In a state of flux: new insight into the transport processes that maintain bacterial metal homeostasis.

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.

The molecular mechanisms of the bacterial iron sensor IdeR.

Life came to depend on iron as a cofactor for many essential enzymatic reactions. However, once the atmosphere was oxygenated, iron became both scarce and toxic. Therefore, complex mechanisms have evolved to scavenge iron from an environment in which it is poorly bioavailable, and to tightly regulate intracellular iron contents. In bacteria, this is typically accomplished with the help of one key regulator, an iron-sensing transcription factor. While Gram-negative bacteria and Gram-positive species with low guanine-cytosine (GC) content generally use Fur (ferric uptake regulator) proteins to regulate iron homeostasis, Gram-positive species with high GC content use the functional homolog IdeR (iron-dependent regulator). IdeR controls the expression of iron acquisition and storage genes, repressing the former, and activating the latter in an iron-dependent manner. In bacterial pathogens such as Corynebacterium diphtheriae and Mycobacterium tuberculosis, IdeR is also involved in virulence, whereas in non-pathogenic species such as Streptomyces, it regulates secondary metabolism as well. Although in recent years the focus of research on IdeR has shifted towards drug development, there is much left to learn about the molecular mechanisms of IdeR. Here, we summarize our current understanding of how this important bacterial transcriptional regulator represses and activates transcription, how it is allosterically activated by iron binding, and how it recognizes its DNA target sites, highlighting the open questions that remain to be addressed.

Optimized porous carbon-fibre microelectrode for multiplexed, highly reproducible and repeatable detection of heavy metals in real water samples.

This work demonstrates the simultaneous identification of four hazardous heavy metals in water samples, namely copper, lead, cadmium, and mercury. A simple yet selective electrode with the simplest fabrication procedure was used. The modified porous carbon threads coated with gold nanoparticles (AuNPs) was employed as a working electrode. The surface chemistry and morphology of the AuNPs deposited porous carbon thread surface were examined. The electrocatalytic activity of the metals on the Au-modified thread surface was observed using the differential pulse voltammetry (DPV) technique. Furthermore, all four metal ions were detected simultaneously, and no interference was observed. Individual and simultaneous experiments to test the impact of concentration revealed that the limit of detection (LoD) was observed to be 1.126 µM, 1.419 µM, 0.966 µM, 0.736 µM for the Cd2+, Pb2+, Cu2+, and Hg2+ metal ions respectively in a linear concentration range of 10-110 µM of each. Subsequently, the study of pH, interference with coexisting metal ions, repeatability study, and stability analysis was also performed. A real sample analysis utilising three different lake water samples is also carried out to further understand the application of the proposed sensor. A good recovery rate is achieved, and the results are reported. This work paves way for the on-field applicability of the present heavy metal detection platform.

Electrochemical Nano-Roughening of Gold Microstructured Electrodes for Enhanced Sensing in Biofluids.

A key challenge for sensor miniaturization is to create electrodes with smaller footprints, while maintaining or increasing sensitivity. In this work, the electroactive surface of gold electrodes was enhanced 30-fold by wrinkling followed by chronoamperometric (CA) pulsing. Electron microscopy showed increased surface roughness in response to an increased number of CA pulses. The nanoroughened electrodes also showed excellent fouling resistance when submerged in solutions containing bovine serum albumin. The nanoroughened electrodes were used for electrochemical detection of Cu2+ in tap water and of glucose in human blood plasma. In the latter case, the nanoroughened electrodes allowed highly sensitive enzyme-free sensing of glucose, with responses comparable to those of two commercial enzyme-based sensors. We anticipate that this methodology to fabricate nanostructured electrodes can accelerate the development of simple, cost-effective, and high sensitivity electrochemical platforms.

Synthesis and Chemosensory Properties of New Cyanosubstituted 2,2'-Bipyridine Derivatives.

A series of novel 6-oxo-1,6-dihydro-[2,2'-bipyridine]-5-carbonitriles has been synthesized and characterized. Their photophysical properties in DMSO solution and aqueous medium as well as fluorescence response to the presence of metal ions have been investigated. The obtained 4-(4-methoxyphenyl)-6-oxo-1,6-dihydro-[2,2'-bipyridine]-5-carbonitrile has been shown as a selective fluorescent "turn-ON" probe for Cd2+ ions with LoD 0.359 µM, 1:1 metal-ligand ratio and binding constant of 5.2 × 104 M-1.

