Metal speciation in natural waters with emphasis on reduced sulfur groups as strong metal binding sites

The proper application of biotic ligand models to predict metal toxicity depends on accurate prediction of metal binding to sites on natural organic matter (NOM) which compete with the 'biotic' ligand for available metal. A hard and soft metal classification along with associated ligand groups (carboxyl, phenolic, amino, sulfidic) are used as a basis to predict metal speciation in the presence of aqueous organic matter. Compilation of conditional metal formation constants (log K') are made for each ligand type using model ligands. Model ligands were chosen to reflect those found in NOM and bio-organic media. Total ligand concentration (L(T)) estimates for different natural settings and log K' values are then used to generate a L(T)-log K' distributions for a specific metal. A plot for Cu(II) gives a similar trend as a compilation of measured data for natural environments. A log K'-L(T) plot for Ag(I) shows a much more discrete binding pattern than for Cu(II). Estimation of speciation of a specific metal in a specific environmental setting and to design speciation and toxicological experiments requires accurate knowledge of the functional groups in NOM.     

COPPER‐UPTAKE KINETICS OF COASTAL AND OCEANIC DIATOMS1

We investigated copper (Cu) acquisition mechanisms and uptake kinetics of the marine diatoms Thalassiosira oceanica Hasle, an oceanic strain, and Thalassiosira pseudonana Hasle et Heimdal, a coastal strain, grown under replete and limiting iron (Fe) and Cu availabilities. The Cu‐uptake kinetics of these two diatoms followed classical Michaelis–Menten kinetics. Biphasic uptake kinetics as a function of Cu concentration were observed, suggesting the presence of both high‐ and low‐affinity Cu‐transport systems. The half‐saturation constants (K_m) and the maximum Cu‐uptake rates (V_max) of the high‐affinity Cu‐transport systems (～7–350 nM and 1.5–17 zmol · μm^?2 · h^?1, respectively) were significantly lower than those of the low‐affinity systems (>800 nM and 30–250 zmol · μm^?2 · h^?1, respectively). The two Cu‐transport systems were controlled differently by low Fe and/or Cu. The high‐affinity Cu‐transport system of both diatoms was down‐regulated under Fe limitation. Under optimal‐Fe and low‐Cu growth conditions, the K_m of the high‐affinity transport system of T. oceanica was lower (7.3 nM) than that of T. pseudonana (373 nM), indicating that T. oceanica had a better ability to acquire Cu at subsaturating concentrations. When Fe was sufficient, the low‐affinity Cu‐transport system of T. oceanica saturated at 2,000 nM Cu, while that of T. pseudonana did not saturate, indicating different Cu‐transport regulation by these two diatoms. Using CuEDTA as a model organic complex, our results also suggest that diatoms might be able to access Cu bound within organic Cu complexes.     

Re-evaluating the effects of organic ligands on copper toxicity to barley root elongation in culture solution

Abstract

In the presence of weak ligands, both free ion activity and organic complexes of Cu should b considered when predicting Cu toxicity in aquatic and soil-plant systems. However, there is littl information about the quantitative contribution of Cu that is organically complexed to Cu toxicity. In thi study, a bioassay using barley root elongation in culture solution was used to investigate the effects o organic ligands with different conditional stability constants on Cu toxicity and the quantitativ contribution of the organically complexed Cu to the Cu toxicity. The results indicated that a significan decrease (p<0.05) in Cu toxicity, assessed by barley root elongation, was observed in response to th addition of organic ligands. The decrease differed, to some extent, with different organic ligands o disodium ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), oxalate and malate at low and constant free Cu²⁺ activity. Addition of EDTA or NTA resulted in strong reduction of Cu toxicity while modest reduction of Cu toxicity was observed for the addition of malate as the relatively wea ligand. Furthermore, the results of the present study revealed that the CuNTA^? and CuEDTA^2? complexes were not toxic, while the Cu–malate complexes were mildly toxic to barley root elongation More importantly, it was found that the toxicity of Cu–malate complexes were nearly 0.5-fold less than that of free Cu²⁺ ions.     

