Cluster Biogenesis Research Articles

Clinical study of ferredoxin-reductase-related mitochondriopathy: Genotype-phenotype correlation and proposal of ancestry-based carrier screening in the Mexican population

PurposeFerredoxin reductase (FDXR) is a flavoprotein that functions in both iron sulfur cluster biogenesis and steroid biosynthesis pathways in the mitochondria. Not surprisingly, loss of FDXR function causes severe mitochondrial diseases in humans. Although several FDXR-related mitochondriopathy (FRM) cohorts have been reported in the literature, further characterization of the natural history of FRM is warranted. MethodsTo better understand the spectrum of FRM, a natural history study of FRM was performed. New cases were added to previously reported FRM cases for analysis (n = 62 cases). ResultsOptic atrophy, movement disorder, and developmental delay were frequent findings. Mortality is high, with 18% of patients, often infants, passing from complications. Notably, 25% of cases were homozygous or compound heterozygous for the previously reported p.Arg386Trp “hotspot” variant. Of the obtained ancestry, all but 1 individual heterozygous for the p.Arg386Trp variant was Hispanic, with many reporting Mexican heritage. Utilizing recent large-scale genome sequencing surveys, the carrier frequency of the p.Arg386Trp variant was estimated as 1 of 185 in the Mexican population. ConclusionGiven the high mortality of FRM and carrier frequency of the common variant, consideration of a new approach for population carrier screening and development of therapeutics for affected individuals is needed.

Evolutionary flexibility and rigidity in the bacterial methylerythritol phosphate (MEP) pathway.

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron-sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron-sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

Mrp and SufT, Two Bacterial Homologs of Eukaryotic CIA Factors Involved in Fe-S Clusters Biogenesis

Fe-S clusters are essential cofactors for the activity of a large variety of metalloproteins that play important roles in respiration, photosynthesis, nitrogen fixation, regulation of gene expression, and numerous metabolic pathways, including biosynthesis of other protein cofactors. Assembly of iron and sulfur atoms into a cluster, followed by its insertion into the polypeptide chain, is a complex process ensured by multiproteic systems. Through evolution, eukaryotes have acquired two Fe-S protein biogenesis systems by endosymbiosis from bacteria. These systems, ISC and SUF, are compartmentalized in mitochondria and plastids, respectively. The eukaryotic Fe-S protein biogenesis system (CIA) is dedicated to the biogenesis of cytosolic and nuclear Fe-S proteins. While the CIA system is absent in bacteria, at least two of its components share homologies with bacterial Fe-S protein biogenesis factors, Mrp and SufT. Here, we provide an overview of the role of Mrp and SufT in Fe-S protein biogenesis in bacteria, aiming to put forward specific but also common features with their eukaryotic CIA counterparts.

Mechanisms involved in the sequestration and resistance of cadmium for a plant-associated Pseudomonas strain

Understanding Cd-resistant bacterial cadmium (Cd) resistance systems is crucial for improving microremediation in Cd-contaminated environments. However, these mechanisms are not fully understood in plant-associated bacteria. In the present study, we investigated the mechanisms underlying Cd sequestration and resistance in the strain AN-B15. These results showed that extracellular Cd sequestration by complexation in strain AN-B15 was primarily responsible for the removal of Cd from the solution. Transcriptome analyses have shown that the mechanisms of Cd resistance at the transcriptional level involve collaborative processes involving multiple metabolic pathways. The AN-B15 strain upregulated the expression of genes related to exopolymeric substance synthesis, metal transport, Fe-S cluster biogenesis, iron recruitment, reactive oxygen species oxidative stress defense, and DNA and protein repair to resist Cd-induced stress. Furthermore, inoculation with AN-B15 alleviated Cd-induced toxicity and reduced Cd uptake in the shoots of wheat seedlings, indicating its potential for remediation. Overall, the results improve our understanding of the mechanisms involved in Cd resistance in bacteria and thus have important implications for improving microremediation.

Mechanisms controlling cellular and systemic iron homeostasis.