Photoluminescence quenching of thermally treated waste-derived carbon dots for selective metal ion sensing.

In the present study, carbon-dots (CDs) were derived from the thermal oxidation of an agricultural waste, bitter tea residue, to obtain different sp2/sp3 ratios and electronic structures for metal sensing. The CDs obtained from calcination at 700 °C exhibited the highest photoluminescence (PL) quantum yield (QY) of 11.8% among all the samples treated at different temperatures. These CDs had a high degree of graphitization, which resulted in a strong π-π* electron transition, and hence in a high QY. The strong photoluminescence of the CDs could be used to sense the metal ions Ag+, Sr2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, and Sn2+ by monitoring their PL intensity at an excitation wavelength of 320 nm. The metals inhibited the PL intensity in the order Ag+ > Fe2+, Fe3+, Ni2+ > Sr2+, Co2+, Cu2+, Sn2+, which demonstrated that the CDs exhibited high metal ion detection capability and selectivity. The detection of Fe3+ using CDs was performed in the range of 10-100 ppm with a LOD (limit of detection) value of 0.380 ppm. Theoretical calculations demonstrated that Ag+, Sr2+, and Sn2+ induced charge transfer excitation and that Fe2+ and Ni2+ induced d-d transitions via complexation with the sp2 clusters. The charge transfer excitation and d-d transitions hindered the π-π* transition of the sp2 clusters, leading to a quenching effect. On the other hand, Li+, Na+, and K+ ions did not alter the π-π* transition of the sp2 clusters, resulting in a negligible quenching effect. In summary, the oxidation level and electronic structure of CDs derived from bitter tea residue could be tailored, and the CDs were shown to be a facile, sustainable, and eco-friendly material for metal sensing.

Switching-On Fluorescence by Copper (II) and Basic Anions: A Case Study with a Pyrene-Functionalized Squaramide.

The new symmetric acyclic N,N'-bis(1-pyrenyl) squaramide (H2L) functionalized with the pyrene moiety as a fluorogenic fragment has been designed and its ability to selectively detect specific anions and metals investigated. H2L selectively binds Cl- both in solution (DMSO 0.5% H2O and MeCN) and in the solid state, and allows to selectively detect Cu2+ in MeCN with the formation of a 2:1 metal-receptor complex, with a green intense emission appreciable by naked eye under the UV lamp. The H2L copper complex preserves its emission properties in the presence of Cl-. The addition of basic anions (OH-, CN-, and F-) up to 10 equivalents caused the deprotonation of the squaramide NHs and a dramatic change of the emission properties of the H2L copper complex.

The copper-sensing transcription factor Mac1, the histone deacetylase Hst1, and nicotinic acid regulate de novo NAD+ biosynthesis in budding yeast.

NADH (NAD+) is an essential metabolite involved in various cellular biochemical processes. The regulation of NAD+ metabolism is incompletely understood. Here, using budding yeast (Saccharomyces cerevisiae), we established an NAD+ intermediate-specific genetic system to identify factors that regulate the de novo branch of NAD+ biosynthesis. We found that a mutant strain (mac1Δ) lacking Mac1, a copper-sensing transcription factor that activates copper transport genes during copper deprivation, exhibits increases in quinolinic acid (QA) production and NAD+ levels. Similar phenotypes were also observed in the hst1Δ strain, deficient in the NAD+-dependent histone deacetylase Hst1, which inhibits de novo NAD+ synthesis by repressing BNA gene expression when NAD+ is abundant. Interestingly, the mac1Δ and hst1Δ mutants shared a similar NAD+ metabolism-related gene expression profile, and deleting either MAC1 or HST1 de-repressed the BNA genes. ChIP experiments with the BNA2 promoter indicated that Mac1 works with Hst1-containing repressor complexes to silence BNA expression. The connection of Mac1 and BNA expression suggested that copper stress affects de novo NAD+ synthesis, and we show that copper stress induces both BNA expression and QA production. Moreover, nicotinic acid inhibited de novo NAD+ synthesis through Hst1-mediated BNA repression, hindered the reuptake of extracellular QA, and thereby reduced de novo NAD+ synthesis. In summary, we have identified and characterized novel NAD+ homeostasis factors. These findings will expand our understanding of the molecular basis and regulation of NAD+ metabolism.

