Biological metals are vital trace elements required by metalloproteins, which are involved in virtually every cellular, structural, and catalytic function of the bacterial cell. Bacterial pathogenesis involves a tug-of-war between the host's nutritional immunity sequestering essential metals and the invading pathogens that deploy adapted high-metal affinity uptake strategies, such as metallophores, in order to efficiently circumvent these defense mechanisms. Pseudopaline is a metallophore produced and secreted by Pseudomonas aeruginosa to acquire zinc when the bioavailability of this metal is severely restricted, as in the presence of a strong metal chelator such as EDTA, or during infections when the nutritional immunity of the host is active. We show that when facing strong metal chelation, the general Znu zinc uptake pathway becomes ineffective and only the pseudopaline pathway is capable of supplying the bacteria with the necessary zinc to maintain their growth, establishing that the pseudopaline pathway is the last-resort pathway for the bacteria to acquire zinc under such restricted growth conditions. Based on this statement, the present study explores the pleiotropic role of pseudopaline-mediated zinc acquisition on clinically relevant phenotypes such as biofilm formation and associated antibiotic tolerance, as well as its capacity to determine infection outcomes using cell-culture and murine models. The expression of pseudopaline-dependent phenotypes in such a diversity of biological contexts demonstrates the essentiality of this specific metal uptake system for P. aeruginosa pathogenicity during infection. We therefore identify this machinery as a promising therapeutic target for P. aeruginosa infections.

The biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) typically relies on the interaction of a leader sequence at the N-terminus of the precursor peptide and a RiPP precursor recognition element (RRE). Bufferins are a newly discovered family of bacterial RiPP metallophores featuring 5-thiooxazole motifs, which are installed by a multinuclear nonheme iron-dependent oxidative enzyme (MNIO) together with its partner protein. As an original feature, bufferin precursors possess Sec-dependent signal peptides. Using a model system from Caulobacter vibrioides, this work investigated key features of bufferin biosynthesis. Biophysical characterization demonstrated the binding of the signal peptide to modification enzymes, and structural modeling showed that the interaction is via the RRE domain. This implies that the signal peptide doubles up as a leader sequence. Mutagenesis in the native C. vibrioides host indicated that the bufferin signal peptide mediates optimal coupling of post-translational modification and export to ensure bufferin function in vivo and that its composition is key in this process. Using heterologous expression in Escherichia coli, where export is bypassed, the substrate scope of the MNIO/partner pair was probed, allowing us to define features of non-native substrates. This study provides a paradigm for signal-peptide-dependent RiPP biogenesis and paves the way for bioengineering of thiooxazole-containing peptides.

Copper is an essential metal notably found in respiration complexes for its redox properties. It is also toxic hence its cellular trafficking is tightly controlled. Bacteria have developed a number of defense systems against copper excess, but its acquisition pathways remain poorly characterized. Ubiquitous in Gram-negative bacteria, TonB-dependent transporters (TBDTs) are outer membrane β-barrel proteins that mediate the proton motive force-dependent import of various nutrients to the periplasm. Here, we characterized a TBDT that imports copper in the whooping cough agent Bordetella pertussis, CrtABp (formerly BfrG), which is a prototype of a new subfamily of TBDTs. Our data indicate that CrtABp is dedicated to the import of copper for heme-copper respiratory oxidoreductases. We revealed that CrtABp imports chelated rather than free copper, solved the crystal structure of CrtABp and identified a conserved ligand binding site. By combining bacterial growth experiments, biophysical approaches and AlphaFold3 modeling we sketched out the features of copper-ligand complexes for CrtABp. In contrast with ferrisiderophore-specific TBDTs, no high-affinity chalkophore ligand of CrtABp could be identified, implying two nonmutually exclusive models. In the host, CrtABp might use a xenometallophore produced by another species present in the same niche to acquire copper. In vitro however, CrtA appears not to have high-affinity ligands but to import copper chelated by small molecules notably harboring carboxylate groups, which might represent a paradigm of ‘scavenger’ TBDTs with low ligand selectivity. We identified an essential, invariant histidine residue that might serve as a selectivity filter for copper-chelate complexes.