In mammals, hundreds of proteins use iron in a multitude of cellular functions, including vital processes such as mitochondrial respiration, gene regulation and DNA synthesis or repair. Highly orchestrated regulatory systems control cellular and systemic iron fluxes ensuring sufficient iron delivery to target proteins is maintained, while limiting its potentially deleterious effects in iron-mediated oxidative cell damage and ferroptosis. In this Review, we discuss how cells acquire, traffick and export iron and how stored iron is mobilized for iron-sulfur cluster and haem biogenesis. Furthermore, we describe how these cellular processes are fine-tuned by the combination of various sensory and regulatory systems, such as the iron-regulatory protein (IRP)-iron-responsive element (IRE) network, the nuclear receptor co-activator 4 (NCOA4)-mediated ferritinophagy pathway, the prolyl hydroxylase domain (PHD)-hypoxia-inducible factor (HIF) axis or the nuclear factor erythroid 2-related factor 2 (NRF2) regulatory hub. We further describe how these pathways interact with systemic iron homeostasis control through the hepcidin-ferroportin axis to ensure appropriate iron fluxes. This knowledge is key for the identification of novel therapeutic opportunities to prevent diseases of cellular and/or systemic iron mismanagement.

Interaction between Cfd1 and Nbp35 proteins involved in cytosolic Fe[sbnd]S cluster assembly machinery deciphers a stable complexation in Leishmania donovani

Leishmania donovani is the causative unicellular parasite for visceral leishmaniasis (VL); and FeS proteins are likely to be very essential for their survival and viability. Cytosolic FeS cluster assembly (CIA) machinery is one of the four systems for the biosynthesis and transfer of FeS clusters among eukaryotes; Cfd1 and Nbp35 are the scaffold components for cytosolic FeS cluster biogenesis. We investigated the role of CIA machinery components and purified Cfd1 and Nbp35 proteins of L. donovani. We also investigated the interactive nature between LdCfd1 and LdNbp35 proteins by in silico analysis, in vitro co-purification, pull down assays along with in vivo immuno-precipitation; which inferred that both LdCfd1 and LdNbp35 proteins are interacting with each other. Thus, our collective data revealed the interaction between these two proteins which forms a stable complex that can be attributed to the cellular process of FeS clusters biogenesis, and transfer to target apo-proteins of L. donovani. The expression of Cfd1 and Nbp35 proteins in Amp B resistant parasites is up-regulated leading to increased amount of FeS proteins. Hence, it favors increased tolerance towards ROS level, which helps parasites survival under drug pressure contributing in Amphotericin B resistance.

Analysis of Reconstituted Tripartite Complex Supports Avidity-based Recruitment of Hsp70 by Substrate Bound J-domain Protein

Hsp70 are ubiquitous, versatile molecular chaperones that cyclically interact with substrate protein(s). The initial step requires synergistic interaction of a substrate and a J-domain protein (JDP) cochaperone, via its J-domain, with Hsp70 to stimulate hydrolysis of its bound ATP. This hydrolysis drives conformational changes in Hsp70 that stabilize substrate binding. However, because of the transient nature of substrate and JDP interactions, this key step is not well understood. Here we leverage a well characterized Hsp70 system specialized for iron-sulfur cluster biogenesis, which like many systems, has a JDP that binds substrate on its own. Utilizing an ATPase-deficient Hsp70 variant, we isolated a Hsp70-JDP-substrate tripartite complex. Complex formation and stability depended on residues previously identified as essential for bipartite interactions: JDP-substrate, Hsp70-substrate and J-domain-Hsp70. Computational docking based on the established J-domain-Hsp70(ATP) interaction placed the substrate close to its predicted position in the peptide-binding cleft, with the JDP having the same architecture as when in a bipartite complex with substrate. Together, our results indicate that the structurally rigid JDP-substrate complex recruits Hsp70(ATP) via precise positioning of J-domain and substrate at their respective interaction sites - resulting in functionally high affinity (i.e., avidity). The exceptionally high avidity observed for this specialized system may be unusual because of the rigid architecture of its JDP and the additional JDP-Hsp70 interaction site uncovered in this study. However, functionally important avidity driven by JDP-substrate interactions is likely sufficient to explain synergistic ATPase stimulation and efficient substrate trapping in many Hsp70 systems.

