Electrochemically coupled CH4 and CO2 consumption driven by microbial processes.

The chemical transformations of methane (CH4) and carbon dioxide (CO2) greenhouse gases typically have high energy barriers. Here we present an approach of strategic coupling of CH4 oxidation and CO2 reduction in a switched microbial process governed by redox cycling of iron minerals under temperate conditions. The presence of iron minerals leads to an obvious enhancement of carbon fixation, with the minerals acting as the electron acceptor for CH4 oxidation and the electron donor for CO2 reduction, facilitated by changes in the mineral structure. The electron flow between the two functionally active microbial consortia is tracked through electrochemistry, and the energy metabolism in these consortia is predicted at the genetic level. This study offers a promising strategy for the removal of CH4 and CO2 in the natural environment and proposes an engineering technique for the utilization of major greenhouse gases.

Coupled Aerobic Methane Oxidation and Arsenate Reduction Contributes to Soil-Arsenic Mobilization in Agricultural Fields.

Microbial oxidation of organic compounds can promote arsenic release by reducing soil-associated arsenate to the more mobile form arsenite. While anaerobic oxidation of methane has been demonstrated to reduce arsenate, it remains elusive whether and to what extent aerobic methane oxidation (aeMO) can contribute to reductive arsenic mobilization. To fill this knowledge gap, we performed incubations of both microbial laboratory cultures and soil samples from arsenic-contaminated agricultural fields in China. Incubations with laboratory cultures showed that aeMO could couple to arsenate reduction, wherein the former bioprocess was carried out by aerobic methanotrophs and the latter by a non-methanotrophic bacterium belonging to a novel and uncultivated representative of Burkholderiaceae. Metagenomic analyses combined with metabolite measurements suggested that formate served as the interspecies electron carrier linking aeMO to arsenate reduction. Such coupled bioprocesses also take place in the real world, supported by a similar stoichiometry and gene activity in the incubations with natural paddy soils, and contribute up to 76.2% of soil-arsenic mobilization into pore waters in the top layer of the soils where oxygen was present. Overall, this study reveals a previously overlooked yet significant contribution of aeMO to reductive arsenic mobilization.

Ecology and molecular targets of hypermutation in the global microbiome.

Changes in the sequence of an organism's genome, i.e., mutations, are the raw material of evolution. The frequency and location of mutations can be constrained by specific molecular mechanisms, such as diversity-generating retroelements (DGRs). DGRs have been characterized from cultivated bacteria and bacteriophages, and perform error-prone reverse transcription leading to mutations being introduced in specific target genes. DGR loci were also identified in several metagenomes, but the ecological roles and evolutionary drivers of these DGRs remain poorly understood. Here, we analyze a dataset of >30,000 DGRs from public metagenomes, establish six major lineages of DGRs including three primarily encoded by phages and seemingly used to diversify host attachment proteins, and demonstrate that DGRs are broadly active and responsible for >10% of all amino acid changes in some organisms. Overall, these results highlight the constraints under which DGRs evolve, and elucidate several distinct roles these elements play in natural communities.

Expression, purification and properties of the enzymes involved in lanthanide-dependent alcohol oxidation: XoxF4, XoxF5, ExaF/PedH, and XoxG4.

In this chapter we describe logistics, protocols and conditions for expression, purification and characterization of Ln3+-dependent alcohol dehydrogenases representing three distinct phylogenetic clades of these enzymes, classified as XoxF4, XoxF5 and ExaF/PedH. We present data on the biochemical properties of a dozen enzymes, all generated by our group, in a comparative fashion. These enzymes display a range of properties in terms of substrate and metal specificities, pH and ammonium requirement, as well as catalytic constants. In addition, we describe a single novel cytochrome, XoxG4, that likely serves as a natural electron acceptor from XoxF5 in methanotrophs of the Gammaproteobacteria class.

Metagenomic Insight into Environmentally Challenged Methane-Fed Microbial Communities.

In this study, we aimed to investigate, through high-resolution metagenomics and metatranscriptomics, the composition and the trajectories of microbial communities originating from a natural sample, fed exclusively with methane, over 14 weeks of laboratory incubation. This study builds on our prior data, suggesting that multiple functional guilds feed on methane, likely through guild-to-guild carbon transfer, and potentially through intraguild and intraspecies interactions. We observed that, under two simulated dioxygen partial pressures-low versus high-community trajectories were different, with considerable variability among the replicates. In all microcosms, four major functional guilds were prominently present, representing Methylococcaceae (the true methanotrophs), Methylophilaceae (the nonmethanotrophic methylotrophs), Burkholderiales, and Bacteroidetes. Additional functional guilds were detected in multiple samples, such as members of Opitutae, as well as the predatory species, suggesting additional complexity for methane-oxidizing communities. Metatranscriptomic analysis suggested simultaneous expression of the two alternative types of methanol dehydrogenases in both Methylococcaceae and Methylophilaceae, while high expression of the oxidative/nitrosative stress response genes suggested competition for dioxygen among the community members. The transcriptomic analysis further suggested that Burkholderiales likely feed on acetate that is produced by Methylococcaceae under hypoxic conditions, while Bacteroidetes likely feed on biopolymers produced by both Methylococcaceae and Methylophilaceae.

