Search

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.)

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.)

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.)

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.)

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.)

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.)

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.

Mycothiol (MSH) is a small thiol molecule with a cysteine-ligated disaccharide structure found in actinomycetes that include streptomycetes and mycobacteria. In Streptomyces coelicolor, a model organism for antibiotic production and differentiation, the amount of MSH is under the control of a sigma factor sigma(R), which is regulated by an antisigma factor RsrA with a thiol-disulphide redox switch. We found that the first gene (mshA) in the biosynthetic pathway for MSH and the gene for amidase (mca) that participates in detoxifying mycothiol-reactive drugs are under direct control of sigma(R). The sigma(R) target genes are induced not only by a thiol oxidant diamide, but also by alkylating agents that cause a rapid decrease in MSH. Expression of the sigma(R) regulon was also elevated in MSH-deficient mutants, suggesting that a decrease in the level of MSH is a natural intracellular trigger for sigma(R) activation. We found that MSH was capable of reducing RsrA to bind sigma(R), whereas glutathione was not. These results support a proposal that the RsrA-sigma(R) system senses the intracellular level of reduced MSH, and that MSH serves as a natural modulator of the transcription system for its own replenishment in addition to being a redox buffer and drug detoxifier.

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.

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 Deltanur 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.

The differentiating bacterium Streptomyces coelicolor harbours some 66 sigma factors, which support its complex life cycle. sigma(B), a functional homologue of sigma(S) from Escherichia coli, controls both osmoprotection and differentiation in S. coelicolor A3(2). Microarray analysis revealed sigma(B)-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. sigma(B) controlled induction of itself and its two paralogues (sigma(L) and sigma(M)) in a hierarchical order of sigma(B)-->sigma(L)-->sigma(M), as revealed by S1 mapping and Western blot analyses. The phenotype of each sigma mutant suggested a sequential action in morphological differentiation; sigma(B) in forming aerial mycelium, sigma(L) in forming spores and sigma(M) for efficient sporulation. sigma(B) 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 sigma(B) 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.

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 DeltasoxRS and DeltaoxyR 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 DeltaoxyR 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.

A gene (sigB) encoding an alternative sigma factor sigmaB in Streptomyces coelicolor A3(2) was isolated and characterized. It encodes a polypeptide of 281 amino acids (31 546 Da) and is highly homologous to Bacillus subtilis sigmaB. The sigB coding region is preceded by four open reading frames (ORFs): dpsA, orfA, rsbB and rsbA in sequential order. RNA analyses revealed that rsbB, rsbA and sigB constitute an operon (sigB operon). Transcripts were produced constitutively from a promoter (sigBp2) upstream of the rsbB coding region, contributing to the basal level expression of sigmaB protein. An inducible promoter (sigBp1) resembling the catB promoter (catBp) was located between the rsbA and sigB coding regions. Transcripts from sigBp1 dramatically increased as cells differentiated on solid media, at the stationary phase in liquid media or by osmotic stresses similar to the behaviour of catBp transcripts. Both catBp and sigBp1 promoters were recognized specifically by sigmaB-containing RNA polymerase in vitro. Disruption of the sigB gene abolished not only the differentiation-associated expression but also the osmotic induction of the catB gene, indicating that catBp is under the control of sigmaB. The sigB mutant exhibited a similar phenotype to the catB mutant, being sensitive to hyperosmolarity, blocked in forming aerial mycelium and with skewed antibiotic production. Therefore, we conclude that sigmaB ensures the proper differentiation and osmoprotection of S. coelicolor cells, primarily via regulation of the expression of catalase B.

In the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called sigmaR in response to changes in the cytoplasmic thiol-disulphide status, and the activity of sigmaR 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 sigmaR-dependent transcription, confirming that RsrA is a negative regulator of sigmaR 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 sigmaR.