The minimal SUF system is not required for Fe–S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans

Iron–sulfur (Fe–S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe–S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe–S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC in Methanosarcina acetivorans, which contains two sufCB gene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or delete sufC1B1 and sufC2B2, respectively. Neither the dual repression of sufC1B1 and sufC2B2 nor the deletion of both sufC1B1 and sufC2B2 affected the growth of M. acetivorans under any conditions tested, including diazotrophy. Interestingly, deletion of only sufC1B1 led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion of sufC2B2 acts as a suppressor mutation in the absence of sufC1B1. In addition, the deletion of sufC1B1 and/or sufC2B2 did not affect the total Fe–S cluster content in M. acetivorans cells. Overall, these results reveal that the minimal SUF system is not required for Fe–S cluster biogenesis in M. acetivorans and challenge the universal role of SufBC in Fe–S cluster biogenesis in methanogens.

A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels.

In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. Here we show that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. We observe a defect in Fe-S cluster biogenesis and increased iron levels in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and we show that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.

Convergent imaging-transcriptomic evidence for disturbed iron homeostasis in Gilles de la Tourette syndrome

Gilles de la Tourette syndrome (GTS) is a neuropsychiatric movement disorder with reported abnormalities in various neurotransmitter systems. Considering the integral role of iron in neurotransmitter synthesis and transport, it is hypothesized that iron exhibits a role in GTS pathophysiology. As a surrogate measure of brain iron, quantitative susceptibility mapping (QSM) was performed in 28 patients with GTS and 26 matched controls. Significant susceptibility reductions in the patients, consistent with reduced local iron content, were obtained in subcortical regions known to be implicated in GTS. Regression analysis revealed a significant negative association of tic scores and striatal susceptibility. To interrogate genetic mechanisms that may drive these reductions, spatially specific relationships between susceptibility and gene-expression patterns from the Allen Human Brain Atlas were assessed. Correlations in the striatum were enriched for excitatory, inhibitory, and modulatory neurochemical signaling mechanisms in the motor regions, mitochondrial processes driving ATP production and iron‑sulfur cluster biogenesis in the executive subdivision, and phosphorylation-related mechanisms affecting receptor expression and long-term potentiation in the limbic subdivision. This link between susceptibility reductions and normative transcriptional profiles suggests that disruptions in iron regulatory mechanisms are involved in GTS pathophysiology and may lead to pervasive abnormalities in mechanisms regulated by iron-containing enzymes.

Copper exerts cytotoxicity through inhibition of iron-sulfur cluster biogenesis on ISCA1/ISCA2/ISCU assembly proteins.

Copper is an essential mineral nutrient that provides the cofactors for some key enzymes. However, excess copper is paradoxically cytotoxic. Wilson's disease is an autosomal recessive hereditary disease characterized by pathological copper accumulation in many organs, with high mortality and disability. Nevertheless, many questions about the molecular mechanism in Wilson's disease remain unknown and there is an imperative need to address these questions to better exploit therapeutic strategy. In this study, we constructed the mouse model of Wilson's disease, ATP7A-/- immortalized lymphocyte cell line and ATP7B knockdown cells to explore whether copper could impair iron-sulfur cluster biogenesis in eukaryotic mitochondria. Through a series of cellular, molecular, and pharmacological analyses, we demonstrated that copper could suppress the assembly of Fe-S cluster, decrease the activity of the Fe-S enzyme and disorder the mitochondrial function both in vivo and in vitro. Mechanistically, we found that human ISCA1, ISCA2 and ISCU proteins have a strong copper-binding activity, which would hinder the process of iron-sulfur assembly. Of note, we proposed a novel mechanism of action to explain the toxicity of copper by providing evidence that iron-sulfur cluster biogenesis may be a primary target of copper toxicity both in cells and mouse models. In summary, the current work provides an in-depth study on the mechanism of copper intoxication and describes a framework for the further understanding of impaired Fe-S assembly in the pathological processes of Wilson's diseases, which helps to develop latent therapeutic strategies for the management of copper toxicity.

Metal regulation of Mycobacterium tuberculosis SufB intein splicing at the host-pathogen crossroad.