A Complex Interplay between Nitric Oxide, Quorum Sensing, and the Unique Secondary Metabolite Tundrenone Constitutes the Hypoxia Response in Methylobacter.

Methylobacter species, members of the Methylococcales, have recently emerged as some of the globally widespread, cosmopolitan species that play a key role in the environmental consumption of methane across gradients of dioxygen tensions. In this work, we approached the question of how Methylobacter copes with hypoxia, via laboratory manipulation. Through comparative transcriptomics of cultures grown under high dioxygen partial pressure versus cultures exposed to hypoxia, we identified a gene cluster encoding a hybrid cluster protein along with sensing and regulatory functions. Through mutant analysis, we demonstrated that this gene cluster is involved in the hypoxia stress response. Through additional transcriptomic analyses, we uncovered a complex interconnection between the NO-mediated stress response, quorum sensing, the secondary metabolite tundrenone, and methanol dehydrogenase functions. This novel and complex hypoxia stress response system is so far unique to Methylobacter species, and it may play a role in the environmental fitness of these organisms and in their cosmopolitan environmental distribution.IMPORTANCE Here, we describe a novel and complex hypoxia response system in a methanotrophic bacterium that involves modules of central carbon metabolism, denitrification, quorum sensing, and a secondary metabolite, tundrenone. This intricate stress response system, so far unique to Methylobacter species, may be responsible for the persistence and activity of these species across gradients of dioxygen tensions and for the cosmopolitan distribution of these organisms in freshwater and soil environments in the Northern Hemisphere, including the fast-melting permafrosts.

Synthetic Methane-Consuming Communities from a Natural Lake Sediment.

The factors and processes that influence the behavior and functionality of ecosystems inhabited by complex microbiomes are still far from being clearly understood. Synthetic microbial communities provide reduced-complexity models that allow an examination of ecological theories under defined and controlled conditions. In this study, we applied a multiphasic approach to study synthetic methane-oxidizing communities and species interactions as proxies to the natural communities. Our results confirm that, under selective pressures, natural-sediment communities of high complexity simplify rapidly, selecting for several major functional guilds, the major partners in methane oxidation being the Methylococcaceae methanotrophs and the Methylophilaceae methylotrophs, along with minor but persistent partners, members of Burkholderiales and Flavobacteriales As a proof of concept, we established minimalist synthetic communities that were representative of the four functional guilds to demonstrate the dependency of the non-methane-utilizing species on the methanotrophs as the primary carbon-providing species. We observed that in communities consisting of multiple representatives of the key guilds, members of the same guild appeared to compete for resources. For example, when two methanotrophs of the same family were present, the two expressed similar key methanotrophy pathways and responded similarly to changing environmental conditions, suggesting that they perform a similar keystone function in situ Similar observations were made for the Methylophilaceae However, differences were noted in the expression of auxiliary and unique genes among strains of the same functional guild, reflecting differential adaptation and suggesting mechanisms for competition. At the same time, differences were also noted in the performances of partners with specific metabolic schemes. For example, a mutant of Methylotenera mobilis impaired in nitrate utilization behaved as a more efficient cooperator in methane consumption, suggesting that the loss of function may lead to changes in communal behavior. Overall, we demonstrate the robust nature of synthetic communities built of native lake sediment strains and their utility in addressing important ecological questions while using a simplified model.IMPORTANCE The metabolism of methane is an important part of the global carbon cycle. While deciphering the community function and the potential role of the different functional guilds is very difficult when considering native complex communities, synthetic communities, built of species originating from a study site in question, present a simplified model and allow specific questions to be addressed as to carbon, nitrogen, and other nutrient transfer among species in a controlled system. This study applies an ecophysiological approach, as a proof of principle, to an already well-studied model system, contributing to a better understanding of microbial community function and microbial ecosystem processes.

Systems Biology Meets Enzymology: Recent Insights into Communal Metabolism of Methane and the Role of Lanthanides.

