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 (MS) approach to analyse 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.

Encapsulins are self-assembling nano-compartments that naturally occur in bacteria and archaea. These nano-compartments encapsulate cargo proteins that bind to the shell\'s interior through specific recognition sequences and perform various metabolic processes. Encapsulation enables organisms to perform chemical reactions without exposing the rest of the cell to potentially harmful substances while shielding cargo molecules from degradation and other adverse effects of the surrounding environment. One particular type of cargo protein, the ferritin-like protein (FLP), is the focus of this review. Encapsulated FLPs are members of the ferritin-like protein superfamily, and they play a crucial role in converting ferrous iron (Fe+2) to ferric iron (Fe+3), which is then stored inside the encapsulin in mineralized form. As such, FLPs regulate iron homeostasis and protect organisms against oxidative stress. Recent studies have demonstrated that FLPs have tremendous potential as biosensors and bioreactors because of their ability to catalyze the oxidation of ferrous iron with high specificity and efficiency. Moreover, they have been investigated as potential targets for therapeutic intervention in cancer drug development and bacterial pathogenesis. Further research will likely lead to new insights and applications for these remarkable proteins in biomedicine and biotechnology.

Klebsiella pneumoniae is an important opportunistic pathogen that causes a variety of infections. It is critical for bacteria to maintain metal homeostasis during infection. By using an isogenic mntP deletion mutant of K. pneumoniae strain NTUH-K2044, we found that MntP was a manganese efflux pump. Manganese increased the tolerance to oxidative stress, and oxidative stress could increase the intracellular manganese concentration. In oxidative stress, the mntP deletion mutant exhibited significantly higher sensitivity to manganese. Furthermore, iron could increase the tolerance of the mntP deletion mutant to manganese. Inductively coupled plasma mass spectrometry analysis revealed that the mntP deletion mutant had higher intracellular manganese and iron concentrations than wild-type and complementary strains. These findings suggested that iron could increase manganese tolerance in K. pneumoniae. This work elucidated the role of MntP in manganese detoxification and Mn/Fe homeostasis in K. pneumoniae.IMPORTANCEMetal homeostasis plays an important role during the process of bacterial infection. Herein, we revealed that MntP was involved in intracellular manganese homeostasis. Manganese promoted resistance to oxidative stress in Klebsiella pneumoniae. Furthermore, we demonstrated that the mntP deletion mutant exhibited significantly lower survival under manganese and H2O2 conditions. Oxidative stress increased the intracellular manganese content of the mntP deletion mutant. MntP played a critical role in maintaining intracellular manganese and iron concentrations. MntP contributed to manganese detoxification and Mn/Fe homeostasis in K. pneumoniae.

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function, and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations, they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the YS deletion mutants results from distinct enzymatic vulnerabilities.IMPORTANCEBacteria require multiple trace metal ions for survival. Metal homeostasis relies on the tightly regulated expression of metal uptake, storage, and efflux proteins. Metal intoxication occurs when metal homeostasis is perturbed and often results from enzyme mis-metalation. In Bacillus subtilis, Mn-dependent superoxide dismutase (MnSOD) is the most abundant Mn-containing protein and is important for oxidative stress resistance. Here, we report novel roles for MnSOD and a co-regulated membrane protein, YqgC, in Mn homeostasis. Loss of both MnSOD and YqgC (but not the individual proteins) prevents the efficient expression of Mn efflux proteins and leads to a large-scale perturbation of the metabolome due to inhibition of Mg-dependent enzymes, including key chorismate-utilizing MST (menaquinone, siderophore, and tryptophan) family enzymes.