Plant acclimation is a growing scientific concept, at molecular, cellular and global scales. All photosynthetic organisms that created an oxic atmosphere on earth possess a gene of unknown function "Acclimation of Photosynthesis to the Environment 1". Here we show that APE1 encodes a thylakoid-bound protein with a unique motif that binds copper and detoxifies the superoxide anion radical, O2•-. Maturation of the recombinant APE1 protein from Chlamydomonas reinhardtii requires formation of cysteine disulfide bonds after copper binding or via a high affinity interaction with a copper chaperone (Plastid Copper Chaperone 1) that boosts its scavenging capacity for O2•-. APE1 co-occurs in evolution with Photosystem II oxygen evolving proteins and it is the archaic O2•- detoxifying enzyme for acclimating photosynthesis to an oxygenic environment.

Yersinopine, a nicotianamine-like metallophore, was recently identified through biochemical analyses, but its in vivo production and functional role remain uncharacterized. In Yersinia pseudotuberculosis and its recent descendant Yersinia pestis, the cnt operon (cntPQRLMI) putatively encodes the biosynthesis and transport of yersinopine. In Y. pestis, however, two frameshift mutations disrupt cntQ, which encodes the predicted permease for yersinopine-metal complexes. This pseudogenization raises critical questions about the functional relevance of yersinopine in these closely related species. Here, we show that cnt operon expression is repressed by the zinc uptake regulator Zur and that both Y. pestis and Y. pseudotuberculosis secrete yersinopine under zinc-limited conditions. Unexpectedly, the operon mediates iron uptake in Y. pseudotuberculosis but supports zinc acquisition in Y. pestis. Moreover, targeted disruption of cntQ in Y. pseudotuberculosis shifts metal specificity from iron to zinc, mimicking the Y. pestis phenotype. Collectively, our results suggest that a single pseudogenization event could rewire metal uptake specificity. Our findings illustrate how evolutionary genome reduction can reshape bacterial physiology.

Copper is typically coordinated by histidine, cysteine, or methionine in proteins, and these residues are particularly sensitive to oxidation. However, it remains unclear whether copper-coordinating residues are more prone to oxidation than non-coordinating ones, and how their susceptibility changes between the apo and copper-bound states. The copper chaperone PcuC, important for cytochrome c oxidase assembly in bacteria, contains a canonical binding site composed of two histidines and two methionines (H51xnM63x22H86xM88), as well as a disordered C-terminal extension enriched in methionine and histidine. To quantify methionine oxidation sensitivity in both apo- and Cu-bound PcuC, we used a methionine-specific oxaziridine probe combined with mass spectrometry and compared labeling patterns to those generated by 18O-labeled hydrogen peroxide. We show that methionine residues display distinct oxidation sensitivities in the apoprotein, and that the oxaziridine reacts similarly to H218O2. Importantly, this probe enables quantification of methionine oxidation independently of hydroxyl radicals generated by copper-driven Fenton chemistry, which lacks residue specificity. In the copper-bound form, Cu binding strongly alters methionine reactivity, with a marked increase in oxidation of the coordinating Met63 and Met88. Structural analysis revealed that two copper ions occupy the canonical site, while the C-terminal extension does not contribute to coordination. Comparison of structural features and oxidation values showed that methionine sensitivity correlates with solvent exposure in the folded domain, but with local positive charge in the disordered region. These findings demonstrate that copper coordination modulates methionine oxidation, and that oxaziridine-based probes provide powerful tools for mapping oxidation sensitivity in (metallo)proteins.

Clusters bound by weak, non-covalent forces, such as van der Waals interactions and hydrogen bonds, are ubiquitous in dilute media ranging from aerosols to molecular fluids and biological structures, their interest being not only fundamental as in astrochemistry but also more applied as in organic electronics. Neutral clusters of up to six 1-hexene molecules produced by supersonic expansion of a gas mixture were ionized, mass selected, and spectroscopically characterized using synchrotron-based VUV photoelectron photoion coincidence technique. Ionization energies inferred from these measurements show decreasing trends as the cluster size increases, by about 0.5 eV over the range of 1–6 molecules. Dedicated theoretical DFT-based calculations were performed to unravel the possible structures of these clusters and determine their vertical and adiabatic ionization energies. Our computational search for stable structures considered the possible chirality effects associated with most conformers of the monomer having enantiomers, in an approach with a broad structural sampling employing classical force fields followed by systematic re-optimization using an efficient quantum chemical method. Vertical and adiabatic ionization energies obtained using wavefunction-based methods exhibit significant dispersion due to conformational flexibility already in the monomer, but these effects are magnified in clusters due to their fluxionality at the experimental temperature of about 130 K. Overall, the trends obtained for the calculated vertical ionization energies agree well with the measured data and suggest that possible chiral recognition effects that could stabilize specific structures are likely to be hampered under the present experimental conditions.