In this review article, we cover the recent developments in understanding the principles and the mechanisms by which microbial communities participating in methane consumption in natural environmental niches are assembled, and the physiological and biochemical mechanisms and regulators that allow efficient carbon transfer within the communities. We first give a brief overview of methanotrophy. We then describe the recent evidence on non-random assembly of bacterial communities that utilize carbon from methane, based on stable isotope probing experiments as well as on results from natural community manipulations followed by metagenomic analysis. We follow up by highlighting results from synthetic methanotophic community manipulations identifying the importance of a lanthanide switch that regulates alternative methanol dehydrogenase enzymes in these communities. We further expand on the recently uncovered significance of lanthanides in methylotrophy and review data on the biochemical properties of representatives of two different clades of lanthanide-dependent enzymes. We also provide an overview of the occurrence and the distribution of the lanthanide-dependent alcohol dehydrogenases in the bacterial domain, these data strongly suggesting significance of these metals beyond methylotrophy.

Rare earth element alcohol dehydrogenases widely occur among globally distributed, numerically abundant and environmentally important microbes.

Lanthanides (Ln3+), known as rare earth elements, have recently emerged as enzyme cofactors, contrary to prior assumption of their biological inertia. Several bacterial alcohol dehydrogenases have been characterized so far that depend on Ln3+ for activity and expression, belonging to the methanol dehydrogenase clade XoxF and the ethanol dehydrogenase clade ExaF/PedH. Here we compile an inventory of genes potentially encoding Ln3+-dependent enzymes, closely related to the previously characterized XoxF and ExaF/PedH enzymes. We demonstrate their wide distribution among some of the most numerically abundant and environmentally important taxa, such as the phylogenetically disparate rhizobial species and metabolically versatile bacteria inhabiting world's oceans, suggesting that reliance on Ln3+-mediated biochemistry is much more widespread in the microbial world than previously assumed. Through protein expression and analysis, we here more than double the extant collection of the biochemically characterized Ln3+-dependent enzymes, demonstrating a range of catalytic properties and substrate and cofactor specificities. Many of these enzymes reveal propensity for oxidation of methanol. This observation, in combination with genome-based reconstruction of methylotrophy pathways for select species suggests a much wider occurrence of this metabolic capability among bacterial species, and thus further suggests the importance of methylated compounds as parts of the global carbon cycling.

New pieces to the lanthanide puzzle.

Recently, rare-earth elements lanthanides (Ln3+ ) have emerged as enzyme cofactors of methanol dehydrogenases of the XoxF type. It is now understood that XoxF enzymes can functionally replace the alternative, calcium-dependent, MxaFI-type methanol dehydrogenases, when Ln3+ are available. These rare-earth metals are not only essential for XoxF activity, but they also regulate gene expression, in a reverse fashion, activating the expression of XoxF and repressing the expression of MxaFI. This type of regulation has created multiple conundrums, including the details of the solubility, transport, sensing and selection mechanisms for Ln3+ by the bacterial cells, as well as the questions relevant to the evolution of the alternative enzymes and their potentially different redox properties. Overall, the newly discovered biological activity of Ln3+ presents a big puzzle. Ochsner et al. add several pieces to this puzzle, utilizing a model phyllosphere colonizer Methylobacterium extorquens PA1. They determine that Ln3+ sensing by this organism can take place via both XoxF-dependent and XoxF-independent mechanisms. They also identify genes for a TonB-dependent transporter and an ABC-type transporter and demonstrate that both are essential for Ln3+ -dependent methanol metabolism. The puzzle still requires multiple additional pieces for completion, but great strides have been made toward the goal of solving it.

Lanthanide-Dependent Methanol Dehydrogenases of XoxF4 and XoxF5 Clades Are Differentially Distributed Among Methylotrophic Bacteria and They Reveal Different Biochemical Properties.

Lanthanide-dependent alcohol dehydrogenases have recently emerged as environmentally important enzymes, most prominently represented in methylotrophic bacteria. The diversity of these enzymes, their environmental distribution, and their biochemistry, as well as their evolutionary relationships with their calcium-dependent counterparts remain virtually untapped. Here, we make important advances toward understanding lanthanide-dependent methylotrophy by assessing the distribution of XoxF4 and XoxF5 clades of lanthanide methanol dehydrogenases among, respectively, Methylophilaceae and non-Methylophilaceae methylotrophs, and we carry out comparative biochemical characterization of XoxF4 and XoxF5 enzymes, demonstrating differences in their properties, including catalytic efficiencies. We conclude that one subtype of the XoxF4 enzyme, XoxF4-1 is the dominant type in nature while other XoxF4 subtypes appear to be auxiliary, representatives of this clade only found in the Methylophilaceae (Betaproteobacteria). In contrast, we demonstrate that XoxF5 enzymes are widespread among Alpha-, Beta-, and Gammaproteobacteria. We purified and biochemically characterized two XoxF4 enzymes (XoxF4-1 and XoxF4-2), both from Methylotenera mobilis, and one XoxF5 enzyme, from Methylomonas sp., after expressing their His-tagged versions in respective natural hosts. All three enzymes showed broad specificities toward alcohols and aldehydes and strict dependence on lighter lanthanides. However, they revealed differences in their properties in terms of optimal pH for in vitro activity, ammonia dependence, the range of lanthanides that could serve as cofactors, and in kinetic properties. Overall, our data advance the understanding of the biochemistry and environmental distribution of these recently discovered enzymes that appear to be key enzymes in lanthanide-dependent methylotrophy.