Intein sequences self-excise from precursor proteins to generate functional proteins in various organisms. Thus, regulation of intein splicing at the host-pathogen interface can determine the fate of infection by controlling generation of essential proteins in microbes. For instance, Mycobacterium tuberculosis (Mtu) SufB intein splicing is crucial for the functionality of SUF complex. This multiprotein system is the sole pathway for [Fe-S] cluster biogenesis in mycobacteria during oxidative stress and Fe starvation. Although metal toxicity and metal starvation are components of host immunity, correlation of metal stress to Mtu SufB intein splicing is missing till date. Current study examines the splicing and N-terminal cleavage reactions of Mtu SufB precursor protein in presence of micronutrient metal ions like Zn+2, Cu+2, and Fe+3/+2. A known intein splicing inhibitor Pt+4 was also tested to support its proposed role as an anti-TB agent. Mtu SufB precursor protein exhibited significant attenuation of splicing and N-terminal cleavage reactions across different concentration ranges for Pt+4, Cu+2, Zn+2, while Fe+3 interaction resulted in precursor accumulation. UV-Vis spectroscopy, inductively coupled plasma-optical emission spectroscopy (ICP-OES), Tryptophan fluorescence assay, and dynamic light scattering (DLS) techniques analyzed metal-protein interaction. Mutagenesis experiments and Ellman's assay identified plausible metal co-ordination sites within Mtu SufB protein. Analyzing the metal effect on Mtu SufB splicing may provide elemental information about the fate of mycobacterial infection, and a probable mechanism to attenuate intracellular survival of Mtu. Current research hints at the host regulatory mechanism on SufB splicing in its native environment and a likely target for developing next-generation anti-TB drugs.

Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3.

Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.287A > G) point mutation, which involves a highly conserved residue, previously identified as a [2Fe-2S] cluster ligand in the BOLA3-[2Fe-2S]-GLRX5 heterocomplex, on the structural and functional properties of BOLA3 protein. The His96Arg mutation has been associated with a severe MMDS2 phenotype, characterized by defects in the activity of mitochondrial respiratory complexes and lipoic acid-dependent enzymes. Size exclusion chromatography, NMR, UV-visible, circular dichroism, and EPR spectroscopy characterization have shown that the His96Arg mutation does not impair the interaction of BOLA3 with its protein partner GLRX5, but leads to the formation of an aberrant BOLA3-[2Fe-2S]-GLRX5 heterocomplex, that is not functional anymore in the assembly of a [4Fe-4S] cluster on NFU1. These results allowed us to rationalize the severe phenotype observed in MMDS2 caused by His96Arg mutation.

The role of thioredoxin proteins in Mycobacterium tuberculosis probed by proteome-wide target profiling

Mycobacterium tuberculosis encounters diverse microenvironments, including oxidative assault (ROS and RNS), when it attempts to establish itself within its human host. Therefore, redox sensory and regulation processes are assumed significant importance, as these are essential processes for M. tuberculosis to survive under these hostile conditions. M. tuberculosis contains thioredoxin system to maintain redox homeostasis, which establish a balance between the thiol/dithiol couple.Still very less is known about it. In the present study, we attempted to capture the targets of all the M. tuberculosis thioredoxin proteins (viz., TrxB and TrxC) and a thioredoxin-like protein, NrdH, under aerobic and hypoxic conditions by performing thioredoxin trapping chromatography followed by mass spectrometry. We found that TrxC captured the maximum number of targets in both the physiological conditions and most of the targets of TrxB and NrdH showing overlap with targets of TrxC, indicating that TrxC acts as main thioredoxin. Further the PANTHER classification system provides involvement of targets in various metabolic processes and Gene Ontology analysis suggests that glutamine biosynthetic process and Fe–S cluster biosynthesis are the most enriched processes in the target list of TrxC and TrxB respectively. Also, we suggest that the thioredoxin system might play an important role under hypoxia by targeting those proteins which are responsible to sense and maintain hypoxic conditions. Furthermore, our studies establish a link between TrxB and iron-sulfur cluster biogenesis in M. tuberculosis. Ultimately, these findings open a new direction to target the thioredoxin system for screening new anti-mycobacterial drug targets.

The impact of genotype on the phenotype of Mycobacterium tuberculosis ΔsufR mutants

Iron-sulphur (FeS) cluster biogenesis is a tightly regulated process in vivo. In Mycobacterium tuberculosis (Mtb), SufR functions as a transcriptional repressor of the operon encoding the primary FeS cluster biogenesis system. Previously, three independently isolated mutants (ΔRv1460stop_1.19, ΔRv1460stop _5.19 and ΔRv1460stop _5.20) harbouring the same deletion in sufR, displayed different growth kinetics in OADC supplemented 7H9 media. To investigate this discrepancy, we performed whole genome sequencing of the 3 mutants and the wild-type progenitor. Single nucleotide polymorphisms (SNPs) were identified in 3 genes in the ΔRv1460stop_1.19 mutant and one gene in the ΔRv1460stop_5.20 mutant. Phenotyping of the ΔRv1460stop_5.19 mutant, which had no additional SNPs, revealed increased susceptibility to clofazimine, DMNQ and menadione, while uptake and survival in THP-1 cells were not significantly different from the wild-type strain. Given that these results differ from those reported for other sufR deletion mutants (ΔSufRMTB and MtbΔSufR), they suggest that the position of the sufR deletion and the genotype of the progenitor strain impact the resulting phenotype.

