The Fe-S cluster biosynthesis in Enterococcus faecium is essential for anaerobic growth and gastrointestinal colonization.

The facultative anaerobic Gram-positive bacterium Enterococcus faecium is a ubiquitous member of the human gut microbiota. However, it has gradually evolved into a pathogenic and multidrug resistant lineage that causes nosocomial infections. The establishment of high-level intestinal colonization by enterococci represents a critical step of infection. The majority of current research on Enterococcus has been conducted under aerobic conditions, while limited attention has been given to its physiological characteristics in anaerobic environments, which reflects its natural colonization niche in the gut. In this study, a high-density transposon mutant library containing 26,620 distinct insertion sites was constructed. Tn-seq analysis identified six genes that significantly contribute to growth under anaerobic conditions. Under anaerobic conditions, deletion of sufB (encoding Fe-S cluster assembly protein B) results in more extensive and significant impairments on carbohydrate metabolism compared to aerobic conditions. Consistently, the pathways involved in this utilization-restricted carbohydrates were mostly expressed at significantly lower levels in mutant compared to wild-type under anaerobic conditions. Moreover, deletion of sufB or pflA (encoding pyruvate formate lyase-activating protein A) led to failure of gastrointestinal colonization in mice. These findings contribute to our understanding of the mechanisms by which E. faecium maintains proliferation under anaerobic conditions and establishes colonization in the gut.

Genetic Analysis of the ts-Lethal Mutant Δpa0665/pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa.

Pa0665 in Pseudomonas aeruginosa shares homologous sequences with that of the essential A-type iron-sulfur (Fe-S) cluster insertion protein ErpA in Escherichia coli. However, its essentiality in P. aeruginosa and its complementation with E. coli erpA has not been experimentally examined. To fulfill this task, we constructed plasmid-based ts-mutant Δpa0665/pTS-pa0665 using a three-step protocol. The mutant displayed growth defects at 42 °C, which were complemented by expressing ec.erpA. Microscopic observations indicated a petite cell phenotype for Δpa0665/pTS-pa0665 at 42 °C, correlated with the downregulation of the oprG gene. RNA sequencing revealed significant transcriptional changes in genes associated with the oxidative phosphorylation (OXPHOS) system, aligning with reduced ATP levels in Δpa0665/pTS-pa0665 under 42 °C. Additionally, the ts-mutant showed heightened sensitivity to H2O2 at 42 °C. Overall, our study demonstrates the essential role of pa0665 for OXPHOS function and is complemented by ec.erpA. We propose that the plasmid-based ts-allele is useful for genetic analysis of essential genes of interest in P. aeruginosa.

Iron-sulfur protein odyssey: exploring their cluster functional versatility and challenging identification.

Iron-sulfur (Fe-S) clusters are an essential and ubiquitous class of protein-bound prosthetic centers that are involved in a broad range of biological processes (e.g. respiration, photosynthesis, DNA replication and repair and gene regulation) performing a wide range of functions including electron transfer, enzyme catalysis, and sensing. In a general manner, Fe-S clusters can gain or lose electrons through redox reactions, and are highly sensitive to oxidation, notably by small molecules such as oxygen and nitric oxide. The [2Fe-2S] and [4Fe-4S] clusters, the most common Fe-S cofactors, are typically coordinated by four amino acid side chains from the protein, usually cysteine thiolates, but other residues (e.g. histidine, aspartic acid) can also be found. While diversity in cluster coordination ensures the functional variety of the Fe-S clusters, the lack of conserved motifs makes new Fe-S protein identification challenging especially when the Fe-S cluster is also shared between two proteins as observed in several dimeric transcriptional regulators and in the mitoribosome. Thanks to the recent development of in cellulo, in vitro, and in silico approaches, new Fe-S proteins are still regularly identified, highlighting the functional diversity of this class of proteins. In this review, we will present three main functions of the Fe-S clusters and explain the difficulties encountered to identify Fe-S proteins and methods that have been employed to overcome these issues.

Genetic dissection of the bacterial Fe-S protein biogenesis machineries.

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.

