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MnxG, a multicopper oxidase, is an enzyme from the marine Bacillus species, which produces manganese oxide minerals through the aerobic oxidation of dissolved Mn2+ − a key process in global manganese geochemical cycling. When isolated in an active form as a part of the MnxEFG protein complex, the enzymatic activity of MnxG is substantially enhanced by mild oxidative priming. Herein, the mechanism for this effect is probed by using direct current (dc) and Fourier transformed alternating current (ac) voltammetric analysis of the MnxEFG complex and the catalytically inactive MnxEF subunit immobilised on a carbon electrode. Analysis of these ac voltammetric data reveals a significant enhancement in the rate of electron transfer in the Type 2 Cu sites upon oxidative priming of the enzyme, which is attributed to the improved catalytic activity of MnxG in the MnxEFG protein complex.

The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O 2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The β-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE 3 F 3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.

The previously uncultured phylum "Candidatus Eremiobacterota" is globally distributed and often abundant in oligotrophic environments. Although it includes lineages with the genetic potential for photosynthesis, one of the most important metabolic pathways on Earth, the absence of pure cultures has limited further insights into its ecological and physiological traits. We report the first successful isolation of a "Ca. Eremiobacterota" strain from a fumarolic ice cave on Mt. Erebus volcano (Antarctica). Polyphasic analysis revealed that this organism is an aerobic anoxygenic photoheterotrophic bacterium with a unique lifestyle, including bacteriochlorophyll a production, CO 2 fixation, a high CO 2 requirement, and phototactic motility using type IV-pili, all of which are highly adapted to polar and fumarolic environments. The cells are rods or filaments with a vesicular type intracytoplasmic membrane system. The genome encodes novel anoxygenic Type II photochemical reaction centers and bacteriochlorophyll synthesis proteins, forming a deeply branched monophyletic clade distinct from known phototrophs. The first cultured strain of the eighth phototrophic bacterial phylum which we name Vulcanimicrobium alpinus gen. nov., sp. nov. advances our understanding of ecology and evolution of photosynthesis., (© 2022. The Author(s).)

Mixing of waters of different ionic strengths induces the geochemical cycling of reactive elements. The most reactive zone is where the gradient in ionic strength is steepest. In oxygenated systems, the redox-active metal manganese cycles between soluble and particulate fractions through three oxidation states, manganese(II), manganese(III) and manganese(IV). This cycling strongly affects the mobility of inorganic and organic chemicals. The most accessible environmental system where waters with different ionic strengths mix are estuaries. During six Eulerian studies in the Columbia River Estuary, each up to 26 h, we measured manganese speciation and concentration across a salinity (S P ) gradient centred around S P  = 0.06-6, equivalent to a seawater ionic strength (I S p ) of 1.2-120 mM. This zone, representing the region between freshwater and the more intensively studied estuarine turbidity maximum, presents a highly dynamic geochemical environment in which the manganese cycle propagates through four steps as I S p increases due to mixing: 1. Before a measurable change in I S p , manganese, as particulate manganese(III/IV) oxides (MnO x ), undergoes reduction, independent of photochemical processes, to soluble manganese(III) stabilized in organic complexes (Mn(III)-L) and manganese(II); 2. As I S p increases between 5 and 80 mM, Mn(III)-L reduction continues and manganese(II) adsorbs onto particle surfaces; 3. As I S p increases further, though remaining below 80 mM (S P ≈ 4), adsorbed manganese(II) desorbs and/or is oxidized and is released as Mn(III)-L or oxidises further to MnO x ; 4. The breakdown of Mn(III)-L complexes leads to higher manganese(II) and MnO x , which at Mid-Estuary-Salinities (I S p  = 320-480 mM) precipitates. This manganese cycling in low I S p waters directly affects a system's redox chemistry and provides a window into understanding the extensive, yet hidden, freshwater/saline water interface in aquifers, soils, sediments and estuaries., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The manganese oxidase complex, Mnx, from Bacillus sp. PL-12 contains a multicopper oxidase (MCO) and oxidizes dissolved Mn(II) to form insoluble manganese oxide (MnO 2 ) mineral. Previous kinetic and spectroscopic analyses have shown that the enzyme's mechanism proceeds through an activation step that facilitates formation of a series of binuclear Mn complexes in the oxidation states II, III, and IV on the path to MnO 2 formation. We now demonstrate that the enzyme is inhibited by first-row transition metals in the order of the Irving-Williams series. Zn(II) strongly (K i ~ 1.5 μM) inhibits both activation and turnover steps, as well as the rate of Mn(II) binding. The combined Zn(II) and Mn(II) concentration dependence establishes that the inhibition is non-competitive. This result is supported by electron paramagnetic resonance (EPR) spectroscopy, which reveals unaltered Mnx-bound Mn(II) EPR signals, both mono- and binuclear, in the presence of Zn(II). We infer that inhibitory metals bind at a site separate from the substrate sites and block the conformation change required to activate the enzyme, a case of allosteric inhibition. The likely biological role of this inhibitory site is discussed in the context of Bacillus spore physiology. While Cu(II) inhibits Mnx strongly, in accord with the Irving-Williams series, it increases Mnx activation at low concentrations, suggesting that weakly bound Cu, in addition to the four canonical MCO-Cu, may support enzyme activity, perhaps as an electron transfer agent., (Copyright © 2021. Published by Elsevier Inc.)

In terms of its oxidative strength, the MnO 2 /Mn 2+ couple is one of the strongest in the aquatic environment. The intermediate oxidation state, manganese(III), is stabilized by a range of organic ligands (Mn(III)-L) and some of these complexes are also strong oxidants or reductants. Here, we present improved methods for quantifying soluble reactive oxidized manganese(III) and particulate reactive oxidized manganese at ultra-low concentrations; the respective detection limits are 6.7 nM and 7 pM (100-cm spectrophotometric path length) and 260 nM and 2.6 nM (1-cm path length). The methods involve a simple, specific, spectrophotometric technique using a water-soluble leuco base (leucoberbelin blue; LBB). LBB is oxidized by manganese through a hydrogen atom transfer reaction forming a colored complex that is stoichiometrically related to the oxidation state of the manganese, either Mn(III)-L or manganese(III,IV) oxides (MnO x ). At the concentration of LBB used in this study, nitrite may be a minor interference, so we provide concentration ranges over which it interferes and suggest potential strategies to mitigate the interference. Unlike previous methods devised to quantify Mn(III)-L, which use ligand exchange reactions, the LBB oxidation requires an electron and therefore needs to physically contact manganese(III) for inner-sphere electron transfer to occur. The method for measuring soluble Mn(III)-L was evaluated in the laboratory, and LBB was found to be oxidized by an extensive suite of weak Mn(III)-L complexes, as it is by MnO x , but could not react with or reacted very slowly with strong Mn(III)-L complexes. According to the molecular structures of the Mn(III)-L complexes tested, LBB can also be used to qualitatively assess the binding strength of Mn(III)-L complexes based on metal-chelate structural considerations. The assays for soluble Mn(III)-L (membrane filtered) and particulate manganese oxides (trapped by membrane filters) were applied to the well-oxygenated estuarine waters of the Saguenay Fjord, a major tributary of the Lower St. Lawrence Estuary, and to Western North Atlantic oceanic waters, off the continental shelf, where there is an oxygen minimum zone (< 67% O 2 saturation). The methods applied can be used in the field or onboard ships and provide important new insights into oxidized manganese speciation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

