[Analysis of the Divalent Cation Blocking in Ion Channels by Crystal Structure and Molecular Dynamics Simulations].

Neural activity generates essential responses, such as thinking, memory formation, and muscle contraction. It is controlled by the well-coordinated activity of various cation-selective channels of the cell membrane. The divalent cation block plays an essential role in various tetrameric ion channels. For example, N-methyl-D-aspartic acid receptors, which are tetrameric ion channels involved in memory formation, are inhibited by magnesium ions. Divalent cations are thought to bind in the ion pathway of the ion channel and as a consequence block the channel current, however, direct observation of such a block has not been reported yet. As a consequence, the behavior of these blocking divalent cations remains poorly understood. NavAb, a similar tetrameric sodium channel cloned from Arcobacter butzleri, is one of the most structurally analyzed tetrameric channels that is not inhibited by divalent cations. In this study, we elucidated the molecular mechanism of the divalent cation block by reproducing the divalent cation block in NavAb. The X-ray crystal structure of divalent-cation-block mutants show electron density in the ion transmission pathway of the divalent cation blocked mutants, indicating that the mutations increasing the hydrophilicity of the inner vestibule of the pore domain enable a divalent cation to stack into the ion pathway. In molecular dynamics simulations, the stacked calcium ion repels the sodium ions near the channel lumen's entrance at the selective filter's bottom. These results suggest the primary process of the divalent cation block mechanism in tetrameric cation channels and suggest a process of functional acquisition in ion channel evolution.

Assembly and catalytic activity of short prion-inspired peptides.

Enzymes play a crucial role in biochemical reactions, but their inherent structural instability limits their performance in industrial processes. In contrast, amyloid structures, known for their exceptional stability, are emerging as promising candidates for synthetic catalysis. This article explores the development of metal-decorated nanozymes formed by short peptides, inspired by prion-like domains. We detail the rational design of synthetic short Tyrosine-rich peptide sequences, focusing on their self-assembly into stable amyloid structures and their metallization with biologically relevant divalent metal cations, such as Cu2+, Ni2+, Co2+ and Zn2+. The provided experimental framework offers a step-by-step guide for researchers interested in exploring the catalytic potential of metal-decorated peptides. By bridging the gap between amyloid structures and catalytic function, these hybrid molecules open new avenues for developing novel metalloenzymes with potential applications in diverse chemical reactions.

Synthesis and characterization of ion-induced sodium alginate/soy protein isolate microgels for the controlled release.

In this study, sodium alginate/ soy protein isolate (SPI) microgels cross-linked by various divalent cations including Cu2+, Ba2+, Ca2+, and Zn2+ were fabricated. Cryo-scanning electron microscopy observations revealed distinctive structural variations among the microgels. In the context of gastric pH conditions, the degree of shrinkage of the microgels followed the sequence of Ca2+ > Ba2+ > Cu2+ > Zn2+. Meanwhile, under intestinal pH conditions, the degree of swelling was ranked as Zn2+ > Ca2+ > Ba2+ > Cu2+. The impact of these variations was investigated through in vitro digestion studies, revealing that all microgels successfully delayed the release of ß-carotene within the stomach. Within the simulated intestinal fluid, the microgel cross-linked with Zn2+ exhibited an initial burst release, while those cross-linked with Cu2+, Ba2+, or Ca2+ displayed a sustained release pattern. This research underscores the potential of sodium alginate/SPI microgels cross-linked with different divalent cations as efficient controlled-release delivery systems.

Orange luminescence of α-Al2O3 related to clusters consisting of F centers and divalent cations.