Ferrostatin-1 specifically targets mitochondrial iron-sulfur clusters and aconitase to improve cardiac function in Sirtuin 3 cardiomyocyte knockout mice.

AIMS: Ferroptosis is a form of iron-regulated cell death implicated in ischemic heart disease. Our previous study revealed that Sirtuin 3 (SIRT3) is associated with ferroptosis and cardiac fibrosis. In this study, we tested whether the knockout of SIRT3 in cardiomyocytes (SIRT3cKO) promotes mitochondrial ferroptosis and whether the blockade of ferroptosis would ameliorate mitochondrial dysfunction. METHODS AND RESULTS: Mitochondrial and cytosolic fractions were isolated from the ventricles of mice. Cytosolic and mitochondrial ferroptosis were analyzed by comparison to SIRT3loxp mice. An echocardiography study showed that SIRT3cKO mice developed heart failure as evidenced by a reduction of EF% and FS% compared to SIRT3loxp mice. Comparison of mitochondrial and cytosolic fractions of SIRT3cKO and SIRT3loxp mice revealed that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased. Similarly, acetylated p53 was significantly upregulated only in the mitochondria. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase. Most importantly, loss of SIRT3 resulted in significant reductions of frataxin, aconitase, and glutathione peroxidase 4 (GPX4) in the mitochondria. This was accompanied by a significant increase in levels of mitochondrial 4-hydroxynonenal. Treatment of SIRT3cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) for 14 days significantly improved preexisting heart failure. Mechanistically, Fer-1 treatment significantly increased GPX4 and aconitase expression/activity, increased mitochondrial iron­sulfur clusters, and improved mitochondrial membrane potential and Complex IV activity. CONCLUSIONS: Inhibition of ferroptosis ameliorated cardiac dysfunction by specifically targeting mitochondrial aconitase and iron­sulfur clusters. Blockade of mitochondrial ferroptosis may be a novel therapeutic target for mitochondrial cardiomyopathies.

On the path to [Fe-S] protein maturation: A personal perspective.

Azotobacter vinelandii is a genetically tractable Gram-negative proteobacterium able to fix nitrogen (N2) under aerobic growth conditions. This narrative describes how biochemical-genetic approaches using A. vinelandii to study nitrogen fixation led to the formulation of the "scaffold hypothesis" for the assembly of both simple and complex [Fe-S] clusters associated with biological nitrogen fixation. These studies also led to the discovery of a parallel, but genetically distinct, pathway for maturation of [Fe-S] proteins that support central metabolic processes.

Fe-S cluster homeostasis and beyond: The multifaceted roles of IscR.

The role of IscR in regulating the transcription of genes involved in Fe-S cluster homeostasis has been well established for the model organism Escherichia coli K12. In this bacterium, IscR coordinates expression of the Isc and Suf Fe-S cluster assembly pathways to meet cellular Fe-S cluster demands shaped by a variety of environmental cues. However, since its initial discovery nearly 25 years ago, there has been growing evidence that IscR function extends well beyond Fe-S cluster homeostasis, not only in E. coli, but in bacteria of diverse lifestyles. Notably, pathogenic bacteria have exploited the ability of IscR to respond to changes in oxygen tension, oxidative and nitrosative stress, and iron availability to navigate their trajectory in their respective hosts as changes in these cues are frequently encountered during host infection. In this review, we highlight these broader roles of IscR in different cellular processes and, in particular, discuss the importance of IscR as a virulence factor for many bacterial pathogens.

The nematode effector Mj-NEROSs interacts with Rieske's iron-sulfur protein influencing plastid ROS production to suppress plant immunity.