The kinetics of siderophore‐mediated olivine dissolution

Silicate minerals represent an important reservoir of nutrients at Earth's surface and a source of alkalinity that modulates long‐term geochemical cycles. Due to the slow kinetics of primary silicate mineral dissolution and the potential for nutrient immobilization by secondary mineral precipitation, the bioavailability of many silicate‐bound nutrients may be limited by the ability of micro‐organisms to actively scavenge these nutrients via redox alteration and/or organic ligand production. In this study, we use targeted laboratory experiments with olivine and the siderophore deferoxamine B to explore how microbial ligands affect nutrient (Fe) release and the overall rate of mineral dissolution. Our results show that olivine dissolution rates are accelerated in the presence of micromolar concentrations of deferoxamine B. Based on the non‐linear decrease in rates with time and formation of a Fe³⁺‐ligand complex, we attribute this acceleration in dissolution rates to the removal of an oxidized surface coating that forms during the dissolution of olivine at circum‐neutral pH in the presence of O₂ and the absence of organic ligands. While increases in dissolution rates are observed with micromolar concentrations of siderophores, it remains unclear whether such conditions could be realized in natural environments due to the strong physiological control on microbial siderophore production. So, to contextualize our experimental results, we also developed a feedback model, which considers how microbial physiology and ligand‐promoted mineral dissolution kinetics interact to control the extent of biotic enhancement of dissolution rates expected for different environments. The model predicts that physiological feedbacks severely limit the extent to which dissolution rates may be enhanced by microbial activity, though the rate of physical transport modulates this limitation.     

Dynamic structure of DNA complexes. Fluorometric measurement of hydrogen-deuterium exchange kinetics of dna-bound ethidium dimer and acridine-ethidium dimer

The hydrogen-deuterium (H-D) exchange kinetics of free and DNA-bound ethidium dimer and acridine-ethidium heterodimer were measured by stopped flow using fluorescence detection. This technique allowed a very accurate measurement of the exchange process. The H-D exchange kinetics were measured in various environments. In some cases, it was observed that the H-D exchange was much faster than the dissociation rate of dimer-DNA complexes. This showed that the exchange was taking place directly from the bound state. Furthermore, the action of a catalyst (imidazolium ion) on the rate of H-D exchange showed that a dynamic structural fluctuation of the ligand in its DNA complex was a necessary step on the exchange process.     

Natural organic matter (NOM) has the potential to modify the multixenobiotic resistance (MXR) activity in freshwater amphipods Eulimnogammarus cyaneus and E. verrucosus

Based on the chemical features of natural organic matter (NOM) with its variety of functional groups, we hypothesized that NOM will modify the multixenobiotic-resistance (MXR) of an organism as xenobiotic chemicals do. The MXR system is a general first rather non-specific line of defense against environmental contaminants. The aim of this study was to compare the impacts on MXR activity in amphipod species (Eulimnogammarus cyaneus and E. verrucosus, from Lake Baikal) stressed by cadmium chloride or dissolved NOM for 24 h. NOM exposure concentrations were environmentally realistic. MXR activity was assessed based on rhodamine B efflux; its specificity was proven by a verapamil inhibition assay. It was shown that both NOM and CdCl₂ lead to substantial reduction of the rhodamine B efflux. This suggests that NOM may be regarded as a chemosensor which is able to reduce the efficiency of the MXR system. Possible mechanisms of direct NOM impact on MXR processes are discussed, such as peroxidation of the membranes (including P-glycoproteins) or internal blockage of the MXR pump by bioconcentrated NOM. In general, our results show that well-developed depuration pathways of freshwater organisms in contaminated environments may be impaired by strong chemical stressors and, more important, by natural biogeochemical matrices such as humic substances — humic substances are present in all freshwater systems.     

Copper inclusion in cellulose using sodium d-gluconate complexes

Copper containing cellulose material is of growing interest, e.g. offering alternative in the field of antimicrobials. Solutions of copper d-gluconate complexes (Cu(2+)-DGL) were used to introduce copper ions into a swollen cellulosic matrix. A ligand exchange mechanism forms the chemical basis of the sorption process. Copper sorption in cellulose was studied in the range between pH 6 and 13. An estimate for the complex stabilities of the Cu-cellulose system could be derived from the calculated species distribution of the different Cu(2+)-DGL complexes present. Spectrophotometry and cyclic voltammetry of Cu(2+)-DGL complex solution were used to confirm the presence of different species participating in the ligand exchange reaction. The pH dependent uptake of Cu(2+) ions in the cellulose matrix can be explained on the basis of the relative stabilities of Cu(2+)-DGL complex vs. Cu(2+)-cellulose complexes. In comparison to pH 10, higher copper content was observed at pH 6 and 13. Copper content was limited by carboxyl content of cellulosic materials, thus in analogy to the structure of Cu(2+)-DGL complexes participation of the carboxyl group as complex forming site is proposed. At high Cu(2+)-concentration and longer time of immersion in the copper complex solutions formation of solid deposits was observed on the surface of the treated fibres.     