Unusual Fluorescence Quenching-Based Al3+ Sensing by an Imidazolylpiperazine Derivative. ß-Cyclodextrin Encapsulation-Assisted Augmented Sensing.

We report in this paper an unusual ß-cyclodextrin mediated-aluminum (III) ion sensing based on augmented quenching of fluorescence. The fluorescent sensing of metal ions by a new ligand prepared (L = 4-[{4-(1H-imidazol-1-yl)phenyl]imino}methyl]piperazine-1-carboxaldehyde) has been investigated as well as the effect of the supramolecular complex formation with ß-CD. In aqueous solution, L shows an increase of fluorescence due to the interaction with ß-cyclodextrin with a formation constant of 77 (± 12) M-1. The ROESY NMR spectrum clearly indicates that L is encapsulated by ß-CD. Theoretical calculations show the possible structure both of the L-ß-CD adduct and of the coordination mode of Al3+ ion to L. In the presence of ß-CD, the piperazine adopts a distorted conformation. It leads to an enhanced Al3+ sensing by the compound in its supramolecular complexed form. The lower limit of detection of Al3+ ions is 6.00 × 10-8 mol L-1. This detection limit slightly expands for L in the presence of ß-CD.

Adaptor protein mediates dynamic pump assembly for bacterial metal efflux.

Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the periplasm and link inner and outer membranes, it remains unclear how they operate efficiently without compromising periplasmic plasticity. Combining single-molecule superresolution imaging and genetic engineering, we study in living Escherichia coli cells the tripartite efflux complex CusCBA of the resistance-nodulation-division family that is essential for bacterial resistance to drugs and toxic metals. We find that CusCBA complexes are dynamic structures and shift toward the assembled form in response to metal stress. Unexpectedly, the periplasmic adaptor protein CusB is a key metal-sensing element that drives the assembly of the efflux complex ahead of the transcription activation of the cus operon for defending against metals. This adaptor protein-mediated dynamic pump assembly allows the bacterial cell for efficient efflux upon cellular demand while still maintaining periplasmic plasticity; this could be broadly relevant to other multicomponent efflux systems.

In Vitro and in Cellulo Sensing of Transition Metals Using Time-Resolved Fluorescence Spectroscopy and Microscopy.

In this work we demonstrate that time domain techniques can be used successfully to monitor realtively weak modulations of the fluorescence in sensing applications. The metal sensing complex Newport Green DCF™ can detect selected transition metals in vivo as well as in vitro. Incremental addition of Ni and/or Zn (in vitro) lead to a substantial reduction in the yield of the fast component in a bi-exponential fluorescence decay (τ1 = 150-250 ps) from 60% to 30-35%. This is rationalised as an inhibition of intra-molecular electron transfer in the NPG sensing complex due to metal complexation. In order to explore this effect in cellulo, NIH 3 T3 mouse skin fibroplast cells were pre-incubated with set levels of Ni and Zn, at a constant concentration of NPG. The fluorescence modulation in cellullo was subsequently studied employing both time-resolved fluorescence microscopy and confocal fluorescence microscopy. In correlation with the in vitro observations, similar effects were observed on the fluorescence decay in cellulo.

Manganese and cobalt activate zebrafish ovarian cancer G-protein-coupled receptor 1 but not GPR4.

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) is activated by some metals in addition to extracellular protons and coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebrafish OGR1, zebrafish GPR4, and human GPR4 (zOGR1, zGPR4, and hGPR4, respectively) could sense the metals and activate the intracellular signaling pathways. On one hand, we found that only manganese and cobalt of the tested metals stimulated SRE-promoter activities in zOGR1-overexpressed HEK293T cells. On the other hand, none of the metals tested stimulated the promoter activities in zGPR4- and hGPR4-overexpressed cells. The OGR1 mutant (H4F), which is lost to activation by extracellular protons, did not stimulate metal-induced SRE-promoter activities. These results suggest that zOGR1, but not GPR4, is also a metal-sensing G-protein-coupled receptor in addition to a proton-sensing G-protein-coupled receptor, although not all metals that activate hOGR1 activated zOGR1.