The orange luminescence of α-Al2O3 under UV excitation is characterized by a 2.07-eV orange broadband emission that has not yet been elucidated. This emission is present in natural and synthetic crystals and powders, as well as in Be-treated samples. All orange-luminescent materials have low Fe concentration (mostly <1000 ppm) with traces of divalent cations, mostly Mg, or Be in Be-diffused material (dozens of ppm). Mg2+, Mn2+, and Be2+ cations substitute for trivalent Al. To accommodate the charge deficit, several defects are created, including oxygen vacancies also called F centers. Indeed, our excitation spectra revealed the presence of several different F centers (F, F+, and clustered F2, F2 +, F2 2+) in those samples. However, the thermal stability and the measured luminescence lifetimes do not match with previously reported characteristics of isolated F centers. Based on our experiments, we suggest that a complex aggregate of two F centers (F2 2+) trapped at divalent cations is a major cause of this uncommon microsecond lifetime emission, even if a variety of other defects, including Cr3+, V3+, or interstitial Al3+, are present.

Reversible pH-Gated MXene Membranes with Ultrahigh Mono-/Divalent-Ion Selectivity.

Artificial ion channel membranes hold high promise in water treatment, nanofluidics, and energy conversion, but it remains a great challenge to construct such smart membranes with both reversible ion-gating capability and desirable ion selectivity. Herein, we constructed a smart MXene-based membrane via p-phenylenediamine functionalization (MLM-PPD) with highly stable and aligned two-dimensional subnanochannels, which exhibits reversible ion-gating capability and ultrahigh metal ion selectivity similar to biological ion channels. The pH-sensitive groups within the MLM-PPD channel confers excellent reversible Mg2+-gating capability with a pH-switching ratio of up to 100. The mono/divalent metal-ion selectivity up to 1243.8 and 400.9 for K+/Mg2+ and Li+/Mg2+, respectively, outperforms other reported membranes. Theoretical calculations combined with experimental results reveal that the steric hindrance and stronger PPD-ion interactions substantially enhance the energy barrier for divalent metal ions passing through the MLM-PPD, and thus leading to ultrahigh mono/divalent metal-ion selectivity. This work provides a new strategy for developing artificial-ion channel membranes with both reversible ion-gating functionality and high-ion selectivity for various applications.

Effects of monovalent and divalent cations on the rheology of entangled DNA.

In this paper we investigate the effects of varying cation valency and concentration on the rheology of entangled λDNA solutions. We show that monovalent cations moderately increase the viscoelasticty of the solutions mainly by stabilising linear concatenation of λDNA "monomers" via hybridisation of their sticky ends. On the contrary, divalent cations have a far more complex and dramatic effect on the rheology of the solution and we observe evidence of inter-molecular DNA-DNA bridging by Mg2+. We argue that these results may be interesting in the context of dense solutions of single and double stranded DNA, e.g. in vivo or in biotechnology applications such as DNA origami and DNA hydrogels.

Divergent redistribution behavior of divalent metal cations associated with Fe(II)-mediated jarosite phase transformation.

The Fe(II)/Fe(III) cycle is an important driving force for dissolution and transformation of jarosite. Divalent heavy metals usually coexist with jarosite; however, their effects on Fe(II)-induced jarosite transformation and different repartitioning behavior during mineral dissolution-recrystallization are still unclear. Here, we investigated Fe(II)-induced (1 mM Fe(II)) jarosite conversion in the presence of Cd(II), Mn(II), Co(II), Ni(II) and Pb(II) (denoted as Me(II), 1 mM), respectively, under anaerobic condition at neutral pH. The results showed that all co-existing Me(II) retarded Fe(II)-induced jarosite dissolution. In the Fe(II)-only system, jarosite first rapidly transformed to lepidocrocite (an intermediate product) and then slowly to goethite; lepidocrocite was the main product. In Fe(II)-Cd(II), -Mn(II), and -Pb(II) systems, coexisting Cd(II), Mn(II) and Pb(II) retarded the above process and lepidocrocite was still the dominant conversion product. In Fe(II)-Co(II) system, coexisting Co(II) promoted lepidocrocite transformation into goethite. In Fe(II)-Ni(II) system, jarosite appeared to be directly converted into goethite, although small amounts of lepidocrocite were detected in the final product. In all treatments, the appearance or accumulation of lepidocrocite may be also related to the re-adsorption of released sulfate. By the end of reaction, 6.0 %, 4.0 %, 76.0 % 11.3 % and 19.2 % of total Cd(II), Mn(II), Pb(II) Co(II) and Ni(II) were adsorbed on the surface of solid products. Up to 49.6 %, 44.3 %, and 21.6 % of Co(II), Ni(II), and Pb(II) incorporated into solid product, with the reaction indicating that the dynamic process of Fe(II) interaction with goethite may promote the continuous incorporation of Co(II), Ni(II), and Pb(II).

