Using a chemical genetic screen to enhance our understanding of the antimicrobial properties of copper

The competitive toxic and stress inducing nature of copper necessitates systems that sequester and export this metal from the cytoplasm of bacterial cells. Several predicted mechanisms of toxicity include the production of reactive oxygen species, thiol depletion, DNA and iron-sulfur cluster disruption. Accompanying these mechanisms include pathways of homeostasis such as chelation, oxidation, and transport. Still, the mechanisms of copper resistance and sensitivity are not fully understood. Furthermore, studies fail to recognize that the response to copper is likely a result of numerous mechanisms, as in the case for homeostasis, in which proteins and enzymes work as a collective to maintain appropriate copper concentrations. In this study we used the Keio collection, an array of 3985 Escherichia coli mutants, each with a deleted non-essential gene, to gain a better understanding of prolonged copper exposure. In short, we recovered two copper homeostatic gene and genes involved in transporting and assembling to be involved in mediating prolonged copper stress under the conditions assessed. The gene coding for the protein TolC was uncovered as a sensitive hit and we demonstrated that tolC, an outer membrane efflux channel, is key in mitigating copper sensitivity. Additionally, the activity of tRNA processing was enriched and the deletion of several proteins involved in import generated copper tolerance. Lastly, key genes belonging to central carbon metabolism and nicotinamide adenine dinucleotide biosynthesis were uncovered as tolerant hits. Overall, this study shows that copper sensitivity and tolerance are a result of numerous mechanisms acting in combination within the cell.

Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

Electron-bifurcation is a fundamental energy conservation mechanism in nature. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic primarily due to the lack of structural information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure is a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC heterotrimers electrically connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and proton reduction. Symmetry expansion identified two conformations of a flexible iron-sulfur cluster domain: a “closed bridge” and an “open bridge” conformation, where a Zn2+ site may act as a “hinge” allowing domain movement. Based on these structural revelations, we propose two new mechanisms of electron-bifurcation in HydABC.

Modulation of MagR magnetic properties via iron–sulfur cluster binding

Iron–sulfur clusters are essential cofactors found in all kingdoms of life and play essential roles in fundamental processes, including but not limited to respiration, photosynthesis, and nitrogen fixation. The chemistry of iron–sulfur clusters makes them ideal for sensing various redox environmental signals, while the physics of iron–sulfur clusters and its host proteins have been long overlooked. One such protein, MagR, has been proposed as a putative animal magnetoreceptor. It forms a rod-like complex with cryptochromes (Cry) and possesses intrinsic magnetic moment. However, the magnetism modulation of MagR remains unknown. Here in this study, iron–sulfur cluster binding in MagR has been characterized. Three conserved cysteines of MagR play different roles in iron–sulfur cluster binding. Two forms of iron–sulfur clusters binding have been identified in pigeon MagR and showed different magnetic properties: [3Fe–4S]-MagR appears to be superparamagnetic and has saturation magnetization at 5 K but [2Fe–2S]-MagR is paramagnetic. While at 300 K, [2Fe–2S]-MagR is diamagnetic but [3Fe–4S]-MagR is paramagnetic. Together, the different types of iron–sulfur cluster binding in MagR attribute distinguished magnetic properties, which may provide a fascinating mechanism for animals to modulate the sensitivity in magnetic sensing.

N-terminal tyrosine of ISCU2 triggers [2Fe-2S] cluster synthesis by ISCU2 dimerization

Synthesis of iron-sulfur (Fe/S) clusters in living cells requires scaffold proteins for both facile synthesis and subsequent transfer of clusters to target apoproteins. The human mitochondrial ISCU2 scaffold protein is part of the core ISC (iron-sulfur cluster assembly) complex that synthesizes a bridging [2Fe-2S] cluster on dimeric ISCU2. Initial iron and sulfur loading onto monomeric ISCU2 have been elucidated biochemically, yet subsequent [2Fe-2S] cluster formation and dimerization of ISCU2 is mechanistically ill-defined. Our structural, biochemical and cell biological experiments now identify a crucial function of the universally conserved N-terminal Tyr35 of ISCU2 for these late reactions. Mixing two, per se non-functional ISCU2 mutant proteins with oppositely charged Asp35 and Lys35 residues, both bound to different cysteine desulfurase complexes NFS1-ISD11-ACP, restores wild-type ISCU2 maturation demonstrating that ionic forces can replace native Tyr-Tyr interactions during dimerization-induced [2Fe-2S] cluster formation. Our studies define the essential mechanistic role of Tyr35 in the reaction cycle of de novo mitochondrial [2Fe-2S] cluster synthesis.