Yersinopine is a nicotianamine-like metallophore recently inferred from a biochemical study, however its production in vivo and functional role have not been evaluated. Intriguingly, the Yersinia pestis cnt operon ( cntPQRLMI ) encoding yersinopine biosynthesis and transport (with cntPQR encoding a predicted ABC transport system, cntLM the two biosynthetic enzymes and cntI the predicted yersinopine exporter) shows two frameshift mutations in the cntQ gene encoding the permease, whereas this gene appears intact in Yersinia pseudotuberculosis . This pseudogenization, which occurred during the emergence of Y. pestis from Y. pseudotuberculosis , questions the role of yersinopine (if any) in both species. Here we show that yersinopine is secreted by both Y. pestis and Y. pseudotuberculosis in metal scarce conditions, and expression of the operon is repressed by the zinc uptake regulator (Zur). Surprisingly, the cnt operon was found to be involved in iron uptake in Y. pseudotuberculosis whereas it played a role in zinc acquisition in Y. pestis . Furthermore, mutation of cntQ in Y. pseudotuberculosis triggered a switch from iron import to zinc import, therefore recapitulating the phenotype observed in Y. pestis . This work demonstrates the production of yersinopine in two closely related bacteria in zinc scarce conditions, and highlights the effect of a pseudogenization event triggering a global change in metal uptake specificity. ### Competing Interest Statement The authors have declared no competing interest.

Strict management of intracellular heme pools, which are both toxic and beneficial, is crucial for bacterial survival during infection. The human pathogen Staphylococcus aureus uses a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme-sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, a heme-binding site is present in the HssS dimer between the membrane and extracellular domains. Heme is embedded in the membrane bilayer with its two protruding porphyrin propionates interacting with two conserved Arg94 and Arg163 that are located extracellularly. Single substitutions of these arginines and two highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic pocket limited the ability of HssS to induce HrtBA synthesis. Combination of the four substitutions abolished HssS activation. Wild-type (WT) HssS copurified with heme from Escherichia coli, whereas heme binding was strongly attenuated in the variants. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation and induce HrtBA-mediated heme extrusion from the membrane. This "gatekeeper" mechanism could limit intracellular diffusion of exogenous heme in S. aureus and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors. IMPORTANCE In the host blood, pathogenic bacteria are exposed to the red pigment heme that concentrates in their lipid membranes, generating cytotoxicity. To overcome heme toxicity, Staphylococcus aureus expresses a membrane sensor protein, HssS. Activation of HssS by heme triggers a phosphotransfer mechanism leading to the expression of a heme efflux system, HrtBA. This detoxification system prevents intracellular accumulation of heme. Our structural and functional data reveal a heme-binding hydrophobic cavity in HssS within the transmembrane domains (TM) helices at the interface with the extracellular domain. This structural pocket is important for the function of HssS as a heme sensor. Our findings provide a new basis for the elucidation of pathogen-sensing mechanisms as a prerequisite to the discovery of inhibitors.

Bacterial genomes are a huge reservoir of genes encoding J-domain protein co-chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J-domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C-terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J-domain proteins that (i) are found in several environmental, mostly aquatic, β- or ɣ-proteobacteria and (ii) contain a conserved PX7 W motif in their C-terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX7 W motif determines the strong AtcJ-AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low-temperature growth and, given the variation in the atc operons, possibly for other biological functions.