Divalent metal ions under low concentration environment improved the thermal gel properties of egg yolk.

To improve the thermal gel properties of egg yolk, the effect of several valence metal ions (K+, Ca2+, Mg2+ and Fe3+) with different concentrations (0-0.72%) on the rheological, gel, and structural properties of egg yolk were investigated. Results showed that monovalent and divalent ions were beneficial to the formation of uniform and dense gel network, especially with the addition of 0.72% magnesium ion, which further improved gel hardness, water holding capacity (WHC) and viscoelastic properties, the properties of egg yolk gel increased with the increase of the concentration of mono-bivalent metal ions. Adding ferric ion remarkably increased the average particle size (d4,3) and apparent viscosity of egg yolk, destroying the disulfide bonds and the hydrophobic interactions in gel. Fourier transform infrared spectroscopy (FT-IR) and fluorescence spectra analysis revealed that metal ions promoted the hydrophobic aggregation among egg yolk proteins and induced the transition of protein secondary structure from ordered to disordered. This work will provide a theoretical reference for the development of low salt and nutrient fortified egg yolk products.

Competition between Stacking and Divalent Cation-Mediated Electrostatic Interactions Determines the Conformations of Short DNA Sequences.

Interplay between divalent cations (Mg2+ and Ca2+) and single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), as well as stacking interactions, is important in nucleosome stability and phase separation in nucleic acids. Quantitative techniques accounting for ion-DNA interactions are needed to obtain insights into these and related problems. Toward this end, we created a sequence-dependent computational TIS-ION model that explicitly accounts for monovalent and divalent ions. Simulations of the rigid 24 base-pair (bp) dsDNA and flexible ssDNA sequences, dT30 and dA30, with varying amounts of the divalent cations show that the calculated excess number of ions around the dsDNA and ssDNA agree quantitatively with ion-counting experiments. Using an ensemble of all-atom structures generated from coarse-grained simulations, we calculated the small-angle X-ray scattering profiles, which are in excellent agreement with experiments. Although ion-counting experiments mask the differences between Mg2+ and Ca2+, we find that Mg2+ binds to the minor grooves and phosphate groups, whereas Ca2+ binds specifically to the minor groove. Both Mg2+ and Ca2+ exhibit a tendency to bind to the minor groove of DNA as opposed to the major groove. The dA30 conformations are dominated by stacking interactions, resulting in structures with considerable helical order. The near cancellation of the favorable stacking and unfavorable electrostatic interactions leads to dT30 populating an ensemble of heterogeneous conformations. The successful applications of the TIS-ION model are poised to confront many problems in DNA biophysics.

Functionalization of a cast NaAl/binary ZnO/SiO2 nanohybrid with amine and Schiff base ligands as an adsorbent of divalent cations in water system.

