RESUMO
Monocercomonoides exilis is the first known amitochondriate eukaryote. Loss of mitochondria in M. exilis ocurred after the replacement of the essential mitochondrial iron-sulfur cluster (ISC) assembly machinery by a unique, bacteria-derived, cytosolic SUF system. It has been hypothesized that the MeSuf pathway, in cooperation with proteins of the cytosolic iron-sulfur protein assembly (CIA) system, is responsible for the biogenesis of FeS clusters in M. exilis, yet biochemical evidence is pending. Here, we address the M. exilis MeSuf system and show that SUF genes, individually or in tandem, support the loading of iron-sulfur (FeS) clusters into the reporter protein IscR in Escherichia coli. The Suf proteins MeSufB, MeSufC, and MeSufDSU interact in vivo with one another and with Suf proteins of E. coli. In vitro, the M. exilis Suf proteins form large complexes of varying composition and hence may function as a dynamic biosynthetic system in the protist. The putative FeS cluster scaffold MeSufB-MeSufC (MeSufBC) forms multiple oligomeric complexes, some of which bind FeS clusters and form selectively only in the presence of adenosine nucleotides. The multi-domain fusion protein MeSufDSU binds a PLP cofactor and can form higher-order complexes with MeSufB and MeSufC. Our work demonstrates the biochemical property of M. exilis Suf proteins to act as a functional FeS cluster assembly system and provides insights into the molecular mechanism of this unique eukaryotic SUF system.
RESUMO
Iron-sulfur (Fe-S) clusters are required for essential biological pathways, including respiration and isoprenoid biosynthesis. Complex Fe-S cluster biogenesis systems have evolved to maintain an adequate supply of this critical protein cofactor. In Escherichia coli, two Fe-S biosynthetic systems, the "housekeeping" Isc and "stress responsive" Suf pathways, interface with a network of cluster trafficking proteins, such as ErpA, IscA, SufA, and NfuA. GrxD, a Fe-S cluster-binding monothiol glutaredoxin, also participates in Fe-S protein biogenesis in both prokaryotes and eukaryotes. Previous studies in E. coli showed that the ΔgrxD mutation causes sensitivity to iron depletion, spotlighting a critical role for GrxD under conditions that disrupt Fe-S homeostasis. Here, we utilized a global chemoproteomic mass spectrometry approach to analyze the contribution of GrxD to the Fe-S proteome. Our results demonstrate that (1) GrxD is required for biogenesis of a specific subset of Fe-S proteins under iron-depleted conditions, (2) GrxD is required for cluster delivery to ErpA under iron limitation, (3) GrxD is functionally distinct from other Fe-S trafficking proteins, and (4) GrxD Fe-S cluster binding is responsive to iron limitation. All these results lead to the proposal that GrxD is required to maintain Fe-S cluster delivery to the essential trafficking protein ErpA during iron limitation conditions.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Glutarredoxinas , Proteínas Ferro-Enxofre , Ferro , Escherichia coli/metabolismo , Escherichia coli/genética , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Ferro/metabolismo , Estresse Fisiológico , LiasesRESUMO
Ironsulfur (Fe-S) clusters are one of the most ancient and versatile inorganic cofactors present in the three domains of life. Fe-S clusters are essential cofactors for the activity of a large variety of metalloproteins that play crucial physiological roles. Fe-S protein biogenesis is a complex process that starts with the acquisition of the elements (iron and sulfur atoms) and their assembly into an Fe-S cluster that is subsequently inserted into the target proteins. The Fe-S protein biogenesis is ensured by multiproteic systems conserved across all domains of life. Here, we provide an overview on how bacterial genetics approaches have permitted to reveal and dissect the Fe-S protein biogenesis process in vivo.