MnO 2 nanoparticles, similar to those found in soils and sediments, have been characterized via their UV-visible and Raman spectra, combined with dynamic light scattering and reactivity measurements. Synthetic colloids were prepared by thiosulfate reduction of permanganate, their sizes controlled with adsorbates acting as capping agents: bicarbonate, phosphate, and pyrophosphate. Biogenic colloids, products of the manganese oxidase, Mnx, were similarly characterized. The band-gap energies of the colloids were found to increase with decreasing hydrodynamic diameter, D h , and were proportional to 1/ D h 2 , as predicted from quantum confinement theory. The intensity ratio of the two prominent Mn-O stretching Raman bands also varied with particle size, consistent with the ratio of edge to bulk Mn atoms. Reactivity of the synthetic colloids toward reduction by Mn 2+ , in the presence of pyrophosphate to trap the Mn 3+ product, was proportional to the surface to volume ratio, but showed surprising complexity. There was also a remnant unreactive fraction, likely attributable to Mn(III)-induced surface passivation. The band gap was similar for biogenic and synthetic colloids of similar size, but decreased when the enzyme solution contained pyrophosphate, which traps the intermediate Mn(III) and slows MnO 2 growth. The band gap/size correlation was used to analyze the growth of the enzymatically produced MnO 2 oxides.

The multi-copper oxidase (MCO) MnxG from marine Bacillus bacteria plays an essential role in geochemical cycling of manganese by oxidizing Mn 2+ (aq) to form manganese oxide minerals at rates that are three to five orders of magnitude faster than abiotic rates. The MCO MnxG protein is isolated as part of a multi-protein complex, denoted as Mnx, which includes one MnxG unit and a hexamer of MnxE 3 F 3 subunit. During the oxidation of Mn 2+ (aq) catalyzed by the Mnx protein complex, an enzyme-bound Mn(III) species was trapped recently in the presence of pyrophosphate (PP) and analyzed using parallel-mode electron paramagnetic resonance (EPR) spectroscopy. Herein, we provide a full analysis of this enzyme-bound Mn(III) intermediate via temperature dependence studies and spectral simulations. This Mnx-bound Mn(III) species is characterized by a hyperfine-coupling value of A( 55 Mn) = 4.2 mT (corresponding to 120 MHz) and a negative zero-field splitting (ZFS) value of D = - 2.0 cm -1 . These magnetic properties suggest that the Mnx-bound Mn(III) species could be either six-coordinate with a 5 B 1g ground state or square-pyramidal five-coordinate with a 5 B 1 ground state. In addition, as a control, Mn(III)PP is also analyzed by parallel-mode EPR spectroscopy. It exhibits distinctly different magnetic properties with a hyperfine-coupling value of A( 55 Mn) = 4.8 mT (corresponding to 140 MHz) and a negative ZFS value of D = - 2.5 cm -1 . The different ZFS values suggest differences in ligand environment of Mnx-bound Mn(III) and aqueous Mn(III)PP species. These studies provide further insights into the mechanism of biological Mn 2+ (aq) oxidation.

Dissolved Mn(III) has been identified at all stages throughout a Water Treatment Works (WTW) receiving inflow from a peaty upland catchment in NE England. Ninety percent of the influent total manganese into the WTW is particulate Mn, in the form of Mn oxide (>0.2 μm). Approximately 9% (mean value, n = 22, range of 0-100%) of the dissolved (<0.2 μm) influent Mn is present as dissolved Mn(III). Mn(III) concentrations are highest (mean of 49% of total dissolved Mn; n = 26, range of 17-89%) within the WTW where water comes into contact with the organic-rich sludges which are produced as waste products in the WTW. These Mn(III)-containing wastewaters are recirculated to the head of the works and constitute a large input of Mn(III) into the WTW. This is the first report of Mn(III) being identified in a WTW. The ability of Mn(III) to act as both an oxidant and a reductant is of interest to the water industry. Understanding the formation and removal of Mn(III) within may help reduce Mn oxide deposits in pipe networks. Further understanding how the ratio of Mn(III) to Mn(II) can be used to optimise dissolved Mn removal would save the water industry significant money in reducing discoloration 'events' at the customers' tap., (Copyright © 2018. Published by Elsevier Ltd.)

The observation of significant concentrations of soluble Mn(III) complexes in oxic, suboxic, and some anoxic waters has triggered a re-evaluation of the previous Mn paradigm which focused on the cycling between soluble Mn(II) and insoluble Mn(III,IV) species as operationally defined by filtration. Though Mn(II) oxidation in aquatic environments is primarily bacterially-mediated, little is known about the effect of Mn(III)-binding ligands on Mn(II) oxidation nor on the formation and removal of Mn(III). Pseudomonas putida GB-1 is one of the most extensively investigated of all Mn(II) oxidizing bacteria, encoding genes for three Mn oxidases (McoA, MnxG, and MopA). P. putida GB-1 and associated Mn oxidase mutants were tested alongside environmental isolates Pseudomonas hunanensis GSL-007 and Pseudomonas sp. GSL-010 for their ability to both directly oxidize weakly and strongly bound Mn(III), and to form these complexes through the oxidation of Mn(II). Using Mn(III)-citrate (weak complex) and Mn(III)-DFOB (strong complex), it was observed that P. putida GB-1, P. hunanensis GSL-007 and Pseudomonas sp. GSL-010 and mutants expressing only MnxG and McoA were able to directly oxidize both species at varying levels; however, no oxidation was detected in cultures of a P. putida mutant expressing only MopA. During cultivation in the presence of Mn(II) and citrate or DFOB, P. putida GB-1, P. hunanensis GSL-007 and Pseudomonas sp. GSL-010 formed Mn(III) complexes transiently as an intermediate before forming Mn(III/IV) oxides with the overall rates and extents of Mn(III,IV) oxide formation being greater for Mn(III)-citrate than for Mn(III)-DFOB. These data highlight the role of bacteria in the oxidative portion of the Mn cycle and suggest that the oxidation of strong Mn(III) complexes can occur through enzymatic mechanisms involving multicopper oxidases. The results support the observations from field studies and further emphasize the complexity of the geochemical cycling of manganese.

Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes. Graphical Abstract ᅟ.

Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase in Bacillus sp. PL-12, Mnx, is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins, MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. Here, we show that native mass spectrometry defines the subunit topology and copper binding of Mnx, while high-resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for understanding Mn biomineralization by such unexplored enzymes.Significant challenges exist for structural characterization of enzymes responsible for biomineralization. Here the authors show that native mass spectrometry and high resolution electron microscopy can define the subunit topology and copper binding of a manganese oxidizing complex, and describe early stage formation of its mineral products.