Casting method was used to synthesize a novel sodium alginate nanohybrid functionalized with aminated ZnO/SiO2 Schiff base for adsorption of nickel (Ni2+) and copper (Cu2+) divalent cations in single and binary water systems. The cast Schiff base nanohybrids were investigated using FESEM, XRD, BET, FTIR, TGA, and XPS analyses. The influence of unfunctionalized binary ZnO/SiO2 nano oxides and aminated Schiff base ligands formed by the reaction between salicylaldehyde and O-phenylenediamine on the adsorption of Ni2+ and Cu2+ cations was evaluated. The results confirmed that the aminated Schiff base ligands led to a higher adsorption ability of the cast nanohybrids containing interaction of divalent cations with nitrogen and oxygen atoms, as well as carboxyl and hydroxyl groups. The adsorption kinetics and isotherm for both cations followed a double-exponential model and the Redlich-Peterson model, respectively. The maximum monolayer capacity was found to be 249.8 mg/g for Cu2+ cation and 96.4 mg/g for Ni2+ cation. Thermodynamic analysis revealed an endothermic and spontaneous adsorption process with an increase in entropy. Furthermore, the synthesized Schiff base adsorbent could be easily reused over five times. The simultaneous adsorption in binary system exhibited a higher adsorption selectivity of the cast Schiff base nanohybrid for Cu2+ cation compared to Ni2+ cation. It was found that the removal percentages of Cu2+ and Ni2+ from industrial electroplating wastewater were 91.3 and 64.5%, respectively. Lastly, cost analysis of the synthesized nanohybrid was investigated.

Microplastics affect membrane biofouling and microbial communities during gravity-driven membrane filtration of primary wastewater.

Recently, gravity-driven membrane (GDM) filtration has been adopted as an alternative solution for decentralized wastewater treatment due to easy installation and maintenance, reduced energy and operation cost, and low global warming impact. This study investigated the influence of microplastic size (0.5-0.8 µm and 40-48 µm) and amount (0.1 and 0.2 g/L) on the membrane performance and microbial community in GDM systems for primary municipal wastewater treatment. The results showed that dosing microplastics in the GDM systems led to 9-54% lower permeate flux than that in the control. This was attributed to more cake formation (up to 6.4-fold) with more deposition of extracellular polymeric substances (EPS, up to 1.5-fold) and divalent cations (up to 2.1-fold) in the presence of microplastics, especially with increasing microplastic amount or size. However, the dosed microplastics promoted formation of heterogeneous cake layers with more porous nature, possibly because microplastics created void space in the cake and also tended to bind with divalent cations to reduce EPS-divalent cations interactions. In the biofilm of the GDM systems, the presence of microplastics could lower the number of total species, but it greatly enhanced the abundance of certain dominant prokaryotes (Phenylobacterium haematophilum, Planctomycetota bacterium, and Flavobacteriales bacterium), eukaryotes (Stylonychia lemnae, Halteria grandinella, and Paramicrosporidium saccamoebae), and virus (phylum Nucleocytoviricota), as well as amino acid and lipid metabolic functions. Especially, the small-size microplastics at a higher dosed amount led to more variations of microbial community structure and microbial metabolic functions.

New Insights into the Dependence of CPEB3 Ribozyme Cleavage on Mn2+ and Mg2.

CPEB3 ribozyme is a self-cleaving RNA that occurs naturally in mammals and requires divalent metal ions for efficient activity. Ribozymes exhibit preferences for specific metal ions, but the exact differences in the catalytic mechanisms of various metal ions on the CPEB3 ribozyme remain unclear. Our findings reveal that Mn2+ functions as a more effective cofactor for CPEB3 ribozyme catalysis compared to Mg2+, as confirmed by its stronger binding affinity to CPEB3 by EPR. Cleavage assays of CPEB3 mutants and molecular docking analyses further showed that excessive Mn2+ ions can bind to a second binding site near the catalytic site, hindering CPEB3 catalytic efficiency and contributing to the Mn2+ bell-shaped curve. These results implicate a pivotal role for the local nucleobase-Mn2+ interactions in facilitating RNA folding and modulating the directed attack of nucleophilic reagents. Our study provides new insights and experimental evidence for exploring the divalent cation dependent cleavage mechanism of the CPEB3 ribozyme.

Hypertonic Aerosols Hydrate Airways Longer and Reduce Acidification Risk with Nonpermeating Cation and Permeating Anion Salts.