Acquisition of Fe from Various Natural Organic Matter Isolates by Aerobic Pseudomonad Bacteria

Iron (Fe) is an essential nutrient to most microorganisms. Aerobic microorganisms exhibit various strategies for acquiring Fe at near-neutral pH conditions, where Fe oxyhydroxides are insoluble. Although much research has focused on microbial acquisition of Fe from minerals, little is known about Fe acquisition from natural organic matter (NOM). Yet, in surface waters, soils and shallow sediments, Fe is often associated with natural organic matter (NOM), and this NOM-associated Fe could represent an important pool of Fe for microorganisms. Here, we investigated the growth of aerobic Pseudomonas mendocina on soil and surface water NOM samples containing Fe, under Fe-limited conditions. In the presence of NOM, bacteria grew to population sizes greater than in no-Fe-added controls, indicating that the bacteria were able to access Fe associated with NOM. Maximum population size correlated with the NOM-associated Fe concentration. In an additional experiment, Pseudomonas putida was able to acquire Fe from an NOM sample, demonstrating that this ability is not limited to P. mendocina. When Fe was added as 30 μ M FeEDTA plus NOM, together in the same reaction flasks, P. mendocina and P. putida growth was less than in the presence of 30 μM FeEDTA alone. The fact that Fe sources are not simply additive and that the presence of NOM inhibits growth in FeEDTA suggests that further study on the responses of bacteria to a combination of Fe sources is needed to understand the complexities of bacterial Fe acquisition in the subsurface.     

Characterization of Silver Nanoparticles under Environmentally Relevant Conditions Using Asymmetrical Flow Field-Flow Fractionation (AF4)

The development of methods to monitor manufactured nanomaterials in the environment is one of the crucial areas for the assessment of their risk. More specifically, particle size analysis is a key element, because many properties of nanomaterial are size dependent. The sizing of nanomaterials in real environments is challenging due to their heterogeneity and reactivity with other environmental components. In this study, the fractionation and characterization of a mixture of polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) of three different sizes were investigated using asymmetrical flow field-flow fractionation (AF4) coupled with UV-Vis spectrophotometry. In particular, the effects of electrolyte composition and natural organic matter (NOM) on the particle size and stability were evaluated. The fractogram peaks (i.e., stability) of three different AgNPs decreased in the presence of both 10 mM NaCl and 10mM CaCl₂, while increased with increasing concentration of humic acid (HA). In addition, the hydrodynamic diameters of AgNPs in both electrolytes slightly increased with an increase of HA concentration, suggesting the adsorption (coating) of HA onto the particle surface. It is also interesting to note that an increase in the particle size depended on the types of electrolyte, which could be explained by the conformational characteristics of the adsorbed HA layers. Consistent these results, AgNPs suspended in lake water containing relatively high concentration of organic carbon (TOC) showed higher particle stability and larger particle size (i.e., by approximately 4nm) than those in river water. In conclusion, the application of AF4 coupled with highly sensitive detectors could be a powerful method to characterize nanoparticles in natural waters.     

Iron Acquisition from Natural Organic Matter by an Aerobic Pseudomonas mendocina Bacterium: Siderophores and Cellular Iron Status

This research investigated the potential role of siderophores in aerobic microbial Fe acquisition from natural organic matter (NOM; XAD-8 isolate and reverse osmosis concentrate pre- and post-Chelex® treatment) through the use of a siderophore-producing Pseudomonas mendocina wild type (WT) bacterium and an engineered mutant (Mt) that was incapable of siderophore production. NOM had complex effects on microbial growth under Fe-limited conditions as measured by optical density, most likely because of the presence of other toxic (trace) metals such as Al, NOM binding interference with additional trace metal nutrients, and/or biofilm development. However, a bioassay for cellular Fe status showed that both WT and Mt readily acquired Fe naturally associated with NOM. Thus, while siderophores may be useful for Fe acquisition from NOM by P. mendocina, they do not appear to be essential for this process.     