Targeting Iron-Sulfur Clusters in Cancer: Opportunities and Challenges for Ferroptosis-Based Therapy.

Iron dysregulation is a hallmark of cancer, characterized by an overexpression of genes involved in iron metabolism and iron-sulfur cluster (ISC) biogenesis. Dysregulated iron homeostasis increases intracellular labile iron, which may lead to the formation of excess cytotoxic radicals and make it vulnerable to various types of regulated cell death, including ferroptosis. The inhibition of ISC synthesis triggers the iron starvation response, increasing lipid peroxidation and ferroptosis in cancer cells treated with oxidative stress-inducing agents. Various methods, such as redox operations, iron chelation, and iron replacement with redox-inert metals, can destabilize or limit ISC formation and function, providing potential therapeutic strategies for cancer treatment. Targeting ISCs to induce ferroptosis represents a promising approach in cancer therapy. This review summarizes the state-of-the-art overview of iron metabolism and ferroptosis in cancer cells, the role of ISC modulation in ferroptosis, and the potential of targeting ISCs for ferroptosis induction in cancer therapy. Further research is necessary to develop and validate these strategies in clinical trials for various cancers, which may ultimately lead to the development of novel and effective treatments for cancer patients.

Essential mitochondrial role in iron-sulfur cluster assembly of the cytoplasmic isopropylmalate isomerase Leu1 in Saccharomyces cerevisiae

Iron-sulfur (Fe-S) cluster assembly in mitochondria and cytoplasm is essential for cell viability. In the yeast S. cerevisiae, Leu1 [4Fe-4S] is the cytoplasmic isopropylmalate isomerase involved in leucine biosynthesis. Using permeabilized Δleu1 cells and recombinant apo-Leu1R, here we show that the [4Fe-4S] cluster assembly on Leu1R can be reconstituted in a physiologic manner requiring both mitochondria and cytoplasm, as judged by conversion of the inactive enzyme to an active form. The mitochondrial contribution to this reconstitution assay is abrogated by inactivating mutations in the mitochondrial ISC (iron-sulfur cluster assembly) machinery components (such as Nfs1 cysteine desulfurase and Ssq1 chaperone) or the mitochondrial exporter Atm1. Likewise, depletion of a CIA (cytoplasmic iron-sulfur protein assembly) component Dre2 leads to impaired Leu1R reconstitution. Mitochondria likely make and export an intermediate, called X-S or (Fe-S)int, that is needed for cytoplasmic Fe-S cluster biosynthesis. Here we show that once exported, the same intermediate can be used for both [2Fe-2S] and [4Fe-4S] cluster biogenesis in the cytoplasm, with no further requirement of mitochondria. Our data also suggest that the exported intermediate can activate defective/latent CIA components in cytoplasm isolated from nfs1 or Δatm1 mutant cells. These findings may provide a way to isolate X-S or (Fe-S)int.

Patient-specific variants of NFU1/NFU-1 disrupt cholinergic signaling in a model of multiple mitochondrial dysfunctions syndrome 1.

Neuromuscular dysfunction is a common feature of mitochondrial diseases and frequently presents as ataxia, spasticity and/or dystonia, all of which can severely impact individuals with mitochondrial diseases. Dystonia is one of the most common symptoms of multiple mitochondrial dysfunctions syndrome 1 (MMDS1), a disease associated with mutations in the causative gene (NFU1) that impair iron-sulfur cluster biogenesis. We have generated Caenorhabditis elegans strains that recreated patient-specific point variants in the C. elegans ortholog (nfu-1) that result in allele-specific dysfunction. Each of these mutants, Gly147Arg and Gly166Cys, have altered acetylcholine signaling at neuromuscular junctions, but opposite effects on activity and motility. We found that the Gly147Arg variant was hypersensitive to acetylcholine and that knockdown of acetylcholine release rescued nearly all neuromuscular phenotypes of this variant. In contrast, we found that the Gly166Cys variant caused predominantly postsynaptic acetylcholine hypersensitivity due to an unclear mechanism. These results are important for understanding the neuromuscular conditions of MMDS1 patients and potential avenues for therapeutic intervention.