Influence of the conditions for aerobic oxidation of Mn2+(aq) catalysed by the MnxEFG protein complex on the morphology, structure and reactivity of the resulting biogenic manganese oxides (MnO x ) is explored. Physical characterisation of MnO x includes scanning and transmission electron microscopy, and X-ray photoelectron and K-edge Mn, Fe X-ray absorption spectroscopy. This characterisation reveals that the MnO x materials share the structural features of birnessite, yet differ in the degree of structural disorder. Importantly, these biogenic products exhibit strikingly different morphologies that can be easily controlled. Changing the substrate-to-protein ratio produces MnO x either as nm-thin sheets, or rods with diameters below 20 nm, or a combination of the two. Mineralisation in solutions that contain Fe2+(aq) makes solids with significant disorder in the structure, while the presence of Ca2+(aq) facilitates formation of more ordered materials. The (photo)oxidation and (photo)electrocatalytic capacity of the MnO x minerals is examined and correlated with their structural properties., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The bacterial protein complex Mnx contains a multicopper oxidase (MCO) MnxG that, unusually, catalyzes the two-electron oxidation of Mn(II) to MnO 2 biomineral, via a Mn(III) intermediate. Although Mn(III)/Mn(II) and Mn(IV)/Mn(III) reduction potentials are expected to be high, we find a low reduction potential, 0.38 V (vs Normal Hydrogen Electrode, pH 7.8), for the MnxG type 1 Cu 2+ , the electron acceptor. Indeed the type 1 Cu 2+ is not reduced by Mn(II) in the absence of molecular oxygen, indicating that substrate oxidation requires an activation step. We have investigated the enzyme mechanism via electronic absorption spectroscopy, using chemometric analysis to separate enzyme-catalyzed MnO 2 formation from MnO 2 nanoparticle aging. The nanoparticle aging time course is characteristic of nucleation and particle growth; rates for these processes followed expected dependencies on Mn(II) concentration and temperature, but exhibited different pH optima. The enzymatic time course is sigmoidal, signaling an activation step, prior to turnover. The Mn(II) concentration and pH dependence of a preceding lag phase indicates weak Mn(II) binding. The activation step is enabled by a pK a > 8.6 deprotonation, which is assigned to Mn(II)-bound H 2 O; it induces a conformation change (consistent with a high activation energy, 106 kJ/mol) that increases Mn(II) affinity. Mnx activation is proposed to decrease the Mn(III/II) reduction potential below that of type 1 Cu(II/I) by formation of a hydroxide-bridged binuclear complex, Mn(II)(μ-OH)Mn(II), at the substrate site. Turnover is found to depend cooperatively on two Mn(II) and is enabled by a pK a 7.6 double deprotonation. It is proposed that turnover produces a Mn(III)(μ-OH) 2 Mn(III) intermediate that proceeds to the enzyme product, likely Mn(IV)(μ-O) 2 Mn(IV) or an oligomer, which subsequently nucleates MnO 2 nanoparticles. We conclude that Mnx exploits manganese polynuclear chemistry in order to facilitate an otherwise difficult oxidation reaction, as well as biomineralization. The mechanism of the Mn(III/IV) conversion step is elucidated in an accompanying paper .

The bacterial manganese oxidase MnxG of the Mnx protein complex is unique among multicopper oxidases (MCOs) in carrying out a two-electron metal oxidation, converting Mn(II) to MnO 2 nanoparticles. The reaction occurs in two stages: Mn(II) → Mn(III) and Mn(III) → MnO 2 . In a companion study , we show that the electron transfer from Mn(II) to the low-potential type 1 Cu of MnxG requires an activation step, likely forming a hydroxide bridge at a dinuclear Mn(II) site. Here we study the second oxidation step, using pyrophosphate (PP) as a Mn(III) trap. PP chelates Mn(III) produced by the enzyme and subsequently allows it to become a substrate for the second stage of the reaction. EPR spectroscopy confirms the presence of Mn(III) bound to the enzyme. The Mn(III) oxidation step does not involve direct electron transfer to the enzyme from Mn(III), which is shown by kinetic measurements to be excluded from the Mn(II) binding site. Instead, Mn(III) is proposed to disproportionate at an adjacent polynuclear site, thereby allowing indirect oxidation to Mn(IV) and recycling of Mn(II). PP plays a multifaceted role, slowing the reaction by complexing both Mn(II) and Mn(III) in solution, and also inhibiting catalysis, likely through binding at or near the active site. An overall mechanism for Mnx-catalyzed MnO 2 production from Mn(II) is presented.

Manganese-oxide minerals (MnO x ) are widely distributed over the Earth's surface, and their geochemical cycling is globally important. A multicopper oxidase (MCO) MnxG protein from marine Bacillus bacteria plays an essential role in producing MnO x minerals by oxidizing Mn 2+ (aq) at rates that are 3 to 5 orders of magnitude faster than abiotic rates. The MnxG protein is isolated as part of a multiprotein complex denoted as "Mnx" that includes accessory protein subunits MnxE and MnxF, with an estimated stoichiometry of MnxE 3 F 3 G and corresponding molecular weight of ≈211 kDa. Herein, we report successful expression and isolation of the MCO MnxG protein without the E 3 F 3 hexamer. This isolated MnxG shows activity for Mn 2+ (aq) oxidation to form manganese oxides. The complement of paramagnetic Cu(II) ions in the Mnx protein complex was examined by electron paramagnetic resonance (EPR) spectroscopy. Two distinct classes of type 2 Cu sites were detected. One class of Cu(II) site (denoted as T2Cu-A), located in the MnxG subunit, is identified by the magnetic parameters g ∥ = 2.320 and A ∥ = 510 MHz. The other class of Cu(II) sites (denoted as T2Cu-B) is characterized by g ∥ = 2.210 and A ∥ = 615 MHz and resides in the putative hexameric MnxE 3 F 3 subunit. These different magnetic properties correlate with the differences in the reduction potentials of the respective Cu(II) centers. These studies provide new insights into the molecular mechanism of manganese biomineralization.

Phylogenetically and metabolically diverse bacterial communities have been found in association with submarine basaltic glass surfaces. The driving forces behind basalt colonization are for the most part unknown. It remains ambiguous if basalt provides ecological advantages beyond representing a substrate for surface colonization, such as supplying nutrients and/or energy. Pseudomonas stutzeri VS-10, a metabolically versatile bacterium isolated from Vailulu'u Seamount, was used as a model organism to investigate the physiological responses observed when biofilms are established on basaltic glasses. In Fe-limited heterotrophic media, P. stutzeri VS-10 exhibited elevated growth in the presence of basaltic glass. Diffusion chamber experiments demonstrated that physical attachment or contact of soluble metabolites such as siderophores with the basaltic glass plays a pivotal role in this process. Electrochemical data indicated that P. stutzeri VS-10 is able to use solid substrates (electrodes) as terminal electron donors and acceptors. Siderophore production and heterotrophic Fe(II) oxidation are discussed as potential mechanisms enhancing growth of P. stutzeri VS-10 on glass surfaces. In correlation with that we discuss the possibility that metabolic versatility could represent a common and beneficial physiological trait in marine microbial communities being subject to oligotrophic and rapidly changing deep-sea conditions.

