John Wiley & Sons Ltd.)

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.

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., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

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.

In the fission yeast Schizosaccharomyces pombe, the stationary phase-specific transcription factor Phx1 contributes to long-term survival, stress tolerance, and meiosis. We identified Phx1-dependent genes through transcriptome analysis, and further analyzed those related with carbohydrate and thiamine metabolism, whose expression decreased in ∆phx1. Consistent with mRNA changes, the level of thiamine pyrophosphate (TPP) and TPP-utilizing pyruvate decarboxylase activity that converts pyruvate to acetaldehyde were also reduced in the mutant. Therefore, Phx1 appears to shift metabolic flux by diverting pyruvate from the TCA cycle and respiration to ethanol fermentation. Among the four predicted genes for pyruvate decarboxylase, only the Phx1-dependent genes (pdc201+ and pdc202+) contributed to long-term survival as judged by mutation and overexpression studies. These findings indicate that the Phx1-mediated long-term survival is achieved primarily through increasing the synthesis and activity of pyruvate decarboxylase. Consistent with this hypothesis, we observed that Phx1 curtailed respiration when cells entered stationary phase. Introduction of Δphx1 mutation compromised the long-lived phenotypes of Δpka1 and Δsck2 mutants that are devoid of pro-aging kinases of nutrient-signalling pathways, and of the Δpyp1 mutant with constitutively activated stress-responsive kinase Sty1. Therefore, achievement of long-term viability through both nutrient limitation and anti-stress response appears to be dependent on Phx1.

Superoxide dismutases (SODs) are widely distributed enzymes that convert superoxides to hydrogen peroxide and molecular oxygen, using various metals as cofactors. Many actinobacteria contain genes for both Ni-containing (sodN) and Fe-containing (sodF) SODs. In Streptomyces coelicolor, expression of the sodF and sodN genes is inversely regulated by nickel-specific Nur, a Fur-family regulator. With sufficient nickel, Nur directly represses sodF transcription, while inducing sodN indirectly. Bioinformatic search revealed that a conserved 19-nt stretch upstream of sodN matches perfectly with the sodF downstream sequence. We found that the sodF gene produced a stable small-sized RNA species (s-SodF) of ∼ 90 nt that harbors the anti-sodN sequence complementary to sodN mRNA from the 5'-end up to the ribosome binding site. Absence of nearby promoters and sensitivity to 5'-phosphate-specific exonuclease indicated that the s-SodF RNA is a likely processed product of sodF mRNA. The s-SodF RNA caused a significant decrease in the half-life of the sodN mRNA. Therefore, Nur activates sodN expression through inhibiting the synthesis of sodF mRNA, from which inhibitory s-SodF RNA is generated. This reveals a novel mechanism by which antagonistic regulation of one gene is achieved by small RNA processed from the 3'UTR of another gene's mRNA.

Fisheries bycatch is a critical source of mortality for rapidly declining populations of leatherback turtles, Dermochelys coriacea. We integrated use-intensity distributions for 135 satellite-tracked adult turtles with longline fishing effort to estimate predicted bycatch risk over space and time in the Pacific Ocean. Areas of predicted bycatch risk did not overlap for eastern and western Pacific nesting populations, warranting their consideration as distinct management units with respect to fisheries bycatch. For western Pacific nesting populations, we identified several areas of high risk in the north and central Pacific, but greatest risk was adjacent to primary nesting beaches in tropical seas of Indo-Pacific islands, largely confined to several exclusive economic zones under the jurisdiction of national authorities. For eastern Pacific nesting populations, we identified moderate risk associated with migrations to nesting beaches, but the greatest risk was in the South Pacific Gyre, a broad pelagic zone outside national waters where management is currently lacking and may prove difficult to implement. Efforts should focus on these predicted hotspots to develop more targeted management approaches to alleviate leatherback bycatch.

SoxR from Escherichia coli and related enterobacteria is activated by a broad range of redox-active compounds through oxidation or nitrosylation of its [2Fe-2S] cluster. Activated SoxR then induces SoxS, which subsequently activates more than 100 genes in response. In contrast, non-enteric SoxRs directly activate their target genes in response to redox-active compounds that include endogenously produced metabolites. We compared the responsiveness of SoxRs from Streptomyces coelicolor (ScSoxR), Pseudomonas aeruginosa (PaSoxR) and E. coli (EcSoxR), all expressed in S. coelicolor, towards natural or synthetic redox-active compounds. EcSoxR responded to all compounds examined, whereas ScSoxR was insensitive to oxidants such as paraquat (Eh -440 mV) and menadione sodium bisulphite (Eh -45 mV) and to NO generators. PaSoxR was insensitive only to some NO generators. Whole-cell EPR analysis of SoxRs expressed in E. coli revealed that the [2Fe-2S](1+) of ScSoxR was not oxidizable by paraquat, differing from EcSoxR and PaSoxR. The mid-point redox potential of purified ScSoxR was determined to be -185 ± 10 mV, higher by approximately 100 mV than those of EcSoxR and PaSoxR, supporting its limited response to paraquat. The overall sensitivity profile indicates that both redox potential and kinetic reactivity determine the differential responses of SoxRs towards various oxidants., (© 2013 John Wiley & Sons Ltd.)