A Diverged Transcriptional Network for Usage of Two Fe-S Cluster Biogenesis Machineries in the Delta-Proteobacterium Myxococcus xanthus.

Myxococcus xanthus possesses two Fe-S cluster biogenesis machineries, ISC (iron-sulfur cluster) and SUF (sulfur mobilization). Here, we show that in comparison to the phylogenetically distant Enterobacteria, which also have both machineries, M. xanthus evolved an independent transcriptional scheme to coordinately regulate the expression of these machineries. This transcriptional response is directed by RisR, which we show to belong to a phylogenetically distant and biochemically distinct subgroup of the Rrf2 transcription factor family, in comparison to IscR that regulates the isc and suf operons in Enterobacteria. We report that RisR harbors an Fe-S cluster and that holo-RisR acts as a repressor of both the isc and suf operons, in contrast to Escherichia coli, where holo-IscR represses the isc operon whereas apo-IscR activates the suf operon. In addition, we establish that the nature of the cluster and the DNA binding sites of RisR, in the isc and suf operons, diverge from those of IscR. We further show that in M. xanthus, the two machineries appear to be fully interchangeable in maintaining housekeeping levels of Fe-S cluster biogenesis and in synthesizing the Fe-S cluster for their common regulator, RisR. We also demonstrate that in response to oxidative stress and iron limitation, transcriptional upregulation of the M. xanthus isc and suf operons was mediated solely by RisR and that the contribution of the SUF machinery was greater than the ISC machinery. Altogether, these findings shed light on the diversity of homeostatic mechanisms exploited by bacteria to coordinately use two Fe-S cluster biogenesis machineries. IMPORTANCE Fe-S proteins are ubiquitous and control a wide variety of key biological processes; therefore, maintaining Fe-S cluster homeostasis is an essential task for all organisms. Here, we provide the first example of how a bacterium from the Deltaproteobacteria branch coordinates expression of two Fe-S cluster biogenesis machineries. The results revealed a new model of coordination, highlighting the unique and common features that have independently emerged in phylogenetically distant bacteria to maintain Fe-S cluster homeostasis in response to environmental changes. Regulation is orchestrated by a previously uncharacterized transcriptional regulator, RisR, belonging to the Rrf2 superfamily, whose members are known to sense diverse environmental stresses frequently encountered by bacteria. Understanding how M. xanthus maintains Fe-S cluster homeostasis via RisR regulation revealed a strategy reflective of the aerobic lifestyle of this organsim. This new knowledge also paves the way to improve production of Fe-S-dependent secondary metabolites using M. xanthus as a chassis.

Analysis of a logical regulatory network reveals how Fe-S cluster biogenesis is controlled in the face of stress.

Iron-sulfur (Fe-S) clusters are important cofactors conserved in all domains of life, yet their synthesis and stability are compromised in stressful conditions such as iron deprivation or oxidative stress. Two conserved machineries, Isc and Suf, assemble and transfer Fe-S clusters to client proteins. The model bacterium Escherichia coli possesses both Isc and Suf, and in this bacterium utilization of these machineries is under the control of a complex regulatory network. To better understand the dynamics behind Fe-S cluster biogenesis in E. coli, we here built a logical model describing its regulatory network. This model comprises three biological processes: 1) Fe-S cluster biogenesis, containing Isc and Suf, the carriers NfuA and ErpA, and the transcription factor IscR, the main regulator of Fe-S clusters homeostasis; 2) iron homeostasis, containing the free intracellular iron regulated by the iron sensing regulator Fur and the non-coding regulatory RNA RyhB involved in iron sparing; 3) oxidative stress, representing intracellular H2O2 accumulation, which activates OxyR, the regulator of catalases and peroxidases that decompose H2O2 and limit the rate of the Fenton reaction. Analysis of this comprehensive model reveals a modular structure that displays five different types of system behaviors depending on environmental conditions, and provides a better understanding on how oxidative stress and iron homeostasis combine and control Fe-S cluster biogenesis. Using the model, we were able to predict that an iscR mutant would present growth defects in iron starvation due to partial inability to build Fe-S clusters, and we validated this prediction experimentally.