Assuntos
Proteínas de Bactérias , Proteínas Ferro-Enxofre , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Bactérias/genética , Bactérias/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Escherichia coli , Proteínas Ferro-Enxofre , Myxococcus xanthus , Proteínas de Escherichia coli/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Redes Reguladoras de Genes , Escherichia coli/genética , Ferro/metabolismo , Enxofre/metabolismo , Proteínas Ferro-Enxofre/químicaRESUMO
Aminoglycosides (AG) have been used against Gram-negative bacteria for decades. Yet, how bacterial metabolism and environmental conditions modify AG toxicity is poorly understood. Here, we show that the level of AG susceptibility varies depending on the nature of the respiratory chain that Escherichia coli uses for growth, i.e., oxygen, nitrate, or fumarate. We show that all components of the fumarate respiratory chain, namely, hydrogenases 2 and 3, the formate hydrogenlyase complex, menaquinone, and fumarate reductase are required for AG-mediated killing under fumarate respiratory conditions. In addition, we show that the AAA+ ATPase RavA and its Von Wildebrand domain-containing partner, ViaA, are essential for AG to act under fumarate respiratory conditions. This effect was true for all AG that were tested but not for antibiotics from other classes. In addition, we show that the sensitizing effect of RavA-ViaA is due to increased gentamicin uptake in a proton motive force-dependent manner. Interestingly, the sensitizing effect of RavA-ViaA was prominent in poor energy conservation conditions, i.e., with fumarate, but dispensable under high energy conservation conditions, i.e., in the presence of nitrate or oxygen. We propose that RavA-ViaA can facilitate uptake of AG across the membrane in low-energy cellular states. IMPORTANCE Antibiotic resistance is a major public health, social, and economic problem. Aminoglycosides (AG) are known to be highly effective against Gram-negative bacteria, but their use is limited to life-threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose. Elucidation of AG-sensitization mechanisms in bacteria would allow reduced effective doses of AG. Here, we have identified the molecular components involved in anaerobic fumarate respiration that are required for AG to kill. In addition to oxidoreductases and menaquinone, this includes new molecular players, RavA, an AAA+ ATPase, and ViaA, its partner that has the VWA motif. Remarkably, the influence of RavA-ViaA on AG susceptibility varies according to the type of bioenergetic metabolism used by E. coli. This is a significant advance because anaerobiosis is well known to reduce the antibacterial activity of AG. This study highlights the critical importance of the relationship between culture conditions, metabolism, and antibiotic susceptibility.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Aminoglicosídeos/farmacologia , Nitratos/metabolismo , Vitamina K 2/metabolismo , Vitamina K 2/farmacologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Metabolismo Energético , Succinato Desidrogenase , Bactérias/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Fumaratos/farmacologia , Fumaratos/metabolismo , Anaerobiose , Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.
Assuntos
Proteínas de Escherichia coli , Proteínas Ferro-Enxofre , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Filogenia , Enxofre/metabolismoRESUMO
Iron-sulfur (Fe-S) clusters are among the oldest protein cofactors, and Fe-S cluster-based chemistry has shaped the cellular metabolism of all living organisms. Over the last 30 years, thanks to molecular biology and genetic approaches, numerous actors for Fe-S cluster assembly and delivery to apotargets have been uncovered. In prokaryotes, Escherichia coli is the best-studied for its convenience of growth and its genetic amenability. During evolution, redundant ways to secure the supply of Fe-S clusters to the client proteins have emerged in E. coli. Disrupting gene expression is essential for gene function exploration, but redundancy can blur the interpretations as it can mask the role of important biogenesis components. This chapter describes molecular biology and genetic strategies that have permitted to reveal the E. coli Fe-S cluster conveying component network, composition, organization, and plasticity. In this chapter, we will describe the following genetic methods to investigate the importance of E. coli Fe-S cluster carriers: one-step inactivation of chromosomal genes in E. coli using polymerase chain reaction (PCR) products, P1 transduction, arabinose-inducible expression system, mevalonate (MVA) genetic by-pass, sensitivity tests to oxidative stress and iron starvation, ß-galactosidase assay, gentamicin survival test, and Hot Fusion cloning method.