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., (Copyright © 2013 Wiley Periodicals, Inc.)

Streptomyces coelicolor produces at least three different catalases (catalases A, B, and C) under different physiological conditions. Catalase B (CatB) is a developmentally regulated catalase required for proper differentiation and osmoprotection of S. coelicolor. We previously observed that the N-terminal 75(2) amino acids (aa) of CatB are cleaved off, with the remaining 75-kDa processed CatB detectable in the extracellular fraction during sporulation. We here report that either the deletion of the N-terminal 75 aa or the arginine-to-alanine substitution (R75A) at the cleavage site, but not the histidine-to-alanine substitution (H131A) at the catalytic site, impaired both the secretion of CatB proteins and the proper differentiation of S. coelicolor cells. The proteolytic activity responsible for the cleavage of CatB was purified and then identified as a metalloprotease, which was named as SmpA (Streptomyces metalloprotease A). The SmpA protein was newly detected after sporulation, coincident with the intracellular appearance of 75-kDa CatB, which was not detected in the smpA null mutant, confirming that SmpA indeed processes CatB in vivo. The smpA mutant was osmosensitive as catB mutant, but it displayed delayed sporulation, with the 75-kDa CatB still detectable in the extracellular milieu. Based on these results, we propose that the post-translational regulation of CatB, which cleaves the N-terminal 75 aa residues through SmpA is crucial for proper differentiation and osmoprotection of S. coelicolor. In the absence of SmpA, an alternative route for CatB processing may function to allow delayed sporulation., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Numerous thiol-reactive compounds cause oxidative stress where cells counteract by activation of survival strategies regulated by thiol-based sensors. In Streptomyces coelicolor, a model actinomycete, a sigma/antisigma pair SigR/RsrA controls the response to thiol-oxidative stress. To unravel its full physiological functions, chromatin immuno-precipitation combined with sequence and transcript analyses were employed to identify 108 SigR target genes in S. coelicolor and to predict orthologous regulons across actinomycetes. In addition to reported genes for thiol homeostasis, protein degradation and ribosome modulation, 64 additional operons were identified suggesting new functions of this global regulator. We demonstrate that SigR maintains the level and activity of the housekeeping sigma factor HrdB during thiol-oxidative stress, a novel strategy for stress responses. We also found that SigR defends cells against UV and thiol-reactive damages, in which repair UvrA takes a part. Using a refined SigR-binding sequence model, SigR orthologues and their targets were predicted in 42 actinomycetes. This revealed a conserved core set of SigR targets to function for thiol homeostasis, protein quality control, possible modulation of transcription and translation, flavin-mediated redox reactions, and Fe-S delivery. The composition of the SigR regulon reveals a robust conserved physiological mechanism to deal with thiol-oxidative stress from bacteria to human., (© 2012 Blackwell Publishing Ltd.)

Background: In the fission yeast Schizosaccharomyces pombe, the phx1+ (pombe homeobox) gene was initially isolated as a multi-copy suppressor of lysine auxotrophy caused by depletion of copper/zinc-containing superoxide dismutase (CuZn-SOD). Overproduction of Phx1 increased the synthesis of homocitrate synthase, the first enzyme in lysine biosynthetic pathway, which is labile to oxidative stress. Phx1 has a well conserved DNA-binding domain called homeodomain at the N-terminal region and is predicted to be a transcription factor in S. pombe. However, its role has not been revealed in further detail. Here we examined its expression pattern and the phenotype of its null mutant to get clues on its function., Results: Fluorescence from the Phx1-GFP expressed from a chromosomal fusion gene demonstrated that it is localized primarily in the nucleus, and is distinctly visible during the stationary phase. When we replaced the N-terminal homeobox domain of Phx1 with the DNA binding domain of Pap1, a well-characterized transcription factor, the chimeric protein caused the elevation of transcripts from Pap1-dependent genes such as ctt1+ and trr1+, suggesting that Phx1 possesses transcriptional activating activity when bound to DNA. The amount of phx1+ transcripts sharply increased as cells entered the stationary phase and was maintained at high level throughout the stationary phase. Nutrient shift down to low nitrogen or carbon sources caused phx1+ induction during the exponential phase, suggesting that cells need Phx1 for maintenance function during nutrient starvation. The Δphx1 null mutant showed decreased viability in long-term culture, whereas overproduction of Phx1 increased viability. Decrease in long-term survival was also observed for Δphx1 under N- or C-starved conditions. In addition, Δphx1 mutant was more sensitive to various oxidants and heat shock. When we examined sporulation of the Δphx1/Δphx1 diploid strain, significant decrease in the formation of meiotic spores was observed., Conclusions: Phx1 is a transcriptional regulator whose synthesis is elevated during stationary phase and by nutrient starvation in S. pombe. It supports long-term survival and stress tolerance against oxidation and heat, and plays a key role in the formation of meiotic spores.

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.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)