Iron is an essential element for proper cell functioning, but unbalanced levels can cause cell death. Iron metabolism is controlled at the blood-tissue barriers provided by microvascular endothelial cells. Dysregulated iron metabolism at these barriers is a factor in both neurodegenerative and cardiovascular diseases. Mammalian iron efflux is mediated by the iron efflux transporter ferroportin (Fpn). Inflammation is a factor in many diseases and correlates with increased tissue iron accumulation. Evidence suggests treatment with interleukin 6 (IL-6) increases intracellular calcium levels and calcium is known to play an important role in protein trafficking. We have shown that calcium increases plasma membrane localization of the iron uptake proteins ZIP8 and ZIP14, but if and how calcium modulates Fpn trafficking is unknown. In this article, we examined the effects of IL-6 and calcium on Fpn localization to the plasma membrane. In HEK cells expressing a doxycycline-inducible GFP-tagged Fpn, calcium increased Fpn-GFP membrane presence by 2 h, while IL-6 increased membrane-localized Fpn-GFP by 3 h. Calcium pretreatment increased Fpn-GFP mediated 55Fe efflux from cells. Endoplasmic reticulum calcium stores were shown to be important for Fpn-GFP localization and iron efflux. Use of calmodulin pathway inhibitors showed that calcium signaling is important for IL-6-induced Fpn relocalization. Studies in brain microvascular endothelial cells in transwell culture demonstrated an initial increase in 55Fe flux with IL-6 that is reduced by 6 h coinciding with upregulation of hepcidin. Overall, this research details one pathway by which inflammatory signaling mediated by calcium can regulate iron metabolism, likely contributing to inflammatory disease mechanisms.

A new study by Nies et al. (J Bacteriol 206:e00080-24, 2024, https://doi.org/10.1128/jb.00080-24) provides a rich, quantitative data set of zinc accumulation by cells of Cupriavidus metallidurans, including of mutant bacterial strains lacking import or efflux genes, and comparison of zinc accumulation by cells previously starved of metal with those of zinc-replete cells. The data surprisingly demonstrate the concomitant activity of both active metal import and metal efflux systems. They present a flow equilibrium model to describe zinc homeostasis in bacteria.

Group B Streptococcus (GBS; also known as Streptococcus agalactiae) is an opportunistic bacterial pathogen that causes sepsis, meningitis, pneumonia, and skin and soft tissue infections in neonates and healthy or immunocompromised adults. GBS is well-adapted to survive in humans due to a plethora of virulence mechanisms that afford responses to support bacterial survival in dynamic host environments. These mechanisms and responses include counteraction of cell death from exposure to excess metal ions that can cause mismetallation and cytotoxicity, and strategies to combat molecules such as reactive oxygen and nitrogen species that are generated as part of innate host defence. Cytotoxicity from reactive molecules can stem from damage to proteins, DNA, and membrane lipids, potentially leading to bacterial cell death inside phagocytic cells or within extracellular spaces within the host. Deciphering the ways in which GBS responds to the stress of cytotoxic reactive molecules within the host will benefit the development of novel therapeutic and preventative strategies to manage the burden of GBS disease. This review summarizes knowledge of GBS carriage in humans and the mechanisms used by the bacteria to circumvent killing by these important elements of host immune defence: oxidative stress, nitrosative stress, and stress from metal ion intoxication/mismetallation.

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the yqgC-sodA deletion mutants results from distinct enzymatic vulnerabilities.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

The impact of particle size of engineered nanoparticles (ENPs) on plant response has marginally been investigated under the foliar application so far. Concerning the significance of particle diameter for their properties and interaction with plants, the effect of size should be considered in the analysis of the effect of micronutrient-based ENPs on plants. It is of particular importance for ENPs containing Cu due to plants needing a relatively low amount of this element, thus there is a risk of overdosing during application as a fertilizer or pesticide. Here, we examined the biochemical and transcriptional response of barley (Hordeum vulgare L.) to Cu nanoparticles (nano-Cu) with different diameters (25 nm, 50 nm, 70 nm), microparticles (micro-Cu), and chelated Cu (EDTA-Cu). The plants suffering from Cu deficiency were foliar sprayed with Cu compounds at 1000 mg/L during the tillering stage. 1- and 7-day plants were analyzed in terms of biomass, Cu content, the activity of enzymes involved with antioxidant response, the content of low molecular weight compounds, and the expression of genes regulated metal homeostasis, aquaporins, and defense. The results showed that the Cu leaf level was differentiated over time and after 7 days it was higher under exposure to the smallest nano-Cu than other particulate Cu. Regardless of the duration of exposure, the Cu content was highest in plants treated with Cu-EDTA. The cluster analysis of all markers revealed a clear distinct response to the smallest nano-Cu and other particulate and ionic treatments. The bigger nano-Cu, depending on the markers, caused the medium effects between the nano-Cu 25 nm and micro-Cu and Cu-EDTA. The found size thresholds at the nanoscale will be useful for the fabrication of safe-by-design agrochemicals to provide crop security and attenuate environmental impact.