Background: Hyperosmolar aerosols appear to promote or suppress upper airway dysfunction caused by dehydration in a composition-dependent manner. We sought to explore this composition dependence experimentally, in an interventional human clinical study, and theoretically, by numerical analysis of upper airway ion and water transport. Methods: In a double-blinded, placebo-controlled clinical study, phonation threshold pressure (PTP) was measured prenasal and postnasal inhalation of hypertonic aerosols of NaCl, KCl, CaCl2, and MgCl2 in seven human subjects. Numerical analysis of water and solute exchanges in the upper airways following deposition of these same aerosols was performed using a mathematical model previously described in the literature. Results: PTP decreased by 9%-22% relative to baseline (p < 0.05) for all salts within the first 30 minutes postadministration, indicating effective laryngeal hydration. Only MgCl2 reduced PTP beyond 90 minutes (21% below baseline at 2 hours postadministration). By numerical analysis, we determined that, while airway water volume up to 15 minutes postdeposition is dictated by osmolarity, after 30 minutes, divalent cation salts, such as MgCl2, better retain airway surface liquid (ASL) volume by slow paracellular clearance of the divalent cation. Fall of CFTR chloride flux with rise in ASL height, a promoter of airway acidification, appears to be a signature of permeating cation (NaCl) and nonpermeating anion (mannitol) aerosol deposition. For hypertonic aerosols that lack permeating cation and include permeating anion (CaCl2 and MgCl2), this acid-trigger signature does not exist. Conclusions: Nonpermeating cation and permeating anion hypertonic aerosols appear to hydrate upper airways longer and, rather than provoke, may reduce laryngeal dysfunction such as cough and bronchoconstriction.

Magnesium alginate as a low-viscosity (intramolecularly cross-linked) system for the sustained and neuroprotective release of magnesium.

The administration of Mg ions is advantageous in pathological scenarios such as pre-enclampsia and forms of neuroinflammation (e.g. stroke or injury); yet, few systems exist for their sustained delivery. Here, we present the (static light scattering and diffusing-wave spectroscopy) characterization of magnesium alginate (MgAlg) as a potentially injectable vehicle ifor the delivery of Mg. Differently from other divalent cations, Mg does not readily induce gelation: it acts within MgAlg coils, making them more rigid and less prone to entangle. As a result, below a threshold concentration (notionally below 0.5 % wt.) MgAlg are inherently less viscous than those of sodium alginate (NaAlg), which is a major advantage for injectables; at higher concentrations, however, (stable, Mg-based) aggregation starts occurring. Importantly, Mg can then be released e.g. in artificial cerebrospinal fluid, via a slow (hours) process of ion exchange. Finally, we here show that MgAlg protects rat neural stem cells from the consequence of an oxidative insult (100 µM H2O2), an effect that we can only ascribe to the sustained liberation of Mg ions, since it was not shown by NaAlg, MgSO4 or the NaAlg/MgSO4 combination. Our results therefore indicate that MgAlg is a promising vehicle for Mg delivery under pathological (inflammatory) conditions.

Sorption of cadmium by layered double hydroxides: Performance, structure-related mechanisms, and sequestration stability assessment.

Layered double hydroxides (LDHs) have been recognized to have great potential for the treatment of heavy metals in wastewater and soil through various mechanisms. Isomorphic substitution is an important mechanism for the sorption of heavy metal cations with LDH reconstruction and highly stable product formation. However, sorption performance, structure-related relationships, and, more importantly, stability are still poorly understood. In this study, a series of LDHs with different structures were synthesized to evaluate their cadmium (Cd) sorption performance and stability concerning the isomorphic substitution mechanism. Divalent cation types in the LDH lattice determined the Cd sorption capacity as well as the isomorphic substitution possibility, following the order of hydroxide solubility of divalent cations (MII): Ca2+>Mg2+>(Cd2+) > Ni2+>Zn2+. In addition, CaAl-LDH exhibited a super-high Cd sorption capacity of 625.0 mg g-1. Cd sorption by LDHs with different interlayer anion types and divalent/trivalent cation molar ratios varied due to crystallite size-related MII release through cation-exchange/isomorphic substitution. Coexisting cations (e.g., Zn2+, Ni2+, Mg2+) influence the sorption performance of MII-LDH mainly through isomorphic substitution mechanism, largely depending on the solubility of MII(OH)2 with a trend of stable product formation. Furthermore, Mg2.9Cd0.1AlCl-LDH was fabricated, and limited Cd dissolution without destruction of the LDH structure was observed under various conditions. For example, only 7.69%, 2.16% and 0.96% of Cd was released from as-prepared Mg2.9Cd0.1AlCl-LDH in NaCl solution (0.02 mol L-1, pH 5), soil extract, and soil matrix, respectively. The very low leaching of Cd from Cd-containing LDHs indicated the high stability of LDH-sorbed Cd via isomorphic substitution and feasible practical application in Cd sequestration in wastewater treatment and soil remediation.