Iron, Heme Synthesis and Erythropoietic Porphyrias: A Complex Interplay

Erythropoietic porphyrias are caused by enzymatic dysfunctions in the heme biosynthetic pathway, resulting in porphyrins accumulation in red blood cells. The porphyrins deposition in tissues, including the skin, leads to photosensitivity that is present in all erythropoietic porphyrias. In the bone marrow, heme synthesis is mainly controlled by intracellular labile iron by post-transcriptional regulation: translation of ALAS2 mRNA, the first and rate-limiting enzyme of the pathway, is inhibited when iron availability is low. Moreover, it has been shown that the expression of ferrochelatase (FECH, an iron-sulfur cluster enzyme that inserts iron into protoporphyrin IX to form heme), is regulated by intracellular iron level. Accordingly, there is accumulating evidence that iron status can mitigate disease expression in patients with erythropoietic porphyrias. This article will review the available clinical data on how iron status can modify the symptoms of erythropoietic porphyrias. We will then review the modulation of heme biosynthesis pathway by iron availability in the erythron and its role in erythropoietic porphyrias physiopathology. Finally, we will summarize what is known of FECH interactions with other proteins involved in iron metabolism in the mitochondria.

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention.

NorA, HmpX, and NorB cooperate to reduce NO toxicity during denitrification and plant pathogenesis in Ralstonia solanacearum

Ralstonia solanacearum, which causes bacterial wilt disease of many crops, needs denitrifying respiration to succeed in hypoxic plant xylem vessels. Inside its host this pathogen confronts toxic oxidative radicals like nitric oxide (NO) generated by both bacterial denitrification and host defenses. R. solanacearum has multiple distinct mechanisms that could mitigate this stress, including Repair of Iron Cluster (RIC) homolog NorA, nitric oxide reductase NorB, and flavohaemoglobin HmpX. R. solanacearum upregulated norA, norB, and hmpX in response to exogenous NO, denitrification, and tomato pathogenesis. Single mutants lacking any of these genes accumulated NO during denitrification and were more susceptible to oxidative stress. Plant defense genes were upregulated in tomatoes infected with the NO-overproducing ΔnorB mutant, suggesting bacterial detoxification of NO reduces pathogen visibility. Expression of many iron and sulfur metabolism genes increased in the ΔnorB, ΔnorA, and ΔhmpX mutants, suggesting that losing even one NO detoxification system demands metabolic compensation. Single mutants suffered only moderate fitness reductions in host plants, possibly because they upregulated their remaining detoxification genes. However, ΔnorA/norB, ΔnorB/hmpX, and ΔnorA/hmpX double mutants grew poorly in denitrifying culture and in planta. Loss of norA, norB, and hmpX may be lethal as the methods used to construct the double mutants did not generate a triple mutant. Aconitase activity assays showed that NorA, HmpX and especially NorB are important for maintaining iron-sulfur cluster proteins. Thus, R. solanacearum's three NO detoxification systems each contribute to and are collectively essential for overcoming oxidative stress during denitrification and growth in a host plant.

Minor Alterations in Core Promoter Element Positioning Reveal Functional Plasticity of a Bacterial Transcription Factor

Transcription regulation is a key process in all living organisms, involving a myriad of transcription factors. In E. coli , the regulator of the iron-sulfur cluster biogenesis pathway, IscR, acts as a global transcription factor, activating the transcription of some pathways and repressing others.

Sulfur Administration in Fe–S Cluster Homeostasis

Mitochondria are the key organelles of Fe–S cluster synthesis. They contain the enzyme cysteine desulfurase, a scaffold protein, iron and electron donors, and specific chaperons all required for the formation of Fe–S clusters. The newly formed cluster can be utilized by mitochondrial Fe–S protein synthesis or undergo further transformation. Mitochondrial Fe–S cluster biogenesis components are required in the cytosolic iron–sulfur cluster assembly machinery for cytosolic and nuclear cluster supplies. Clusters that are the key components of Fe–S proteins are vulnerable and prone to degradation whenever exposed to oxidative stress. However, once degraded, the Fe–S cluster can be resynthesized or repaired. It has been proposed that sulfurtransferases, rhodanese, and 3-mercaptopyruvate sulfurtransferase, responsible for sulfur transfer from donor to nucleophilic acceptor, are involved in the Fe–S cluster formation, maturation, or reconstitution. In the present paper, we attempt to sum up our knowledge on the involvement of sulfurtransferases not only in sulfur administration but also in the Fe–S cluster formation in mammals and yeasts, and on reconstitution-damaged cluster or restoration of enzyme’s attenuated activity.