Manganese(ii) oxidation in the environment is thought to be driven by bacteria because enzymatic catalysis is many orders of magnitude faster than the abiotic processes. The heterologously purified Mn oxidase (Mnx) from marine Bacillus sp. PL-12 is made up of the multicopper oxidase (MCO) MnxG and two small Cu and heme-binding proteins of unknown function, MnxE and MnxF. Mnx binds Cu and oxidizes both Mn(ii) and Mn(iii), generating Mn(iv) oxide minerals that resemble those found on the Bacillus spore surface. Spectroscopic techniques have illuminated details about the metallo-cofactors of Mnx, but very little is known about their requirement for catalytic activity, and even less is known about the substrate specificity of Mnx. Here we quantify the canonical MCO Cu and persistent peripheral Cu bound to Mnx, and test Mnx oxidizing ability toward different substrates at varying pH. Mn(ii) appears to be the best substrate in terms of k cat , but its oxidation does not follow Michaelis-Menten kinetics, instead showing a sigmoidal cooperative behavior. Mnx also oxidizes Fe(ii) substrate, but in a Michaelis-Menten manner and with a decreased activity, as well as organic substrates. The reduced metals are more rapidly consumed than the larger organic substrates, suggesting the hypothesis that the Mnx substrate site is small and tuned for metal oxidation. Of biological relevance is the result that Mnx has the highest catalytic efficiency for Mn(ii) at the pH of sea water, especially when the protein is loaded with greater than the requisite four MCO copper atoms, suggesting that the protein has evolved specifically for Mn oxidation.

In a natural geochemical cycle, manganese-oxide minerals (MnO x ) are principally formed through a microbial process, where a putative multicopper oxidase MnxG plays an essential role. Recent success in isolating the approximately 230 kDa, enzymatically active MnxEFG protein complex, has advanced our understanding of biogenic MnO x mineralization. Here, the kinetics of MnO x formation catalyzed by MnxEFG are examined using a quartz crystal microbalance (QCM), and the first electrochemical characterization of the MnxEFG complex is reported using Fourier transformed alternating current voltammetry. The voltammetric studies undertaken using near-neutral solutions (pH 7.8) establish the apparent reversible potentials for the Type 2 Cu sites in MnxEFG immobilized on a carboxy-terminated monolayer to be in the range 0.36-0.40 V versus a normal hydrogen electrode. Oxidative priming of the MnxEFG protein complex substantially enhances the enzymatic activity, as found by in situ electrochemical QCM analysis. The biogeochemical significance of this enzyme is clear, although the role of an oxidative priming of catalytic activity might be either an evolutionary advantage or an ancient relic of primordial existence., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes., Importance: The identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a regulator involved in both flagellum synthesis and biofilm synthesis in Pseudomonas aeruginosa Therefore, these results are also an important step toward understanding the regulation of Mn(II) oxidation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Bacteria are one of the premier biological forces that, in combination with chemical and physical forces, drive metal availability in the environment. Bacterial spores, when found in the environment, are often considered to be dormant and metabolically inactive, in a resting state waiting for favorable conditions for them to germinate. However, this is a highly oversimplified view of spores in the environment. The surface of bacterial spores represents a potential site for chemical reactions to occur. Additionally, proteins in the outer layers (spore coats or exosporium) may also have more specific catalytic activity. As a consequence, bacterial spores can play a role in geochemical processes and may indeed find uses in various biotechnological applications. The aim of this review is to introduce the role of bacteria and bacterial spores in biogeochemical cycles and their potential use as toxic metal bioremediation agents.

Multicopper oxidases (MCOs) catalyze the oxidation of a diverse group of metal ions and organic substrates by successive single-electron transfers to O2 via four bound Cu ions. MnxG, which catalyzes MnO2 mineralization by oxidizing both Mn(II) and Mn(III), is unique among multicopper oxidases in that it carries out two energetically distinct electron transfers and is tightly bound to accessory proteins. There are two of these, MnxE and MnxF, both approximately 12kDa. Although their sequences are similar to those found in the genomes of several Mn-oxidizing Bacillus species, they are dissimilar to those of proteins with known function. Here, MnxE and MnxF are co-expressed independent of MnxG and are found to oligomerize into a higher order stoichiometry, likely a hexamer. They bind copper and heme, which have been characterized by electron paramagnetic resonance (EPR), X-ray absorption spectroscopy (XAS), and UV-visible (UV-vis) spectrophotometry. Cu is found in two distinct type 2 (T2) copper centers, one of which appears to be novel; heme is bound as a low-spin species, implying coordination by two axial ligands. MnxE and MnxF do not oxidize Mn in the absence of MnxG and are the first accessory proteins to be required by an MCO. This may indicate that Cu and heme play roles in electron transfer and/or Cu trafficking., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lateral bays of the lower Columbia River estuary are areas of enhanced water retention that influence net ecosystem metabolism through activities of their diverse microbial communities. Metagenomic characterization of sediment microbiota from three disparate sites in two brackish lateral bays (Baker and Youngs) produced ∼100 Gbp of DNA sequence data analyzed subsequently for predicted SSU rRNA and peptide-coding genes. The metagenomes were dominated by Bacteria. A large component of Eukaryota was present in Youngs Bay samples, i.e., the inner bay sediment was enriched with the invasive New Zealand mudsnail, Potamopyrgus antipodarum, known for high ammonia production. The metagenome was also highly enriched with an archaeal ammonia oxidizer closely related to Nitrosoarchaeum limnia. Combined analysis of sequences and continuous, high-resolution time series of biogeochemical data from fixed and mobile platforms revealed the importance of large-scale reciprocal particle exchanges between the mainstem estuarine water column and lateral bay sediments. Deposition of marine diatom particles in sediments near Youngs Bay mouth was associated with a dramatic enrichment of Bacteroidetes (58% of total Bacteria) and corresponding genes involved in phytoplankton polysaccharide degradation. The Baker Bay sediment metagenome contained abundant Archaea, including diverse methanogens, as well as functional genes for methylotrophy and taxonomic markers for syntrophic bacteria, suggesting that active methane cycling occurs at this location. Our previous work showed enrichments of similar anaerobic taxa in particulate matter of the mainstem estuarine water column. In total, our results identify the lateral bays as both sources and sinks of biogenic particles significantly impacting microbial community composition and biogeochemical activities in the estuary.