Influence of Divalent Cation Inhibition and Dissolved Organic Matter Enhancement on Phenol Oxidation Kinetics by Manganese Oxides.

Manganese oxides can oxidize organic compounds, such as phenols, and may potentially be used in passive water treatment applications. However, the impact of common water constituents, including cations and dissolved organic matter (DOM), on this reaction is poorly understood. For example, the presence of DOM can increase or decrease phenol oxidation rates with manganese oxides. Furthermore, the interactions of DOM and cations and their impact on the phenol oxidation rates have not been examined. Therefore, we investigated the oxidation kinetics of six phenolic contaminants with acid birnessite in ten whole water samples. The oxidation rate constants of 4-chlorophenol, 4-tert-octylphenol, 4-bromophenol, and phenol consistently decreased in all waters relative to buffered ultrapure water, whereas the oxidation rate of bisphenol A and triclosan increased by up to 260% in some waters. Linear regression analyses and targeted experiments demonstrated that the inhibition of phenol oxidation is largely determined by cations. Furthermore, quencher experiments indicated that radical-mediated interactions from oxidized DOM contributed to enhanced oxidation of bisphenol A. The variable changes between compounds and water samples demonstrate the challenge of accurately predicting contaminant transformation rates in environmentally relevant systems based on experiments conducted in the absence of natural water constituents.

Impact of Divalent Cations on In-Layer Positional Order of DNA-Based Liquid Crystals: Implications for DNA Condensation.

The layered liquid crystalline phases formed by DNA molecules, which include rigid and flexible segments ("gapped DNA"), enable the study of both end-to-end stacking and side-to-side (helix-to-helix) lateral interactions, forming a model system to study such interactions at physiologically relevant DNA and ion concentrations. The observed layer structure exhibits long-range interlayer and in-layer positional correlations. In particular, the in-layer order has implications for DNA condensation, as it reflects whether these normally repulsive interactions become attractive under certain ionic conditions. Using synchrotron small-angle X-ray scattering measurements, we investigate the impact of divalent Mg2+ cations (in addition to a constant 150 mM Na+) on the stability of the inter- and in-layer DNA ordering as a function of temperature between 5 and 65 °C. DNA constructs with different terminal base pairings were created to mediate the strength of the attractive end-to-end stacking interactions between the blunt ends of the gapped DNA constructs. We demonstrate that the stabilities at a fixed DNA concentration of both interlayer and in-layer order are significantly enhanced even at a few mM Mg2+ concentration. The stabilities are even higher at 30 mM Mg2+; however, a marked decrease is observed at 100 mM Mg2+, suggesting a change in the nature of side-by-side interactions within this Mg2+ concentration range. We discuss the implications of these results in terms of counterion-mediated DNA-DNA attraction and DNA condensation.

Dimeric structures of DNA ATTTC repeats promoted by divalent cations.