Excited-State Intramolecular Proton-Transfer Properties of Three Tris(N-Salicylideneaniline)-Based Chromophores with Extended Conjugation.

The synthesis and photophysical properties of three tris(N-salicylideneaniline) (TSA) compounds containing 1,3,5-triarylbenzene, -tristyrylbenzene, and -tris(arylethynyl)benzene core units are reported. The TSA compounds underwent efficient excited-state intramolecular proton transfer (ESIPT) in solution and in solid state due to the preformed C=N⋅⋅⋅H-O hydrogen-bonded motifs of the structures. Steady-state fluorescence emission spectra of the TSA molecules revealed dual bands only in DMSO, and large Stokes shifts in other polar aprotic and protic solvents. Femtosecond transient absorption spectroscopic measurements in THF revealed lifetime values in the range of 14-16 ps for the excited-state keto-tautomer. The TSA compounds are also responsive to metal ions (Cu2+ and Zn2+ ) in DMSO, exhibit enhanced aggregate-induced emission (AIE) properties in DMSO/water mixtures, and are highly luminescent in the solid state.

Bismuth nanoparticles integration into heavy metal electrochemical stripping sensor.

Between their many applications bismuth nanoparticles (BiNPs) are showing interest as pre-concentrators in heavy metals detection while being applied as working electrode modifiers used in electrochemical stripping analysis. From the different reported methods to synthesize BiNPs we are focused on the typical polyol method, largely used in these types of metallic and semi-metallic nanoparticles. This study presents the strategy for an easy control of the shape and size of BiNPs including nanocubes, nanosferes and triangular nanostructures. To improve the BiNP size and shape, different reducing agents (ethylene glycol or sodium hypophosphite) and stabilizers (polyvinyl pyrrolidone, PVP, in different amounts) have been studied. The efficiency of BiNPs for heavy metals analysis in terms of detection sensitivity while being used as modifiers of screen-printed carbon electrodes including the applicability of the developed device in real sea water samples is shown. A parallel study between the obtained nanoparticles and their performance in heavy metal sensing has been described in this communication.

A metal-organic framework containing unusual eight-connected Zr-oxo secondary building units and orthogonal carboxylic acids for ultra-sensitive metal detection.

Two metal-organic frameworks (MOFs) with Zr-oxo secondary building units (SBUs) were prepared by using p,p'-terphenyldicarboxylate (TPDC) bridging ligands pre-functionalized with orthogonal succinic acid (MOF-1) and maleic acid groups (MOF-2). Single-crystal X-ray structure analysis of MOF-1 provides the first direct evidence for eight-connected SBUs in UiO-type MOFs. In contrast, MOF-2 contains twelve-connected SBUs as seen in the traditional UiO MOF topology. These structural assignments were confirmed by extended X-ray absorption fine structure (EXAFS) analysis. The highly porous MOF-1 is an excellent fluorescence sensor for metal ions with the detection limit of <0.5 ppb for Mn(2+) and three to four orders of magnitude greater sensitivity for metal ions than previously reported luminescent MOFs.

The hierarchic network of metal-response transcription factors in Escherichia coli.

Enterobacteria such as Escherichia coli are able to survive under various environments within host animals by changes of the expression pattern of its genome. The selective expression of genes in its genome takes place by controlling the promoter recognition properties of RNA polymerase by protein-protein interplays with transcription factors. In this review, I describe the regulatory network formed by the metal-sensing transcription factors in E. coli. Comprehensive analyses identify the set of regulation targets for a total of 13 metal-response transcription factors, indicating that nine species of transcription factors are local regulators while four species of transcription factors are global regulators. The signal transduction pathways for these metal-response regulons show not only the complex cross-talks but also the hierarchic multi-regulatory network. This regulatory network seems to play a role for E. coli survival to colonize in a large intestine within host animals.