The dynamics of manganese solid formation (as MnOx) by the multicopper oxidase (MCO)-containing Mnx protein complex were examined by electron paramagnetic resonance (EPR) spectroscopy. Continuous-wave (CW) EPR spectra of samples of Mnx, prepared in atmosphere and then reacted with Mn(II) for times ranging from 7 to 600 s, indicate rapid oxidation of the substrate manganese (with two-phase pseudo-first-order kinetics modeled using rate coefficients of: k(1obs) = 0.205 ± 0.001 s(-1) and k(2obs) = 0.019 ± 0.001 s(-1)). This process occurs on approximately the same time scale as in vitro solid MnOx formation when there is a large excess of Mn(II). We also found CW and pulse EPR spectroscopic evidence for at least three classes of Mn(II)-containing species in the reaction mixtures: (i) aqueous Mn(II), (ii) a specifically bound mononuclear Mn(II) ion coordinated to the Mnx complex by one nitrogenous ligand, and (iii) a weakly exchange-coupled dimeric Mn(II) species. These findings provide new insights into the molecular mechanism of manganese mineralization.

The Earth's crust hosts a subsurface, dark, and oligotrophic biosphere that is poorly understood in terms of the energy supporting its biomass production and impact on food webs at the Earth's surface. Dark oligotrophic volcanic ecosystems (DOVEs) are good environments for investigations of life in the absence of sunlight as they are poor in organics, rich in chemical reactants and well known for chemical exchange with Earth's surface systems. Ice caves near the summit of Mt. Erebus (Antarctica) offer DOVEs in a polar alpine environment that is starved in organics and with oxygenated hydrothermal circulation in highly reducing host rock. We surveyed the microbial communities using PCR, cloning, sequencing and analysis of the small subunit (16S) ribosomal and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RubisCO) genes in sediment samples from three different caves, two that are completely dark and one that receives snow-filtered sunlight seasonally. The microbial communities in all three caves are composed primarily of Bacteria and fungi; Archaea were not detected. The bacterial communities from these ice caves display low phylogenetic diversity, but with a remarkable diversity of RubisCO genes including new deeply branching Form I clades, implicating the Calvin-Benson-Bassham (CBB) cycle as a pathway of CO2 fixation. The microbial communities in one of the dark caves, Warren Cave, which has a remarkably low phylogenetic diversity, were analyzed in more detail to gain a possible perspective on the energetic basis of the microbial ecosystem in the cave. Atmospheric carbon (CO2 and CO), including from volcanic emissions, likely supplies carbon and/or some of the energy requirements of chemoautotrophic microbial communities in Warren Cave and probably other Mt. Erebus ice caves. Our work casts a first glimpse at Mt. Erebus ice caves as natural laboratories for exploring carbon, energy and nutrient sources in the subsurface biosphere and the nutritional limits on life.

Our previous study showed that formation of TcS2-like phases is favored over TcO2 under sulfidic conditions stimulated by nano zerovalent iron. This study further investigates the stability of Tc(IV) sulfide upon reoxidation by solution chemistry, solid phase characterization, and X-ray absorption spectroscopy. Tc dissolution data showed that Tc(VII) reduced by sulfide-transformed nZVI has substantially slower reoxidation kinetics than Tc(VII) reduced by nZVI only. The initial inhibition of Tc(IV) dissolution at S/Fe = 0.112 is due to the redox buffer capacity of FeS, which is evidenced by the parallel trends in oxidation-reduction potentials (ORP) and Tc dissolution kinetics. The role of FeS in inhibiting Tc oxidation is further supported by the Mössbauer spectroscopy and micro X-ray diffraction data at S/Fe = 0.112, showing persistence of FeS after 24-h oxidation but complete oxidation after 120-h oxidation. X-ray absorption spectroscopy data for S/Fe = 0.011 showed significantly increasing percentages of TcS2 in the solid phase after 24-h oxidation, indicating stronger resistance of TcS2 to oxidation. At S/Fe = 0.112, the XAS results revealed significant transformation of Tc speciation from TcS2 to TcO2 after 120-h oxidation. Given that no apparent Tc dissolution occurred during this period, the speciation transformation might play a secondary role in hindering Tc oxidation. Collectively, the results indicate that sequestrating Tc as TcS2 under stimulated sulfate reduction is a promising strategy to improve the long-term stability of reduced Tc in subsurface remediation.

Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium that produces pyoverdine-type siderophores (PVDs), which facilitate the uptake of Fe(III) but also influence MnO2 formation. Recently, a non-ribosomal peptide synthetase mutant that does not synthesize PVD was described. Here we identified a gene encoding the PVDGB-1 (PVD produced by strain GB-1) uptake receptor (PputGB1&#95;4082) of strain GB-1 and confirmed its function by in-frame mutagenesis. Growth and other physiological responses of these two mutants and of wild type were compared during cultivation in the presence of three chemically distinct sets of PVDs (siderotypes n°1, n°2, and n°4) derived from various pseudomonads. Under iron-limiting conditions, Fe(III) complexes of various siderotype n°1 PVDs (including PVDGB-1) allowed growth of wild type and the synthetase mutant, but not the receptor mutant, confirming that iron uptake with any tested siderotype n°1 PVD depended on PputGB1&#95;4082. Fe(III) complexes of a siderotype n°2 PVD were not utilized by any strain and strongly induced PVD synthesis. In contrast, Fe(III) complexes of siderotype n°4 PVDs promoted the growth of all three strains and did not induce PVD synthesis by the wild type, implying these complexes were utilized for iron uptake independent of PputGB1&#95;4082. These differing properties of the three PVD types provided a way to differentiate between effects on MnO2 formation that resulted from iron limitation and others that required participation of the PVDGB-1 receptor. Specifically, MnO2 production was inhibited by siderotype n°1 but not n°4 PVDs indicating PVD synthesis or PputGB1&#95;4082 involvement rather than iron-limitation caused the inhibition. In contrast, iron limitation was sufficient to explain the inhibition of Mn(II) oxidation by siderotype n°2 PVDs. Collectively, our results provide insight into how competition for iron via siderophores influences growth, iron nutrition and MnO2 formation in more complex environmental systems.

Groundwater composition and coupled redox cycles can affect the long-term stability of U(IV) products from bioremediation. The effects of Mn(II), a redox active cation present at uranium-contaminated sites, on UO2 dissolution in both oxic and anoxic systems were investigated using batch and continuous-flow reactors. Under anoxic conditions Mn(II) inhibited UO2 dissolution, which was probably due to adsorption of Mn(II) and precipitation of MnCO3 that decreased exposure of U(IV) surface sites to oxidants. In contrast, Mn(II) promoted UO2 dissolution under oxic conditions through Mn redox cycling. Oxidation of Mn(II) by O2 produced reactive Mn species, possibly short-lived Mn(III) in solution or at the surface, that oxidatively dissolved the UO2 more rapidly than could the O2 alone. At pH 8 the Mn cycling was such that there was no measurable accumulation of particulate Mn oxides. At pH 9 Mn oxides could be produced and accumulate, while they were continuously reduced by UO2, with Mn(II) returning to the aqueous phase. With the rapid turnover of Mn in the redox cycle, concentrations of Mn as low as 10 μM could maintain an enhanced UO2 dissolution rate. The presence of the siderophore desferrioxamine B (a strong Mn(III)-complexing ligand) effectively decoupled the redox interactions of uranium and manganese to suppress the promotional effect of Mn(II).

When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS, and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase (NRPS), coded by the operon PputGB1&#95;4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1&#95;4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1&#95;4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group, and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III).