Assuntos
Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Biologia MolecularRESUMO
The level of antibiotic resistance exhibited by bacteria can vary as a function of environmental conditions. Here, we report that phenazine-methosulfate (PMS), a redox-cycling compound (RCC) enhances resistance to fluoroquinolone (FQ) norfloxacin. Genetic analysis showed that E. coli adapts to PMS stress by making Fe-S clusters with the SUF machinery instead of the ISC one. Based upon phenotypic analysis of soxR, acrA, and micF mutants, we showed that PMS antagonizes fluoroquinolone toxicity by SoxR-mediated up-regulation of the AcrAB drug efflux pump. Subsequently, we showed that despite the fact that SoxR could receive its cluster from either ISC or SUF, only SUF is able to sustain efficient SoxR maturation under exposure to prolonged PMS period or high PMS concentrations. This study furthers the idea that Fe-S cluster homeostasis acts as a sensor of environmental conditions, and because its broad influence on cell metabolism, modifies the antibiotic resistance profile of E. coli.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/fisiologia , Proteínas Ferro-Enxofre/metabolismo , Fatores de Transcrição/metabolismo , Antibacterianos/uso terapêutico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Antagonismo de Drogas , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Metilfenazônio Metossulfato/farmacologia , Testes de Sensibilidade Microbiana , Norfloxacino/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genéticaRESUMO
Iron sulfur (Fe-S) clusters rank among the most ancient and conserved prosthetic groups. Fe-S clusters containing proteins are present in most, if not all, organisms. Fe-S clusters containing proteins are involved in a wide range of cellular processes, from gene regulation to central metabolism, via gene expression, RNA modification or bioenergetics. Fe-S clusters are built by biogenesis machineries conserved throughout both prokaryotes and eukaryotes. We focus mostly on bacterial ISC machinery, but not exclusively, as we refer to eukaryotic ISC system when it brings significant complementary information. Besides covering the structural and regulatory aspects of Fe-S biogenesis, this review aims to highlight Fe-S biogenesis facets remaining matters of discussion, such as the role of frataxin, or the link between fatty acid metabolism and Fe-S homeostasis. Last, we discuss recent advances on strategies used by different species to make and use Fe-S clusters in changing redox environmental conditions.
Assuntos
Escherichia coli/fisiologia , Proteínas Ferro-Enxofre/fisiologia , Ferro/metabolismo , Enxofre/metabolismo , Proteína de Transporte de Acila/fisiologia , Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica , Homeostase , Humanos , Proteínas de Ligação ao Ferro , Proteínas Ferro-Enxofre/química , Oxirredução , Saccharomyces cerevisiae/fisiologia , FrataxinaRESUMO
Iron-sulfur (Fe-S) clusters are considered one of the most ancient and versatile inorganic cofactors present in the three domains of life. Fe-S clusters can act as redox sensors or catalysts and are found to be used by a large number of functional and structurally diverse proteins. Here, we cover current knowledge of the SUF multiprotein machinery that synthesizes and inserts Fe-S clusters into proteins. Specific focus is put on the ABC ATPase SufC, which contributes to building Fe-S clusters, and appeared early on during evolution.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Ferro/metabolismo , Enxofre/metabolismoRESUMO
Despite recent advances in understanding the biogenesis of iron-sulfur (Fe-S) proteins, most studies focused on aerobic bacteria as model organisms. Accordingly, multiple players have been proposed to participate in the Fe-S delivery step to apo-target proteins, but critical gaps exist in the knowledge of Fe-S proteins biogenesis in anaerobic organisms. Mrp/NBP35 ATP-binding proteins are a subclass of the soluble P-loop containing nucleoside triphosphate hydrolase superfamily (P-loop NTPase) known to bind and transfer Fe-S clusters in vitro. Here, we report investigations of a novel atypical two-domain Mrp/NBP35 ATP-binding protein named MrpORP associating a P-loop NTPase domain with a dinitrogenase iron-molybdenum cofactor biosynthesis domain (Di-Nase). Characterization of full length MrpORP, as well as of its two domains, showed that both domains bind Fe-S clusters. We provide in vitro evidence that the P-loop NTPase domain of the MrpORP can efficiently transfer its Fe-S cluster to apo-target proteins of the ORange Protein (ORP) complex, suggesting that this novel protein is involved in the maturation of these Fe-S proteins. Last, we showed for the first time, by fluorescence microscopy imaging a polar localization of a Mrp/NBP35 protein.
Assuntos
Proteínas de Bactérias/metabolismo , Desulfovibrio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Proteínas AAA/genética , Proteínas AAA/metabolismo , Proteínas de Bactérias/genética , Citosol , Desulfovibrio/classificação , Desulfovibrio/genética , Proteínas de Ligação ao GTP/genética , Proteínas Ferro-Enxofre/genética , Molibdoferredoxina/metabolismo , Nitrogenase/genética , Nitrogenase/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
The spread of antimicrobial resistance has become a serious public health concern, making once-treatable diseases deadly again and undermining the achievements of modern medicine1,2. Drug combinations can help to fight multi-drug-resistant bacterial infections, yet they are largely unexplored and rarely used in clinics. Here we profile almost 3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in six strains from three Gram-negative pathogens-Escherichia coli, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa-to identify general principles for antibacterial drug combinations and understand their potential. Despite the phylogenetic relatedness of the three species, more than 70% of the drug-drug interactions that we detected are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs that target different cellular processes, whereas synergies are more conserved and are enriched in drugs that target the same process. We provide mechanistic insights into this dichotomy and further dissect the interactions of the food additive vanillin. Finally, we demonstrate that several synergies are effective against multi-drug-resistant clinical isolates in vitro and during infections of the larvae of the greater wax moth Galleria mellonella, with one reverting resistance to the last-resort antibiotic colistin.
Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/efeitos dos fármacos , Animais , Benzaldeídos/farmacologia , Colistina/farmacologia , Combinação de Medicamentos , Interações Medicamentosas , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Aditivos Alimentares/farmacologia , Larva/efeitos dos fármacos , Larva/microbiologia , Testes de Sensibilidade Microbiana , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Filogenia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , Salmonella typhimurium/classificação , Salmonella typhimurium/efeitos dos fármacos , Especificidade da EspécieRESUMO
Fe-S cluster-containing proteins occur in most organisms, wherein they assist in myriad processes from metabolism to DNA repair via gene expression and bioenergetic processes. Here, we used both in vitro and in vivo methods to investigate the capacity of the four Fe-S carriers, NfuA, SufA, ErpA, and IscA, to fulfill their targeting role under oxidative stress. Likewise, Fe-S clusters exhibited varying half-lives, depending on the carriers they were bound to; an NfuA-bound Fe-S cluster was more stable (t½ = 100 min) than those bound to SufA (t½ = 55 min), ErpA (t½ = 54 min), or IscA (t½ = 45 min). Surprisingly, the presence of NfuA further enhanced stability of the ErpA-bound cluster to t½ = 90 min. Using genetic and plasmon surface resonance analyses, we showed that NfuA and ErpA interacted directly with client proteins, whereas IscA or SufA did not. Moreover, NfuA and ErpA interacted with one another. Given all of these observations, we propose an architecture of the Fe-S delivery network in which ErpA is the last factor that delivers cluster directly to most if not all client proteins. NfuA is proposed to assist ErpA under severely unfavorable conditions. A comparison with the strategy employed in yeast and eukaryotes is discussed.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Redes e Vias Metabólicas , Estresse Oxidativo , Oxigênio/metabolismo , Regulação Bacteriana da Expressão Gênica , OxirreduçãoRESUMO
Fe-S bound proteins are ubiquitous and contribute to most basic cellular processes. A defect in the ISC components catalyzing Fe-S cluster biogenesis leads to drastic phenotypes in both eukaryotes and prokaryotes. In this context, the Frataxin protein (FXN) stands out as an exception. In eukaryotes, a defect in FXN results in severe defects in Fe-S cluster biogenesis, and in humans, this is associated with Friedreich's ataxia, a neurodegenerative disease. In contrast, prokaryotes deficient in the FXN homolog CyaY are fully viable, despite the clear involvement of CyaY in ISC-catalyzed Fe-S cluster formation. The molecular basis of the differing importance in the contribution of FXN remains enigmatic. Here, we have demonstrated that a single mutation in the scaffold protein IscU rendered E. coli viability strictly dependent upon a functional CyaY. Remarkably, this mutation changed an Ile residue, conserved in prokaryotes at position 108, into a Met residue, conserved in eukaryotes. We found that in the double mutant IscUIM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes. Biochemical analyses of the "eukaryotic-like" IscUIM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters. Finally, bioinformatic studies of prokaryotic IscU proteins allowed us to trace back the source of FXN-dependency as it occurs in present-day eukaryotes. We propose an evolutionary scenario in which the current mitochondrial Isu proteins originated from the IscUIM version present in the ancestor of the Rickettsiae. Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108th position of the scaffold.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Biologia Computacional , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Proteínas de Ligação ao Ferro/genética , Proteínas Ferro-Enxofre/metabolismo , Viabilidade Microbiana , Mutação , Filogenia , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , FrataxinaRESUMO
Since their discovery in the 50's, Fe-S cluster proteins have attracted much attention from chemists, biophysicists and biochemists. However, in the 80's they were joined by geneticists who helped to realize that in vivo maturation of Fe-S cluster bound proteins required assistance of a large number of factors defining complex multi-step pathways. The question of how clusters are formed and distributed in vivo has since been the focus of much effort. Here we review how genetics in discovering genes and investigating processes as they unfold in vivo has provoked seminal advances toward our understanding of Fe-S cluster biogenesis. The power and limitations of genetic approaches are discussed. As a final comment, we argue how the marriage of classic strategies and new high-throughput technologies should allow genetics of Fe-S cluster biology to be even more insightful in the future. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Ferro-Enxofre/genética , Epistasia Genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Previsões , Proteínas Ferro-Enxofre/metabolismo , Biologia Molecular/métodos , Biologia Molecular/tendências , MutaçãoRESUMO