Meglumine-based Sustainable Three-component Deep Eutectic Solvent Applicable for the Synthesis of Pyrazolo[5,1-b]quinazoline-3-carboxylates as a Sensing Probe for Cu2+ Ions.

An unprecedented meglumine-based three-component deep eutectic solvent (3c-DES) (MegPAc) was synthesized using meglumine, p-toluenesulfonic acid (PTSA), and acetic acid as a renewable, and non-toxic solvent. The exploitation of the MegPAc as an eco-friendly reaction media to construct a selective and sensitive small organic molecular sensing probe, namely, pyrazolo[5,1-b]quinazoline-3-carboxylates (PQCs) was executed. Captivatingly, the MegPAc served the dual role of solvent and catalyst, and it delivered the title components with 69-94 % yields within 67-150 minutes. Furthermore, a UV-visible study unfolds the selective detection of Cu2+ ions with our synthetic probe 4 ba and resulted in hypsochromic shift due to electrostatic interactions. Additionally, 1H NMR titration study and density functional theory (DFT) calculations were performed to attest the binding mechanism of sensing probe 4 ba and Cu2+ ions. Worthy of mention, this protocol unveils the efficacy of meglumine-based 3c-DES for the first time as a bio-renewable system to synthesize the PQCs.

Ultra-lightweight living structural material for enhanced stiffness and environmental sensing.

Natural materials such as bone, wood, and bamboo can inspire the fabrication of stiff, lightweight structural materials. Biofilms are one of the most dominant forms of life in nature. However, little is known about their physical properties as a structural material. Here we report an Escherichia coli biofilm having a Young's modulus close to 10 â€‹GPa with ultra-low density, indicating a high-performance structural material. The mechanical and structural characterization of the biofilm and its components illuminates its adaptable bottom-up design, consisting of lightweight microscale cells covered by a dense network of amyloid nanofibrils on the surface. We engineered E. coli such that 1) carbon nanotubes assembled on the biofilm, enhancing its stiffness to over 30 â€‹GPa, or that 2) the biofilm sensitively detected heavy metal as an example of an environmental toxin. These demonstrations offer new opportunities for developing responsive living structural materials to serve many real-world applications.

Fluorescent Aptaswitch for Detection of Lead Ions.

Detection of metal ions has essential roles in biology, food industry, and environmental sciences. In this work, we developed a Pb2+ detection strategy based on a fluorophore-tagged Pb2+-binding aptamer. The DNA aptamer changes its conformation on binding Pb2+, switching from an "off" state (low fluorescence) to an "on" state (high fluorescence). This method provides a quantitative readout with a detection limit of 468 nM, is highly specific to Pb2+ when tested against other metal ions, and is functional in complex biofluids. Such metal sensing DNA aptamers could be coupled with other biomolecules for sense-and-actuate mechanisms in biomedical and environmental applications.

Sustainable Preparation of Graphene Quantum Dots for Metal Ion Sensing Application.

Over the past several years, graphene quantum dots (GQDs) have been extensively studied in water treatment and sensing applications because of their exceptional structure-related properties, intrinsic inert carbon property, eco-friendly nature, etc. This work reported on the preparation of GQDs from the ethanolic extracts of eucalyptus tree leaves by a hydrothermal treatment technique. Different heat treatment times and temperatures were used during the hydrothermal treatment technique. The optical, morphological, and compositional analyses of the green-synthesized GQDs were carried out. It can be noted that the product yield of GQDs showed the maximum yield at a reaction temperature of 300 °C. Further, it was noted that at a treatment period of 480 min, the greatest product yield of about 44.34% was attained. The quantum yields of prepared GQDs obtained after 480 min of treatment at 300 °C (named as GQD/300) were noted to be 0.069. Moreover, the D/G ratio of GQD/300 was noted to be 0.532 and this suggested that the GQD/300 developed has a nano-crystalline graphite structure. The TEM images demonstrated the development of GQD/300 with sizes between 2.0 to 5.0 nm. Furthermore, it was noted that the GQD/300 can detect Fe3+ in a very selective manner, and hence the developed GQD/300 was successfully used for the metal ion sensing application.