Structural studies of repetitive DNA sequences may provide insights why and how certain repeat instabilities in their number and nucleotide sequence are managed or even required for normal cell physiology, while genomic variability associated with repeat expansions may also be disease-causing. The pentanucleotide ATTTC repeats occur in hundreds of genes important for various cellular processes, while their insertion and expansion in noncoding regions are associated with neurodegeneration, particularly with subtypes of spinocerebellar ataxia and familial adult myoclonic epilepsy. We describe a new striking domain-swapped DNA-DNA interaction triggered by the addition of divalent cations, including Mg2+ and Ca2+. The results of NMR characterization of d(ATTTC)3 in solution show that the oligonucleotide folds into a novel 3D architecture with two central C:C+ base pairs sandwiched between a couple of T:T base pairs. This structural element, referred to here as the TCCTzip, is characterized by intercalative hydrogen-bonding, while the nucleobase moieties are poorly stacked. The 5'- and 3'-ends of TCCTzip motif are connected by stem-loop segments characterized by A:T base pairs and stacking interactions. Insights embodied in the non-canonical DNA structure are expected to advance our understanding of why only certain pyrimidine-rich DNA repeats appear to be pathogenic, while others can occur in the human genome without any harmful consequences.

Molecular determinants of ASIC1 modulation by divalent cations.

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the nervous system. ASIC gating is modulated by divalent cations as well as small molecules; however, the molecular determinants of gating modulation by divalent cations are not well understood. Previously, we identified two small molecules that bind to ASIC1a at a novel site in the acidic pocket and modulate ASIC1 gating in a manner broadly resembling divalent cations, raising the possibility that these small molecules may help to illuminate the molecular determinants of gating modulation by divalent cations. Here, we examined how these two groups of modulators might interact as well as mutational effects on ASIC1a gating and its modulation by divalent cations. Our results indicate that binding of divalent cations to an acidic pocket site plays a key role in gating modulation of the channel.

Klebsiella pneumoniae DedA family proteins have redundant roles in divalent cation homeostasis and resistance to phagocytosis.

The DedA superfamily is a highly conserved family of membrane proteins. Deletion of Escherichia coli yqjA and yghB, encoding related DedA family proteins, results in sensitivity to elevated temperature, antibiotics, and alkaline pH. The human pathogen Klebsiella pneumoniae possesses genes encoding DedA family proteins with >90% amino acid identity to E. coli YqjA and YghB. We hypothesized that the deletion of K. pneumoniae yqjA and yghB will impact its physiology and may reduce its virulence. The K. pneumoniae ΔyqjA ΔyghB mutant (strain VT101) displayed a growth defect at 42°C and alkaline pH sensitivity, not unlike its E. coli counterpart. However, VT101 retained mostly wild-type resistance to antibiotics. We found VT101 was sensitive to the chelating agent EDTA, the anionic detergent SDS, and agents capable of alkalizing the bacterial cytoplasm such as bicarbonate or chloroquine. We could restore growth at alkaline pH and at elevated temperature by addition of 0.5-2 mM Ca2+ or Mg2+ to the culture media. VT101 displayed a slower uptake of calcium, which was dependent upon calcium channel activity. VT201, with similar deletions as VT101 but derived from a virulent K. pneumoniae strain, was highly susceptible to phagocytosis by alveolar macrophages and displayed a defect in the production of capsule. These findings suggest divalent cation homeostasis and virulence are interlinked by common functions of the DedA family.IMPORTANCEKlebsiella pneumoniae is a dangerous human pathogen. The DedA protein family is found in all bacteria and is a membrane transporter often required for virulence and antibiotic resistance. K. pneumoniae possesses homologs of E. coli YqjA and YghB, with 60% amino acid identity and redundant functions, which we have previously shown to be required for tolerance to biocides and alkaline pH. A K. pneumoniae strain lacking yqjA and yghB was found to be sensitive to alkaline pH, elevated temperature, and EDTA/SDS and displayed a defect in calcium uptake. Sensitivity to these conditions was reversed by addition of calcium or magnesium to the growth medium. Introduction of ΔyqjA and ΔyghB mutations into virulent K. pneumoniae resulted in the loss of capsule, increased phagocytosis by macrophages, and a partial loss of virulence. These results show that targeting the Klebsiella DedA family results in impaired divalent cation transport and, in turn, loss of virulence.