Uranium is a naturally occurring radionuclide. Its redistribution, primarily due to human activities, can have adverse effects on human and non-human biota, which poses environmental concerns. The molecular mechanisms of uranium tolerance and the cellular response induced by uranium exposure in bacteria are not yet fully understood. Here, we carried out a comparative analysis of four actinobacterial strains isolated from metal and radionuclide-rich soils that display contrasted uranium tolerance phenotypes. Comparative proteogenomics showed that uranyl exposure affects 39–47% of the total proteins, with an impact on phosphate and iron metabolisms and membrane proteins. This approach highlighted a protein of unknown function, named UipA, that is specific to the uranium-tolerant strains and that had the highest positive fold-change upon uranium exposure. UipA is a single-pass transmembrane protein and its large C-terminal soluble domain displayed a specific, nanomolar binding affinity for UO22+ and Fe3+. ATR-FTIR and XAS-spectroscopy showed that mono and bidentate carboxylate groups of the protein coordinated both metals. The crystal structure of UipA, solved in its apo state and bound to uranium, revealed a tandem of PepSY domains in a swapped dimer, with a negatively charged face where uranium is bound through a set of conserved residues. This work reveals the importance of UipA and its PepSY domains in metal binding and radionuclide tolerance.

Spectroscopic investigation of membrane proteins in their native environment is a challenging task. Earlier we demonstrated the feasibility to measure precise distances within outer membrane proteins in E. coli and native membranes using methanethiosulfonate (MTS) functionalized labels combined with pulsed electron-electron double resonance spectroscopy. Here we show the application of maleimide functionalized Gd(III), nitroxide, and trityl labels for in situ distance measurement using the cobalamin transporter BtuB. These labels enabled distance measurements for BtuB in E. coli and native outer membranes and in the membranes maleimide-Gd-DOTA also is effective. Further, we show that the observable dipolar evolution time can be significantly prolonged in the native environments using the Carr-Purcell 5-pulse electron double resonance sequence. For a nitroxide-nitroxide pair, application of sech/tanh inversion pulses substantially suppressed the 4-pulse artifact at the Q-band frequency. In the case of a nitroxide-trityl pair, Gaussian pump pulses of varying amplitude are sufficient to suppress the artifact to the typical noise level. The feasibility of a range of bioresistant spin labels and the 5-pulse electron double resonance offers promising tools for investigating heterooligomeric membrane protein complexes in their native environment.

Metal sulfides are a common group of extracellular bacterial biominerals. Only few cases of intracellular biomineralization have been reported in this group, mostly limited to greigite (Fe3S4) in magnetotactic bacteria. Here, we report the intracellular but periplasmic biomineralization of copper sulfide by the magnetotactic bacterium Desulfamplus magnetovallimortis (strain BW-1) that is known to mineralize greigite and magnetite (Fe3O4) in the cytoplasm. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide that remains in a thermodynamically unstable state. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization.

Enterococcus faecalis is a commensal Gram-positive pathogen found in the intestines of mammals and is also a leading cause of severe infections occurring mainly among antibiotic-treated dysbiotic hospitalized patients. Like most intestinal bacteria, E. faecalis does not synthesize heme (in this report, heme refers to iron protoporphyrin IX regardless of the iron redox state). Nevertheless, environmental heme can improve E. faecalis fitness by activating respiration metabolism and a catalase that limits hydrogen peroxide stress. Since free heme also generates toxicity, its intracellular levels need to be strictly controlled. Here, we describe a unique transcriptional regulator, FhtR (named FhtR for faecalis heme transport regulator), which manages heme homeostasis by controlling an HrtBA-like efflux pump (named HrtBAEf for the HrtBA from E. faecalis). We show that FhtR, by managing intracellular heme concentration, regulates the functional expression of the heme-dependent catalase A (KatA), thus participating in heme detoxification. The biochemical features of FhtR binding to DNA, and its interaction with heme that induces efflux, are characterized. The FhtR-HrtBAEf system is shown to be relevant in a mouse intestinal model. We further show that FhtR senses heme from blood and hemoglobin but also from crossfeeding by Escherichia coli. These findings bring to light the central role of heme sensing by FhtR in response to heme fluctuations within the gastrointestinal tract, which allow this pathogen to limit heme toxicity while ensuring expression of an oxidative defense system. IMPORTANCE Enterococcus faecalis, a normal and harmless colonizer of the human intestinal flora can cause severe infectious diseases in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote its resistance and its virulence. E. faecalis, which cannot synthesize heme, an essential but toxic metabolite, needs to scavenge this molecule from the host to respire and fight stress generated by oxidants. Here, we report a new mechanism used by E. faecalis to sense heme and trigger the synthesis of a heme efflux pump that balances the amount of heme inside the bacteria. We show in a mouse model that E. faecalis uses this mechanisms within the gastrointestinal tract.

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