Bradley M. Tebo

The oxidation of manganese from Mn(II) to Mn(III) or Mn(IV) is a common trait in the Pseudomonas genus. Many pseudomonads oxidize Mn enzymatically using one or more multicopper oxidases (MnxG and McoA) and in some cases a heme peroxidase (MopA). The oxidation of Mn leads to the extracellular precipitation of reactive Mn oxides in association with extracellular polymeric substances (EPS). Here, we review the current state of knowledge on Mn oxidation and biomineralization in pseudomonads. While most of the genetic, biochemical, and physiological insights we present derive from studies on Pseudomonas putida GB-1, we also examine the mechanisms of Mn oxidation in other Mn-oxidizing pseudomonads and survey the potential for Mn oxidation across the genus through phylogenetic analysis. Although the physiological and microbial community function(s) for Mn oxidation and biomineralization have yet to be elucidated, we discuss possible roles for Mn oxidation and identify key knowledge gaps regarding the mechanism and function of this trait in the Pseudomonas genus.

In oxic environments, the stable form of manganese is an insoluble MnO2 mineral. However, the uncatalyzed oxidation of soluble Mn(II) is very slow. Consequently, this oxidation, a key part of the global manganese cycle, is largely under biological control, being carried out by bacteria and fungi. Dormant spores of many Bacillus species employ multicopper oxidase enzymes (MCOs) that use oxygen directly to oxidize Mn(II) and form MnO2 minerals. This presents a biochemical conundrum, because MCOs work through single electron transfers from their substrates, whereas conversion of Mn(II) to MnO2 is a 2-electron oxidation, involving one-electron steps with high kinetic barriers due to high reduction potentials. How is this accomplished? Expression and purification of the first bacterial manganese oxidizing MCO complex Mnx created a new chapter in the field of inorganic biochemistry: protein-controlled manganese biomineralization. This review documents the history of genetic studies in Bacillus sp. that led to production of the enzymatic complex Mnx responsible for Mn biomineralization. Biochemical and cryoelectron microscopy structural studies then showed how Mnx has solved the problem of high kinetic barriers to Mn oxidation by exploiting the stabilizing effect of hydroxo- and oxo-bridging in binuclear Mn complexes in all three oxidation states, II, III, and IV. We offer an outlook for future directions of manganese biomineralization research, connecting it to broader quests in the fields of geochemistry and geomicrobiology.

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 O₂ 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 MnxE₃F₃ 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.

Multi-subunit enzymes function as coordinated assemblies. Yet most enzymatic assays measure the summed output of all populations in solution and cannot easily differentiate contributions of individual subunits. Native mass spectrometry detects intact protein complexes in the gas phase. Surface induced dissociation further releases subunits from protein complexes while retaining compact conformations and bound ligands. Combined with ion mobility, the released subunits can then be carefully monitored for more in-depth structural analysis. Mnx is a unique bacterial multicopper oxidase complex that oxidizes Mn(II) to form MnO2 minerals, and is composed of three subunits: MnxG, a multicopper oxidase containing the active site, and two accessory proteins, MnxE and MnxF which also bind copper ions. Other known multicopper oxidases do not require accessory proteins, therefore the functions of MnxE and MnxF are not well understood. Here, we use native mass spectrometry with surface induced dissociation and ion mobility to characterize the metal binding properties of Mnx with two metals, catalytic Cu(II) and Mn(II) substrate. We demonstrate our assay can detect subtle structural changes within each subunit, which are presumably related to the allosteric mechanism. We also noticed that ionic strength and solution composition can impact metal binding and must be carefully investigated for such experiments.

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, CO₂ fixation, a high CO₂ 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.

Manganese (Mn) is an essential element for life. Although its concentration is at (sub)nanomolar levels throughout the ocean, it affects the oxygen concentration of the ocean because it is central to the photosynthetic formation of dioxygen, O₂, in photosystem center II. Mn inputs into the ocean are from atmospheric transport of particles and their dissolution to form dissolved Mn, and from the flux of dissolved Mn from rivers, sediments, and hydrothermal vents. The main removal mechanism is transport of particulate Mn from dust and organic matter to the sediments. The environmental chemistry of manganese centers on its +2, +3, and +4 oxidation states. Most recent data show that Mn(II) is dissolved, that Mn(IV) is particulate MnO₂, and that Mn(III) can be particulate or dissolved when bound to organic complexes [denoted as Mn(III)‐L]. Mn(II) is oxidized primarily by microbial processes whereas MnO₂is reduced by abiotic and biotic processes. Photochemical processing aids redox cycling in surface waters. In suboxic zones, which are defined as areas where both dissolved O₂and H₂S concentrations are below 3 μM, both oxidation and reduction processes can occur but usually at different depths. In suboxic zones, dissolved Mn is also released from organic matter during its decomposition and from MnO₂reduction.

MnO₂ 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 thiosulphate 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 bandgap energies of the colloids were found to increase with decreasing hydrodynamic diameter, D_h, and were proportional to 1/D_h², 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 Mn²⁺, in the presence of pyrophosphate to trap the Mn³⁺ 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 bandgap 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 MnO₂ growth. The band-gap/size correlation was used to analyze the growth of the enzymatically produced MnO₂ oxides.

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.