The stability of UO2 is critical to the success of reductive bioremediation of uranium. When reducing conditions are no longer maintained, Mn redox cycling may catalytically mediate the oxidation of UO2 and remobilization of uranium. Ligand-stabilized soluble Mn(III) was recently recognized as an important redox-active intermediate in Mn biogeochemical cycling. This study evaluated the kinetics of oxidative UO2 dissolution by soluble Mn(III) stabilized by pyrophosphate (PP) and desferrioxamine B (DFOB). The Mn(III)-PP complex was a potent oxidant that induced rapid UO2 dissolution at a rate higher than that by a comparable concentration of dissolved O2. However, the Mn(III)-DFOB complex was not able to induce oxidative dissolution of UO2. The ability of Mn(III) complexes to oxidize UO2 was probably determined by whether the coordination of Mn(III) with ligands allowed the attachment of the complexes to the UO2 surface to facilitate electron transfer. Systematic investigation into the kinetics of UO2 oxidative dissolution by the Mn(III)-PP complex suggested that Mn(III) could directly oxidize UO2 without involving particulate Mn species (e.g., MnO2). The expected 2:1 reaction stoichiometry between Mn(III) and UO2 was observed. The reactivity of soluble Mn(III) in oxidizing UO2 was higher at lower ratios of pyrophosphate to Mn(III) and lower pH, which is probably related to differences in the ligand-to-metal ratio and/or protonation states of the Mn(III)-pyrophosphate complexes. Disproportionation of Mn(III)-PP occurred at pH 9.0, and the oxidation of UO2 was then driven by both MnO2 and soluble Mn(III). Kinetic models were derived that provided excellent fits of the experimental results.

Soluble manganese(III) [Mn(III)] can potentially serve as both oxidant and reductant in one-electron-transfer reactions with other redox species. In near-surface sediment porewater, it is often overlooked as a major component of Mn cycling. Applying a spectrophotometric kinetic method to hemipelagic sediments from the Laurentian Trough (Quebec, Canada), we found that soluble Mn(III), likely stabilized by organic or inorganic ligands, accounts for up to 90% of the total dissolved Mn pool. Vertical profiles of dissolved oxygen and dissolved and solid Mn suggest that soluble Mn(III) is primarily produced via oxidation of Mn(II) diffusing upwards from anoxic sediments with lesser contributions from biotic and abiotic reductive dissolution of MnO2. The conceptual model of the sedimentary redox cycle should therefore explicitly include dissolved Mn(III).

Reactive Mn(IV) oxide minerals are ubiquitous in the environment and control the bioavailability and distribution of many toxic and essential elements and organic compounds. Their formation is thought to be dependent on microbial enzymes, because spontaneous Mn(II) to Mn(IV) oxidation is slow. Several species of marine Bacillus spores oxidize Mn(II) on their exosporium, the outermost layer of the spore, encrusting them with Mn(IV) oxides. Molecular studies have identified the mnx (Mn oxidation) genes, including mnxG, encoding a putative multicopper oxidase (MCO), as responsible for this two-electron oxidation, a surprising finding because MCOs only catalyze single-electron transfer reactions. Characterization of the enzymatic mechanism has been hindered by the lack of purified protein. By purifying active protein from the mnxDEFG expression construct, we found that the resulting enzyme is a blue (absorption maximum 590 nm) complex containing MnxE, MnxF, and MnxG proteins. Further, by analyzing the Mn(II)- and (III)-oxidizing activity in the presence of a Mn(III) chelator, pyrophosphate, we found that the complex facilitates both electron transfers from Mn(II) to Mn(III) and from Mn(III) to Mn(IV). X-ray absorption spectroscopy of the Mn mineral product confirmed its similarity to Mn(IV) oxides generated by whole spores. Our results demonstrate that Mn oxidation from soluble Mn(II) to Mn(IV) oxides is a two-step reaction catalyzed by an MCO-containing complex. With the purification of active Mn oxidase, we will be able to uncover its mechanism, broadening our understanding of Mn mineral formation and the bioinorganic capabilities of MCOs.

Lithotrophic iron-oxidizing bacteria (FeOB) form microbial mats at focused flow or diffuse flow vents in deep-sea hydrothermal systems where Fe(II) is a dominant electron donor. These mats composed of biogenically formed Fe(III)-oxyhydroxides include twisted stalks and tubular sheaths, with sheaths typically composing a minor component of bulk mats. The micron diameter Fe(III)-oxyhydroxide-containing tubular sheaths bear a strong resemblance to sheaths formed by the freshwater FeOB, Leptothrix ochracea. We discovered that veil-like surface layers present in iron-mats at the Loihi Seamount were dominated by sheaths (40-60% of total morphotypes present) compared with deeper (> 1 cm) mat samples (0-16% sheath). By light microscopy, these sheaths appeared nearly identical to those of L. ochracea. Clone libraries of the SSU rRNA gene from this top layer were dominated by Zetaproteobacteria, and lacked phylotypes related to L. ochracea. In mats with similar morphologies, terminal-restriction fragment length polymorphism (T-RFLP) data along with quantitative PCR (Q-PCR) analyses using a Zetaproteobacteria-specific primer confirmed the presence and abundance of Zetaproteobacteria. A Zetaproteobacteria fluorescence in situ hybridization (FISH) probe hybridized to ensheathed cells (4% of total cells), while a L. ochracea-specific probe and a Betaproteobacteria probe did not. Together, these results constitute the discovery of a novel group of marine sheath-forming FeOB bearing a striking morphological similarity to L. ochracea, but belonging to an entirely different class of Proteobacteria., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Under anoxic conditions, soluble pertechnetate (⁹⁹TcO₄⁻) can be reduced to less soluble TcO₂·nH₂O, but the oxide is highly susceptible to reoxidation. Here we investigate an alternative strategy for remediation of Tc-contaminated groundwater whereby sequestration as Tc sulfide is favored by sulfidic conditions stimulated by nano zerovalent iron (nZVI). nZVI was pre-exposed to increasing concentrations of sulfide in simulated Hanford groundwater for 24 h to mimic the onset of aquifer biotic sulfate reduction. Solid-phase characterizations of the sulfidated nZVI confirmed the formation of nanocrystalline FeS phases, but higher S/Fe ratios (>0.112) did not result in the formation of significantly more FeS. The kinetics of Tc sequestration by these materials showed faster Tc removal rates with increasing S/Fe between 0 and 0.056, but decreasing Tc removal rates with S/Fe > 0.224. The more favorable Tc removal kinetics at low S/Fe could be due to a higher affinity of TcO₄⁻ for FeS than iron oxides, and electron microscopy confirmed that the majority of the Tc was associated with FeS phases. The inhibition of Tc removal at high S/Fe appears to have been caused by excess HS(-). X-ray absorption spectroscopy revealed that as S/Fe increased, the pathway for Tc(IV) formation shifted from TcO₂·nH2₂ to Tc sulfide phases. The most substantial change of Tc speciation occurred at low S/Fe, coinciding with the rapid increase in Tc removal rate. This agreement further confirms the importance of FeS in Tc sequestration.