A conceptual model of organo-mineral interactions in soils: self-assembly of organic molecular fragments into zonal structures on mineral surfaces

In this paper, we propose a structure for organo-mineral associations in soils based on recent insights concerning the molecular structure of soil organic matter (SOM), and on extensive published evidence from empirical studies of organo-mineral interfaces. Our conceptual model assumes that SOM consists of a heterogeneous mixture of compounds that display a range of amphiphilic or surfactant-like properties, and are capable of self-organization in aqueous solution. An extension of this self-organizational behavior in solution, we suggest that SOM sorbs to mineral surfaces in a discrete zonal sequence. In the contact zone, the formation of particularly strong organo-mineral associations appears to be favored by situations where either (i) polar organic functional groups of amphiphiles interact via ligand exchange with singly coordinated mineral hydroxyls, forming stable inner-sphere complexes, or (ii) proteinaceous materials unfold upon adsorption, thus increasing adhesive strength by adding hydrophobic interactions to electrostatic binding. Entropic considerations dictate that exposed hydrophobic portions of amphiphilic molecules adsorbed directly to mineral surfaces be shielded from the polar aqueous phase through association with hydrophobic moieties of other amphiphilic molecules. This process can create a membrane-like bilayer containing a hydrophobic zone, whose components may exchange more easily with the surrounding soil solution than those in the contact zone, but which are still retained with considerable force. Sorbed to the hydrophilic exterior of hemimicellar coatings, or to adsorbed proteins, are organic molecules forming an outer region, or kinetic zone, that is loosely retained by cation bridging, hydrogen bonding, and other interactions. Organic material in the kinetic zone may experience high exchange rates with the surrounding soil solution, leading to short residence times for individual molecular fragments. The thickness of this outer region would depend more on input than on the availability of binding sites, and would largely be controlled by exchange kinetics. Movement of organics into and out of this outer region can thus be viewed as similar to a phase-partitioning process. The zonal concept of organo-mineral interactions presented here offers a new basis for understanding and predicting the retention of organic compounds, including contaminants, in soils and sediments.     

Translocation of botulinum neurotoxin serotype A and associated proteins across the intestinal epithelia

Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins. Botulinum neurotoxins associate with neurotoxin‐associated proteins (NAPs) forming large complexes that are protected from the harsh environment of the gastrointestinal tract. However, it is still unclear how BoNT complexes as large as 900 kDa traverse the epithelial barrier and what role NAPs play in toxin translocation. In this study, we examined the transit of BoNT serotype A (BoNT/A) holotoxin, complex and recombinantly purified NAP complex through cultured and polarized Caco‐2 cells and, for the first time, in the small mouse intestine. Botulinum neurotoxin serotype A and NAPs in the toxin complex were detectable inside intestinal cells beginning at 2 h post intoxication. Appearance of the BoNT/A holotoxin signal was slower, with detection starting at 4–6 h. This indicated that the holotoxin alone was sufficient for entry but the presence of NAPs enhanced the rate of entry. Botulinum neurotoxin serotype A detection peaked at approximately 6 and 8 h for complex and holotoxin, respectively, and thereafter began to disperse with some toxin remaining in the epithelia after 24 h. Purified HA complexes alone were also internalized and followed a similar time course to that of BoNT/A complex internalization. However, recombinant HA complexes did not enhance BoNT/A holotoxin entry in the absence of a physical link with BoNT/A. We propose a model for BoNT/A toxin complex translocation whereby toxin complex entry is facilitated by NAPs in a receptor‐mediated mechanism. Understanding the intestinal uptake of BoNT complexes will aid the development of new measures to prevent or treat oral intoxications.     