Root-knot nematodes (RKN; Meloidogyne species) are plant pathogens that introduce several effectors in their hosts to facilitate infection. The actual targets and functioning mechanism of these effectors largely remain unexplored. This study illuminates the role and interplay of the Meloidogyne javanica nematode effector ROS suppressor (Mj-NEROSs) within the host plant environment. Mj-NEROSs suppresses INF1-induced cell death as well as flg22-induced callose deposition and reactive oxygen species (ROS) production. A transcriptome analysis highlighted the downregulation of ROS-related genes upon Mj-NEROSs expression. NEROSs interacts with the plant Rieske's iron-sulfur protein (ISP) as shown by yeast-two-hybrid and bimolecular fluorescence complementation. Secreted from the subventral pharyngeal glands into giant cells, Mj-NEROSs localizes in the plastids where it interacts with ISP, subsequently altering electron transport rates and ROS production. Moreover, our results demonstrate that isp Arabidopsis thaliana mutants exhibit increased susceptibility to M. javanica, indicating ISP importance for plant immunity. The interaction of a nematode effector with a plastid protein highlights the possible role of root plastids in plant defense, prompting many questions on the details of this process.

CISD3/MiNT is required for complex I function, mitochondrial integrity, and skeletal muscle maintenance.

Mitochondria play a central role in muscle metabolism and function. A unique family of iron-sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3-NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions.

Crystal structure of the iron-sulfur cluster transfer protein ApbC from Escherichia coli.

Iron-sulfur (Fe-S) clusters are ubiquitous and are necessary to sustain basic life processes. The intracellular Fe-S clusters do not form spontaneously and many proteins are required for their biosynthesis and delivery. The bacterial P-loop NTPase family protein ApbC participates in Fe-S cluster assembly and transfers the cluster into apoproteins, with the Walker A motif and CxxC motif being essential for functionality of ApbC in Fe-S protein biogenesis. However, the structural basis underlying the ApbC activity and the motifs' role remains unclear. Here, we report the crystal structure of Escherichia coli ApbC at 2.8 Å resolution. The dimeric structure is in a W shape and the active site is located in the 2-fold center. The function of the motifs can be annotated by structural analyses. ApbC has an additional N-terminal domain that differs from other P-loop NTPases, possibly conferring its inherent specificity in vivo.

Electron-transferring flavoprotein and its dehydrogenase contributed to growth development and virulence in Beauveria bassiana.

Electron-transferring flavoprotein (Etf) and its dehydrogenase (Etfdh) are integral components of the electron transport chain in mitochondria. In this study, we characterize two putative etf genes (Bbetfa and Bbetfb) and their dehydrogenase gene Bbetfdh in the entomopathogenic fungus Beauveria bassiana. Individual deletion of these genes caused a significant reduction in vegetative growth, conidiation, and delayed conidial germination. Lack of these genes also led to abnormal metabolism of fatty acid and increasing lipid body accumulation. Furthermore, the virulence of Bbetfs and Bbetfdh deletion mutants was severely impaired due to decreasing infection structure formation. Additionally, all deletion strains showed reduced ATP synthesis compared to the wild-type strain. Taken together, Bbetfa and Bbetfb, along with Bbetfdh, play principal roles in fungal vegetative growth, conidiation, conidial germination, and pathogenicity of B. bassiana due to their essential functions in fatty acid metabolism.

Protists: Eukaryotic single-celled organisms and the functioning of their organelles.

Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the diversity and function of protistan organelles has increased in recent years, providing a complex panoply of structure/function relationships. This is particularly noticeable with the description of hydrogenosomes, and the diverse array of structures that followed, having hybrid hydrogenosome/mitochondria attributes. These diverse organelles have lost the major, at one time, definitive components of the mitochondrion (tricarboxylic cycle enzymes and cytochromes), however they all contain the machinery for the assembly of Fe-S clusters, which is the single unifying feature they share. The plasticity of organelles, like the mitochondrion, is therefore evident from its ability to lose its identity as an aerobic energy generating powerhouse while retaining key ancestral functions common to both aerobes and anaerobes. It is interesting to note that the apicoplast, a non-photosynthetic plastid that is present in all apicomplexan protozoa, apart from Cryptosporidium and possibly the gregarines, is also the site of Fe-S cluster assembly proteins. It turns out that in Cryptosporidium proteins involved in Fe-S cluster biosynthesis are localized in the mitochondrial remnant organelle termed the mitosome. Hence, different organisms have solved the same problem of packaging a life-requiring set of reactions in different ways, using different ancestral organelles, discarding what is not needed and keeping what is essential. Don't judge an organelle by its cover, more by the things it does, and always be prepared for surprises.