In eukaryotes, frataxin deficiency (FXN) causes severe phenotypes including loss of iron-sulfur (Fe-S) cluster protein activity, accumulation of mitochondrial iron and leads to the neurodegenerative disease Friedreich's ataxia. In contrast, in prokaryotes, deficiency in the FXN homolog, CyaY, was reported not to cause any significant phenotype, questioning both its importance and its actual contribution to Fe-S cluster biogenesis. Because FXN is conserved between eukaryotes and prokaryotes, this surprising discrepancy prompted us to reinvestigate the role of CyaY in Escherichia coli. We report that CyaY (i) potentiates E. coli fitness, (ii) belongs to the ISC pathway catalyzing the maturation of Fe-S cluster-containing proteins and (iii) requires iron-rich conditions for its contribution to be significant. A genetic interaction was discovered between cyaY and iscX, the last gene of the isc operon. Deletion of both genes showed an additive effect on Fe-S cluster protein maturation, which led, among others, to increased resistance to aminoglycosides and increased sensitivity to lambda phage infection. Together, these in vivo results establish the importance of CyaY as a member of the ISC-mediated Fe-S cluster biogenesis pathway in E. coli, like it does in eukaryotes, and validate IscX as a new bona fide Fe-S cluster biogenesis factor.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Aptidão Genética , Proteínas Ferro-Enxofre/metabolismo , Aminoglicosídeos/farmacologia , Bacteriófago lambda/fisiologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Ferro/metabolismo , Mutação , Óperon , Enxofre/metabolismoRESUMO
Iron-sulfur clusters (Fe-S) are ubiquitous cofactors present in numerous proteins of most living organisms. By way of an example, the E. coli bacterium synthesizes more that 130 different types of Fe-S proteins. Fe-S proteins are involved in a great diversity of biological processes, ranging from respiration, photosynthesis, central metabolism, to genetic expression and genomic stability. Proteins can acquire spontaneously Fe-S clusters in vitro, but in vivo, dedicated molecular machineries are necessary. Dysfunction of these machineries alters cellular capacities leading to lethality in bacteria and severe pathologies in humans. In this review we will describe how cells make Fe-S clusters and deliver them to clients proteins. The importance of Fe-S clusters homeostasis will be illustrated by reporting a list of cellular dysfunctions associated with mutations altering either Fe-S proteins or Fe-S biogenesis machineries.
Assuntos
Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/fisiologia , Animais , Bactérias/patogenicidade , Fenômenos Fisiológicos Bacterianos , Escherichia coli/química , Escherichia coli/fisiologia , Humanos , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Mutação , Fenômenos Fisiológicos Vegetais , Enxofre/metabolismoRESUMO
The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.
Assuntos
Liases de Carbono-Enxofre/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Ferredoxinas/química , Proteínas de Ligação ao Ferro/química , Modelos Moleculares , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Estrutura Quaternária de Proteína , FrataxinaRESUMO
All bactericidal antibiotics were recently proposed to kill by inducing reactive oxygen species (ROS) production, causing destabilization of iron-sulfur (Fe-S) clusters and generating Fenton chemistry. We find that the ROS response is dispensable upon treatment with bactericidal antibiotics. Furthermore, we demonstrate that Fe-S clusters are required for killing only by aminoglycosides. In contrast to cells, using the major Fe-S cluster biosynthesis machinery, ISC, cells using the alternative machinery, SUF, cannot efficiently mature respiratory complexes I and II, resulting in impendence of the proton motive force (PMF), which is required for bactericidal aminoglycoside uptake. Similarly, during iron limitation, cells become intrinsically resistant to aminoglycosides by switching from ISC to SUF and down-regulating both respiratory complexes. We conclude that Fe-S proteins promote aminoglycoside killing by enabling their uptake.