Contribution to the reduction‐induced fluorescence enhancement of natural organic matter: Aromatic ketones outweigh quinones

This work involves the comparison of the fluorescence excitation ? emission matrices of different low‐molecular‐weight carbonyl compounds and natural organic matter (NOM). The aim is to determine if quinone or aromatic ketone groups are more responsible for the reduction‐induced fluorescence enhancement of NOM. After reduction, the aromatic ketones showed a significantly greater fluorescence change than the quinones, proving that the former play a more important role. Further analysis of the fluorescence of the NOM samples after re‐oxidization by oxygen with a Cu²⁺ catalyst, provided additional reliable evidence in support of the dominant role of aromatic ketones in the fluorescence change. This work demonstrates that aromatic ketone moieties should be given more attention when considering the physicochemical properties of NOM and related environmental processes.     

The role of reduction in iron uptake processes in a unicellular, planktonic cyanobacterium

In many aquatic environments the essential micronutrient iron is predominantly complexed by a heterogeneous pool of strong organic chelators. Research on iron uptake mechanisms of cyanobacteria inhabiting these environments has focused on endogenous siderophore production and internalization. However, as many cyanobacterial species do not produce siderophores, alternative Fe acquisition mechanisms must exist. Here we present a study of the iron uptake pathways in the unicellular, planktonic, non-siderophore producing strain Synechocystis sp. PCC 6803. By applying trace metal clean techniques and a chemically controlled growth medium we obtained reliable and reproducible short-term (radioactive assays) and long-term (growth experiments) iron uptake rates. We found that Synechocystis 6803 is capable of acquiring iron from exogenous ferrisiderophores (Ferrioxamine-B, FeAerobactin) and that unchelated, inorganic Fe is a highly available source of iron. Inhibition of iron uptake by the Fe(II)-specific ligand, ferrozine, indicated that reduction of both inorganic iron and ferrisiderophore complexes occurs before transport through the plasma membrane. Measurements of iron reduction rates and the inhibitory effect of ferrozine on growth supported this conclusion. The reduction-based uptake strategy is well suited for acquiring iron from multiple complexes in dilute aquatic environments and may play an important role in other cyanobacterial strains.     

Terminal sialic acids on CD44 N‐glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains

Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein‐ligand binding involved in cell–cell and cell–matrix interactions. CD44 is a single‐pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N‐terminal extracellular hyaluronan‐binding domain (HABD); specifically, sialic acid capped N‐glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N‐glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously‐formed charge‐paired hydrogen bonding interactions between the negatively‐charged sialic acid residues and positively‐charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate. Proteins 2014; 82:3079–3089. © 2014 Wiley Periodicals, Inc.     

Iron uptake by the ichthyotoxic Chattonella marina (Raphidophyceae): impact of superoxide generation1

Significant production of superoxide, a known reductant of both inorganic and organically complexed iron(III), occurs in natural systems by both biotic and abiotic pathways. We have investigated the generation of superoxide by Chattonella marina (Subrahman.) Y. Hara et Chihara, a phytoplankton taxon known to produce high levels of this reactive oxygen species, and examined the role of superoxide in the acquisition of iron by this organism. Additionally, a generalized model for iron acquisition by C. marina has been developed, which includes three pathways of iron acquisition from organically complexed iron(III): nondissociative reductive uptake, dissociative reductive uptake, and nonreductive dissociative uptake. The model is shown to be particularly useful in ascertaining the relative importance of these various iron‐uptake pathways as a function of solution parameters including concentration and iron‐binding strength of the organic ligand and superoxide concentration. Our results suggest that superoxide can participate in the C. marina iron‐uptake process when iron is complexed to weak ligands, such as citrate, but plays only a minor role when iron is bound to a strong ligand. It thus appears that facilitation of iron acquisition is not the sole purpose of superoxide production by these organisms.     

Snow vole (Chionomys nivalis Martins) affects the redistribution of soil organic matter and hormone‐like activity in the alpine ecosystem: ecological implications