Peptide Mimics of the Cysteine-Rich Regions of HapX and SreA Bind a [2Fe-2S] Cluster In Vitro.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.

Iron­sulfur clusters in viral proteins: Exploring their elusive nature, roles and new avenues for targeting infections.

Viruses have evolved complex mechanisms to exploit host factors for replication and assembly. In response, host cells have developed strategies to block viruses, engaging in a continuous co-evolutionary battle. This dynamic interaction often revolves around the competition for essential resources necessary for both host cell and virus replication. Notably, iron, required for the biosynthesis of several cofactors, including iron­sulfur (FeS) clusters, represents a critical element in the ongoing competition for resources between infectious agents and host. Although several recent studies have identified FeS cofactors at the core of virus replication machineries, our understanding of their specific roles and the cellular processes responsible for their incorporation into viral proteins remains limited. This review aims to consolidate our current knowledge of viral components that have been characterized as FeS proteins and elucidate how viruses harness these versatile cofactors to their benefit. Its objective is also to propose that viruses may depend on incorporation of FeS cofactors more extensively than is currently known. This has the potential to revolutionize our understanding of viral replication, thereby carrying significant implications for the development of strategies to target infections.

Chaperone function in Fe-S protein biogenesis: Three possible scenarios.

Among the six known iron­sulfur (FeS) cluster biogenesis machineries that function across all domains of life only one involves a molecular chaperone system. This machinery, called ISC for 'iron sulfur cluster', functions in bacteria and in mitochondria of eukaryotes including humans. The chaperone system - a dedicated J-domain protein co-chaperone termed Hsc20 and its Hsp70 partner - is essential for proper ISC machinery function, interacting with the scaffold protein IscU which serves as a platform for cluster assembly and subsequent transfer onto recipient apo-proteins. Despite many years of research, surprisingly little is known about the specific role(s) that the chaperones play in the ISC machinery. Here we review three non-exclusive scenarios that range from involvement of the chaperones in the cluster transfer to regulation of the cellular levels of IscU itself.

Harnessing iron­sulfur enzymes for synthetic biology.

Reactions catalysed by iron-sulfur (Fe-S) enzymes appear in a variety of biosynthetic pathways that produce valuable natural products. Harnessing these biosynthetic pathways by expression in microbial cell factories grown on an industrial scale would yield enormous economic and environmental benefits. However, Fe-S enzymes often become bottlenecks that limits the productivity of engineered pathways. As a consequence, achieving the production metrics required for industrial application remains a distant goal for Fe-S enzyme-dependent pathways. Here, we identify and review three core challenges in harnessing Fe-S enzyme activity, which all stem from the properties of Fe-S clusters: 1) limited Fe-S cluster supply within the host cell, 2) Fe-S cluster instability, and 3) lack of specialized reducing cofactor proteins often required for Fe-S enzyme activity, such as enzyme-specific flavodoxins and ferredoxins. We highlight successful methods developed for a variety of Fe-S enzymes and electron carriers for overcoming these difficulties. We use heterologous nitrogenase expression as a grand case study demonstrating how each of these challenges can be addressed. We predict that recent breakthroughs in protein structure prediction and design will prove well-suited to addressing each of these challenges. A reliable toolkit for harnessing Fe-S enzymes in engineered metabolic pathways will accelerate the development of industry-ready Fe-S enzyme-dependent biosynthesis pathways.

Fe/S proteins in microbial sulfur oxidation.