Streptomyces coelicolor produces at least three catalases, the expression of which varies under different conditions. We characterized a gene (catB) for developmentally controlled catalase of 779 amino acids (83408 Da), homologous to KatE of Escherichia coli and Bacillus subtilis. Expression of the catB gene increased at the stationary phase in liquid culture and after the onset of differentiation on solid culture. It was also increased by osmotic treatments. Transcription was initiated from a promoter (catBp), whose sequence (ATGCCTCG-N13-GGGTAC) resembled promoters recognized by sigmaB of B. subtilis. CatB protein underwent proteolytic cleavage of its N-terminal 95 amino acids and was secreted to the medium when cells sporulated. Disruption of the catB gene caused impairment in the formation of aerial mycelium and reduction in the synthesis of undecylprodigiosin. On the contrary, hyperproduction of actinorhodin was observed in accordance with the increase in actII-ORF4 transcription. In addition, catB mutant became hypersensitive to osmotic stresses. These results suggest that regulated synthesis of CatB protein is necessary to ensure proper differentiation as well as to protect S. coelicolor cells against osmotic stresses.

A novel type of superoxide dismutase containing nickel as a cofactor (NiSOD) has been discovered in several Streptomyces spp. The gene for NiSOD (sodN) was cloned from S. coelicolor Müller using degenerate oligonucleotide probes designed from the N-terminal peptide sequence of the purified enzyme. It encodes a polypeptide of 131 amino acids (14703 Da), without any apparent sequence similarity to other known proteins. The N-terminus of the purified NiSOD was located 14 amino acids downstream from the initiation codon of the deduced open reading frame (ORF), indicating the involvement of protein processing. The molecular mass of the processed polypeptide was predicted to be 13201 Da, in close agreement with that of the purified NiSOD (13.4 kDa). The transcription start site of the sodN gene was determined by S1 mapping and primer extension analysis. Ni2+ regulates the synthesis of NiSOD polypeptide. S1 mapping of both 5' and 3' ends of sodN mRNA revealed that Ni2+ increased the level of monocistronic sodN mRNA by more than ninefold without changing its half-life, thus demonstrating that Ni2+ regulates transcription. Both precursor and processed NiSOD polypeptides with little SOD activity were produced from the cloned sodN gene in S. lividans in the absence of sufficient Ni2+; however, on addition of Ni2+, active NiSOD consisting of only processed polypeptide was formed. Expression of the full-length sodN gene in E. coli produced NiSOD polypeptide without any SOD activity even in the presence of Ni2+. However, deletion of nucleotides encoding the N-terminal 14 amino acids from the sodN gene allowed the production of active NiSOD in E. coli, indicating that N-terminal processing is required to produce active NiSOD. These results reveal the unique role of nickel as a multifaceted regulator in S. coelicolor controlling sodN transcription and protein processing, as well as acting as a catalytic cofactor.

The pqi-5 gene, producing a probable membrane protein of unknown function, has been reported to be a member of the soxRS regulon. The SoxRS-dependent induction of pqi-5 by paraquat occurs only during the exponential phase. The expression of pqi-5 increased in the absence of paraquat during the stationary phase or under conditions of carbon or phosphate starvation. This increase was regulated at the transcriptional level by RpoS (sigma S), which recognized the second promoter (P2) approx. 5 nucleotides upstream from the promoter (P1) used at the exponential phase. Studies with a series of 5' deletions revealed that the paraquat-responsive element resides between -52 and -42 nucleotides upstream from the P1 start site, whose nucleotide sequence matches closely to other SoxS-binding sequences. The stationary-phase induction required sequences up to position -42, which correspond to the 5' border of the putative -35 hexamer for the P2 promoter. The binding of the purified SoxS protein to the pqi-5 promoter upstream sequences was demonstrated by gel mobility-shift and DNase I protection assays. The transcription from P1 promoter by E sigma D was activated by purified SoxS in vitro, as was observed in vivo. The dual regulation of pqi-5 by SoxS at the exponential phase and RpoS at the stationary phase is the first to be reported among the members of the soxRS regulon, suggesting that this gene might indeed play some role under stressful conditions.

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.