In alpine environments, colonies of snow vole (Chionomys nivalis Martins) cause strong pedoturbation, which may affect humification process and soil organic matter (SOM) cycling, with repercussions on the hormone‐like activity of organics. We investigated the effect of snow vole pedoturbation on the chemical and spectroscopic features of soil organic fractions, and the potential hormone‐like activity of humic and fulvic acids (HA, FA). The study site was located on the high‐mountain environment of the Majella massif (central Italy). Pedoturbated and regular soils were morphologically described and characterized for pH and content of total organic carbon, total extractable carbon, HA, and FA. Both HA and FA were extracted and investigated using attenuated total reflectance/Fourier transform infrared (ATR/FTIR), nuclear magnetic resonance with high‐resolution magic angle spinning (HRMAS‐NMR), and ¹H‐¹³C heteronuclear single quantum coherence (HSQC). HA and FA were also tested for their auxin‐like and gibberellin‐like activities. Results provide evidences that bioturbated and regular soils contain a poorly decomposed SOM, but HA and FA with a well‐defined molecular structure. The HA and FA from both bioturbated and regular soils show a hormone‐like activity with a different allocation along the soil profile. In the regular soil, the highest auxin‐like activity was shown by HA and FA from Oe1 horizon, while gibberellin‐like activity was expressed by FA from Oe2 horizon. Burrowing activity determines a redistribution of organics throughout the profile with a relatively high auxin‐like activity in the FA from straw tunnel wall (STW) and gibberellin‐like activity in the HA from vole feces (VF). The relative high presence of carboxylic acids, amides, proteins, and amino acids in the FA from STW and the aromatic moieties in the HA from VF put evidences for their different behavior. The fact that snow vole activity has modified the chemical and biological properties of SOM in these soils otherwise considered governed only by low temperature has important ecological implications such as the preservation of soil fertility and vegetal biodiversity.     

Switches of hydrogen bonds during ligand–protein association processes determine binding kinetics

Revealing the processes of ligand–protein associations deepens our understanding of molecular recognition and binding kinetics. Hydrogen bonds (H‐bonds) play a crucial role in optimizing ligand–protein interactions and ligand specificity. In addition to the formation of stable H‐bonds in the final bound state, the formation of transient H‐bonds during binding processes contributes binding kinetics that define a ligand as a fast or slow binder, which also affects drug action. However, the effect of forming the transient H‐bonds on the kinetic properties is little understood. Guided by results from coarse‐grained Brownian dynamics simulations, we used classical molecular dynamics simulations in an implicit solvent model and accelerated molecular dynamics simulations in explicit waters to show that the position and distribution of the H‐bond donor or acceptor of a drug result in switching intermolecular and intramolecular H‐bond pairs during ligand recognition processes. We studied two major types of HIV‐1 protease ligands: a fast binder, xk263, and a slow binder, ritonavir. The slow association rate in ritonavir can be attributed to increased flexibility of ritonavir, which yields multistep transitions and stepwise entering patterns and the formation and breaking of complex H‐bond pairs during the binding process. This model suggests the importance of conversions of spatiotemporal H‐bonds during the association of ligands and proteins, which helps in designing inhibitors with preferred binding kinetics. Copyright © 2014 John Wiley & Sons, Ltd.     

A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b

Methanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin, mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well as mbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT is truly responsible for methanobactin uptake, a knockout was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT::Gm^r mutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-type M. trichosporium OB3b, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT::Gm^r mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-type M. trichosporium OB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.     

The Effects of Fulvic Acid on Copper Bioavailability to Porcine Oviductal Epithelial Cells

This study assessed the effect of dissolved organic matter on the copper (Cu) bioavailability to mammalian cells, porcine oviductal epithelial cells (POEC), in order to imply its effect onto humans. Cu toxicity was investigated in the presence of with and without fulvic acid (FA). Dissociation and exchange rate constants were calculated by using competing ligand Chelex-100, and optical parameters were employed to help explain the complexation of their aromatic and aliphatic structures. Their morphological change was observed using transmission electron microscope (TEM), and Cu species were calculated using MINTEQA2 program. The results showed that the dissociation rate constant of Cu²⁺–FA was equal to 9.08?×?10^?4?s^?1, which was slower than the exchange rate at 1.95?×?10^?3?s^?1. Although Cu–FA was significantly absorbed into the cells higher than Cu²⁺, it showed less damage than tested with Cu²⁺. TEM and optical studies showed many aggregations around nucleus suggesting the amphipathic character of FA helped binging to the nuclear surfaces of both Cu–FA and FA treatments. Even though the MINTEQA2 calculations showed that there was free Cu²⁺ in the mixed solutions around 39.2%, it could not bind with the cell surface. This suggested that the effect of FA was strong and had a lot of influence on the living surface of POEC, modifying the effect of Cu toxicity.