Iron-sulfur clusters serve as indispensable cofactors within proteins across all three domains of life. Fe/S clusters emerged early during the evolution of life on our planet and the biogeochemical cycle of sulfur is one of the most ancient and important element cycles. It is therefore no surprise that Fe/S proteins have crucial roles in the multiple steps of microbial sulfur metabolism. During dissimilatory sulfur oxidation in prokaryotes, Fe/S proteins not only serve as electron carriers in several steps, but also perform catalytic roles, including unprecedented reactions. Two cytoplasmic enzyme systems that oxidize sulfane sulfur to sulfite are of particular interest in this context: The rDsr pathway employs the reverse acting dissimilatory sulfite reductase rDsrAB as its key enzyme, while the sHdr pathway utilizes polypeptides resembling the HdrA, HdrB and HdrC subunits of heterodisulfide reductase from methanogenic archaea. Both pathways involve components predicted to bind unusual noncubane Fe/S clusters acting as catalysts for the formation of disulfide or sulfite. Mapping of Fe/S cluster machineries on the sulfur-oxidizing prokaryote tree reveals that ISC, SUF, MIS and SMS are all sufficient to meet the Fe/S cluster maturation requirements for operation of the sHdr or rDsr pathways. The sHdr pathway is dependent on lipoate-binding proteins that are assembled by a novel pathway, involving two Radical SAM proteins, namely LipS1 and LipS2. These proteins coordinate sulfur-donating auxiliary Fe/S clusters in atypical patterns by three cysteines and one histidine and act as lipoyl synthases by jointly inserting two sulfur atoms to an octanoyl residue. This article is part of a Special Issue entitled: Biogenesis and Function of Fe/S proteins.

When iron and sulfur met on an anoxic planet and eventually made clusters essential for life.

[FeS] clusters are co-factors that are essential for life and are synthesized by dedicated multiprotein cellular machineries. In this review, we present the current scenario for the emergence and the diversification of the [FeS] cluster biosynthesis machineries. In addition to well-known NIF, ISC and SUF machineries, two alternative minimal systems, SMS, and MIS, were recently identified. Taxonomic distribution and phylogeny analyses indicate that SMS and MIS were present in the Last Universal Common Ancestor (LUCA), well before the increase of oxygen on Earth. ISC, SUF and NIF systems emerged later in the history of life. The possible reasons for the emergence and diversification of these machineries are discussed.

Shared functions of Fe-S cluster assembly and Moco biosynthesis.

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In the recent years it has become evident that the availability of Fe-S clusters play an important role for the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is an enzyme involved in the transfer of sulfur to various acceptor proteins with a main role in the assembly of Fe-S clusters. In this review, we dissect the dependence of the production of active molybdoenzymes in detail, starting from the regulation of gene expression and further explaining sulfur delivery and Fe-S cluster insertion into target enzymes. Further, Fe-S cluster assembly is also linked to iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, we explain that the expression of the genes is dependent on an active FNR protein. FNR is a very important transcription factor that represents the master-switch for the expression of target genes in response to anaerobiosis. Moco biosynthesis is further directly dependent on the presence of ArcA and also on an active Fur protein.

Mitochondria function in cytoplasmic FeS protein biogenesis.

Iron­sulfur (FeS) clusters are cofactors of numerous proteins involved in essential cellular functions including respiration, protein translation, DNA synthesis and repair, ribosome maturation, anti-viral responses, and isopropylmalate isomerase activity. Novel FeS proteins are still being discovered due to the widespread use of cryogenic electron microscopy (cryo-EM) and elegant genetic screens targeted at protein discovery. A complex sequence of biochemical reactions mediated by a conserved machinery controls biosynthesis of FeS clusters. In eukaryotes, a remarkable epistasis has been observed: the mitochondrial machinery, termed ISC (Iron-Sulfur Cluster), lies upstream of the cytoplasmic machinery, termed CIA (Cytoplasmic Iron­sulfur protein Assembly). The basis for this arrangement is the production of a hitherto uncharacterized intermediate, termed X-S or (Fe-S)int, produced in mitochondria by the ISC machinery, exported by the mitochondrial ABC transporter Atm1 (ABCB7 in humans), and then utilized by the CIA machinery for the cytoplasmic/nuclear FeS cluster assembly. Genetic and biochemical findings supporting this sequence of events are herein presented. New structural views of the Atm1 transport phases are reviewed. The key compartmental roles of glutathione in cellular FeS cluster biogenesis are highlighted. Finally, data are presented showing that every one of the ten core components of the mitochondrial ISC machinery and Atm1, when mutated or depleted, displays similar phenotypes: mitochondrial and cytoplasmic FeS clusters are both rendered deficient, consistent with the epistasis noted above.