Effects of Mn redox cycling on the stability of bioreduced U(IV) are evaluated here. U(VI) can be biologically reduced to less soluble U(IV) species and the stimulation of biological activity to that end is a salient remediation strategy; however, the stability of these materials in the subsurface environments where they form remains unproven. Manganese oxides are capable of rapidly oxidizing U(IV) to U(VI) in mixed batch systems where the two solid phases are in direct contact. However, it is unknown whether the same oxidation would take place in a porous medium. To probe that question, U(IV) immobilized in agarose gels was exposed to conditions allowing biological Mn(II) oxidation (HEPES buffer, Mn(II), 5% O2 and Bacillus sp. SG-1 spores). Results show the oxidation of U(IV) to U(VI) is due primarily to O2 rather than to MnO2. U(VI) produced is retained within the gel to a greater extent when Mn oxides are present, suggesting the formation of strong surface complexes. The implication for the long-term stability of U in a bioremediated site is that, in the absence of competing ligands, biological Mn(II) oxidation may promote the immobilization of U(VI) produced by the oxidation of U(IV).

Manganese(IV) oxides, believed to form primarily through microbial activities, are extremely important mineral phases in marine environments where they scavenge a variety of trace elements and thereby control their distributions. The presence of various ions common in seawater are known to influence Mn oxide mineralogy yet little is known about the effect of these ions on the kinetics of bacterial Mn(II) oxidation and Mn oxide formation. We examined factors affecting bacterial Mn(II) oxidation by spores of the marine Bacillus sp. strain SG-1 in natural and artificial seawater of varying ionic conditions. Ca 2+ concentration dramatically affected Mn(II) oxidation, while Mg 2+ , Sr 2+ , K + , Na + and NO 3 - ions had no effect. The rate of Mn(II) oxidation at 10mM Ca 2+ (seawater composition) was four or five times that without Ca 2+ . The relationship between Ca 2+ content and oxidation rate demonstrates that the equilibrium constant is small (on the order of 0.1) and the binding coefficient is 0.5. The pH optimum for Mn(II) oxidation changed depending on the amount of Ca 2+ present, suggesting that Ca 2+ exerts a direct effect on the enzyme perhaps as a stabilizing bridge between polypeptide components. We also examined the effect of varying concentrations of NaCl or KNO 3 (0 mM - 2000 mM) on the kinetics of Mn(II) oxidation in solutions containing 10 mM Ca 2+ . Mn(II) oxidation was unaffected by changes in ionic strength (I) below 0.2, but it was inhibited by increasing salt concentrations above this value. Our results suggest that the critical coagulation concentration is around 200 mM of salt (I = ca. 0.2), and that the ionic strength of seawater (I > 0.2) accelerates the precipitation of Mn oxides around the spores. Under these conditions, the aggregation of Mn oxides reduces the supply of dissolved O 2 and/or Mn 2+ and inhibits the Mn(II) -> Mn(III) step controlling the enzymatic oxidation of Mn(II). Our results suggest that the hardness and ionic strength of the aquatic environment at circumneutral pH strongly influences the rate of biologically mediated Mn(II) oxidation.

The mobility of hexavalent uranium in soil and groundwater is strongly governed by adsorption to mineral surfaces. As strong naturally occurring adsorbents, manganese oxides may significantly influence the fate and transport of uranium. Models for U(VI) adsorption over a broad range of chemical conditions can improve predictive capabilities for uranium transport in the subsurface. This study integrated batch experiments of U(VI) adsorption to synthetic and biogenic MnO(2), surface complexation modeling, ζ-potential analysis, and molecular-scale characterization of adsorbed U(VI) with extended X-ray absorption fine structure (EXAFS) spectroscopy. The surface complexation model included inner-sphere monodentate and bidentate surface complexes and a ternary uranyl-carbonato surface complex, which was consistent with the EXAFS analysis. The model could successfully simulate adsorption results over a broad range of pH and dissolved inorganic carbon concentrations. U(VI) adsorption to synthetic δ-MnO(2) appears to be stronger than to biogenic MnO(2), and the differences in adsorption affinity and capacity are not associated with any substantial difference in U(VI) coordination.

Bacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase in Pseudomonas putida GB-1 has long remained unknown. To identify the P. putida GB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (mnxG, mofA, moxA, and mopA). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11 Pseudomonas species. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1&#95;2447, which encodes an MCO homologous to MnxG, and PputGB1&#95;2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1&#95;2447 and PputGB1&#95;2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes.

Global cycling of environmental manganese requires catalysis by bacteria and fungi for MnO(2) formation, since abiotic Mn(II) oxidation is slow under ambient conditions. Genetic evidence from several bacteria indicates that multicopper oxidases (MCOs) are required for MnO(2) formation. However, MCOs catalyze one-electron oxidations, whereas the conversion of Mn(II) to MnO(2) is a two-electron process. Trapping experiments with pyrophosphate (PP), a Mn(III) chelator, have demonstrated that Mn(III) is an intermediate in Mn(II) oxidation when mediated by exosporium from the Mn-oxidizing bacterium Bacillus SG-1. The reaction of Mn(II) depends on O(2) and is inhibited by azide, consistent with MCO catalysis. We show that the subsequent conversion of Mn(III) to MnO(2) also depends on O(2) and is inhibited by azide. Thus, both oxidation steps appear to be MCO-mediated, likely by the same enzyme, which is indicated by genetic evidence to be the MnxG gene product. We propose a model of how the manganese oxidase active site may be organized to couple successive electron transfers to the formation of polynuclear Mn(IV) complexes as precursors to MnO(2) formation.

Micro-organisms capable of oxidizing the redox-active transition metal manganese play an important role in the biogeochemical cycle of manganese. In the present mini-review, we focus specifically on Mn(II)-oxidizing bacteria. The mechanisms by which bacteria oxidize Mn(II) include a two-electron oxidation reaction catalysed by a novel multicopper oxidase that produces Mn(IV) oxides as the primary product. Bacteria also produce organic ligands, such as siderophores, that bind to and stabilize Mn(III). The realization that this stabilized Mn(III) is present in many environments and can affect the redox cycles of other elements such as sulfur has made it clear that manganese and the bacteria that oxidize it profoundly affect the Earth's biogeochemistry.

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM (13)C-NaHCO(3), doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.

A novel hydrothermal field has been discovered at the base of Lōihi Seamount, Hawaii, at 5000 mbsl. Geochemical analyses demonstrate that 'FeMO Deep', while only 0.2 °C above ambient seawater temperature, derives from a distal, ultra-diffuse hydrothermal source. FeMO Deep is expressed as regional seafloor seepage of gelatinous iron- and silica-rich deposits, pooling between and over basalt pillows, in places over a meter thick. The system is capped by mm to cm thick hydrothermally derived iron-oxyhydroxide- and manganese-oxide-layered crusts. We use molecular analyses (16S rDNA-based) of extant communities combined with fluorescent in situ hybridizations to demonstrate that FeMO Deep deposits contain living iron-oxidizing Zetaproteobacteria related to the recently isolated strain Mariprofundus ferroxydans. Bioenergetic calculations, based on in-situ electrochemical measurements and cell counts, indicate that reactions between iron and oxygen are important in supporting chemosynthesis in the mats, which we infer forms a trophic base of the mat ecosystem. We suggest that the biogenic FeMO Deep hydrothermal deposit represents a modern analog for one class of geological iron deposits known as 'umbers' (for example, Troodos ophilolites, Cyprus) because of striking similarities in size, setting and internal structures.

Manganese oxide (Mn oxide) minerals from bacterial sources produce electron paramagnetic resonance (EPR) spectral signatures that are mostly distinct from those of synthetic simulants and abiogenic mineral Mn oxides. Biogenic Mn oxides exhibit only narrow EPR spectral linewidths (∼500 G), whereas abiogenic Mn oxides produce spectral linewidths that are 2-6 times broader and range from 1200 to 3000 G. This distinction is consistent with X-ray structural observations that biogenic Mn oxides have abundant layer site vacancies and edge terminations and are mostly of single ionic species [i.e., Mn(IV)], all of which favor narrow EPR linewidths. In contrast, abiogenic Mn oxides have fewer lattice vacancies, larger particle sizes, and mixed ionic species [Mn(III) and Mn(IV)], which lead to the broader linewidths. These properties could be utilized in the search for extraterrestrial physicochemical biosignatures, for example, on Mars missions that include a miniature version of an EPR spectrometer.

Members of the neutrophilic iron-oxidizing candidate class Zetaproteobacteria have predominantly been found at sites of microbially mediated iron oxidation in marine environments around the Pacific Ocean. Eighty-four full-length (>1,400-bp) and 48 partial-length Zetaproteobacteria small-subunit (SSU) rRNA gene sequences from five novel clone libraries, one novel Zetaproteobacteria isolate, and the GenBank database were analyzed to assess the biodiversity of this burgeoning class of the Proteobacteria and to investigate its biogeography between three major sampling regions in the Pacific Ocean: Loihi Seamount, the Southern Mariana Trough, and the Tonga Arc. Sequences were grouped into operational taxonomic units (OTUs) on the basis of a 97% minimum similarity. Of the 28 OTUs detected, 13 were found to be endemic to one of the three main sampling regions and 2 were ubiquitous throughout the Pacific Ocean. Additionally, two deeply rooted OTUs that potentially dominate communities of iron oxidizers originating in the deep subsurface were identified. Spatial autocorrelation analysis and analysis of molecular variance (AMOVA) showed that geographic distance played a significant role in the distribution of Zetaproteobacteria biodiversity, whereas environmental parameters, such as temperature, pH, or total Fe concentration, did not have a significant effect. These results, detected using the coarse resolution of the SSU rRNA gene, indicate that the Zetaproteobacteria have a strong biogeographic signal.

Bacterially mediated manganese(II) oxidation greatly affects the biogeochemical cycling of Mn and other elements. One species of bacteria that are capable of Mn(II) oxidation is the gamma-proteobacterium Pseudomonas putida GB-1. In this organism, Mn(II) oxidation begins in stationary phase on the outer surface of the cell, forming a layer of insoluble Mn(III,IV) oxides. A random transposon mutagenesis screen isolated 12 mutant strains of P. putida GB-1 that exhibited increased Mn(II) oxidation on solid media relative to wild type. In 8 out of the 12 strains, the transposon had inserted into a putative flagellar gene. Those 8 strains each had motility defects, thus the disrupted genes are part of the P. putida GB-1 flagellar regulon. The flagellar genes identified include putative structural components (FliC, FliD, FlgE, and FlgL) and regulatory proteins (FlgM and FleN). Deletion of either the FleN gene (fleN) or the overlapping gene fliA resulted in increased Mn(II) oxidation, while in-frame deletion of fliF, which encodes an essential component of the basal body, did not. In liquid media, the flagellar mutants exhibited delayed Mn(II) oxidation relative to wild type. These results suggest that bacterial Mn(II) oxidation is regulated in part by flagellar-mediated responses to the surface substrate.

The Columbia River is a major source of dissolved nutrients and trace metals for the west coast of North America. A large proportion of these nutrients are sourced from the Columbia River Estuary, where coastal and terrestrial waters mix and resuspend particulate matter within the water column. As estuarine water is discharged off the coast, it transports the particulate matter, dissolved nutrients and microorganisms forming nutrient-rich and metabolically dynamic plumes. In this study, bacterial manganese oxidation within the plume and estuary was investigated during spring and neap tides. The microbial community proteome was fractionated and assayed for Mn oxidation activity. Proteins from the outer membrane and the loosely bound outer membrane fractions were separated using size exclusion chromatography and Mn(II)-oxidizing eluates were analysed with tandem mass spectrometry to identify potential Mn oxidase protein targets. Multi-copper oxidase (MCO) and haem-peroxidase enzymes were identified in active fractions. T-RFLP profiles and cluster analysis indicates that organisms and bacterial communities capable of oxidizing Mn(II) can be sourced from the Columbia River estuary and nearshore coastal ocean. These organisms are producing up to 10 fM MnO₂ cell⁻¹ day⁻¹. Evidence for the presence of Mn(II)-oxidizing bacterial isolates from the genera Aurantimonas, Rhodobacter, Bacillus and Shewanella was found in T-RFLP profiles. Specific Q-PCR probes were designed to target potential homologues of the Aurantimonas manganese oxidizing peroxidase (Mop). By comparing total Mop homologues, Aurantimonas SSU rRNA and total bacterial SSU rRNA gene copies, it appears that Aurantimonas can only account for ~1.7% of the peroxidase genes quantified. Under the broad assumption that at least some of the peroxidase homologues quantified are involved in manganese oxidation, it is possible that other organisms oxidize manganese via peroxidases., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

A new spectrophotometric protocol was developed for the simultaneous determination of soluble Mn(III), Mn(II) and total Mn [sum of soluble Mn(III) and Mn(II)] in sediment porewaters using a water soluble meso-substituted porphyrin [α,β,γ,δ-tetrakis(4-carboxyphenyl)porphine (T(4-CP)P)]. A simple kinetic rate model is used to quantify soluble Mn(II), Mn(III) and total Mn concentrations during a metal substitution reaction. Under optimized conditions, the method accurately determines soluble Mn(II) and Mn(III) within a concentration range of 100 nM-10 μM. The detection limit of total soluble Mn is 50 nM. Using this method, soluble Mn(II) and Mn(III) concentrations were determined in standard solutions within 0.4-2% of the known values and agreed closely with results of inductively coupled plasma mass spectrometric and voltammetric analyses. The procedure was successfully applied to determine soluble Mn(II), Mn(III) and total Mn in sediment porewaters of the Lower St. Lawrence Estuary. Mn(III) represented up to 85% of the total soluble Mn pool in surface sediments., (Copyright © 2011 Elsevier B.V. All rights reserved.)