Physiological Effect of XoxG(4) on Lanthanide-Dependent Methanotrophy.

A recent surprising discovery of the activity of rare earth metals (lanthanides) as enzyme cofactors as well as transcriptional regulators has overturned the traditional assumption of biological inertia of these metals. However, so far, examples of such activities have been limited to alcohol dehydrogenases. Here we describe the physiological effects of a mutation in xoxG, a gene encoding a novel cytochrome, XoxG(4), and compare these to the effects of mutation in XoxF, a lanthanide-dependent methanol dehydrogenase, at the enzyme activity level and also at the community function level, using Methylomonas sp. strain LW13 as a model organism. Through comparative phenotypic characterization, we establish XoxG as the second protein directly involved in lanthanide-dependent metabolism, likely as a dedicated electron acceptor from XoxF. However, mutation in XoxG caused a phenotype that was dramatically different from the phenotype of the mutant in XoxF, suggesting a secondary function for this cytochrome, in metabolism of methane. We also purify XoxG(4) and demonstrate that this protein is a true cytochrome c, based on the typical absorption spectra, and we demonstrate that XoxG can be directly reduced by a purified XoxF, supporting one of its proposed physiological functions. Overall, our data continue to suggest the complex nature of the interplay between the calcium-dependent and lanthanide-dependent alcohol oxidation systems, while they also suggest that addressing the roles of these alternative systems is essential at the enzyme and community function level, in addition to the gene transcription level.IMPORTANCE The lanthanide-dependent biochemistry of living organisms remains a barely tapped area of knowledge. So far, only a handful of lanthanide-dependent alcohol dehydrogenases have been described, and their regulation by lanthanides has been demonstrated at the transcription level. Little information is available regarding the concentrations of lanthanides that could support sufficient enzymatic activities to support specific metabolisms, and so far, no other redox proteins involved in lanthanide-dependent methanotrophy have been demonstrated. The research presented here provides enzyme activity-level data on lanthanide-dependent methanotrophy in a model methanotroph. Additionally, we identify a second protein important for lanthanide-dependent metabolism in this organism, XoxG(4), a novel cytochrome. XoxG(4) appears to have multiple functions in methanotrophy, one function as an electron acceptor from XoxF and another function remaining unknown. On the basis of the dramatic phenotype of the XoxG(4) mutant, this function must be crucial for methanotrophy.

Current Trends in Methylotrophy.

Methylotrophy is a field of study dealing with microorganisms capable of utilization of compounds devoid of carbon-carbon bonds (C1 compounds). In this review, we highlight several emerging trends in methylotrophy. First, we discuss the significance of the recent discovery of lanthanide-dependent alcohol dehydrogenases for understanding both the occurrence and the distribution of methylotrophy functions among bacteria, and then we discuss the newly appreciated role of lanthanides in biology. Next, we describe the detection of other methylotrophy pathways across novel bacterial taxa and insights into the evolution of methylotrophy. Further, data are presented on the occurrence and activity of aerobic methylotrophs in hypoxic and anoxic environments, questioning the prior assumptions on niche separation of aerobic and anaerobic methylotrophy. The concept of communal function in aerobic methane oxidation is also briefly discussed. Finally, we review recent research in engineering methylotrophs for biotechnological applications as well as recent progress in engineering synthetic methylotrophy.

Applications of methylotrophs: can single carbon be harnessed for biotechnology?

This review summarizes developments in the field of applied research involving microbial conversion of single carbon compounds (methane, methanol, CO2). The potential of the microorganisms involved in biotechnological applications could be realized via engineering native C1 utilizers toward higher output of value-added compounds, including biofuels, or via production of value chemicals as parts of novel, heterologously expressed biochemical pathways. Alternatively, C1 metabolism could be implemented in traditional industrial platforms (Escherichia coli, yeast), via introduction of specific metabolic modules. Most recent research spanning both approaches is covered. The potential of C1 utilizers in biomining of rare Earth elements, as well as the potential of C1 consuming microbial consortia in industrial applications are discussed.

Natural Selection in Synthetic Communities Highlights the Roles of Methylococcaceae and Methylophilaceae and Suggests Differential Roles for Alternative Methanol Dehydrogenases in Methane Consumption.

We describe experiments that follow species dynamics and gene expression patterns in synthetic bacterial communities including species that compete for the single carbon substrate supplied, methane, and species unable to consume methane, which could only succeed through cooperative interactions. We demonstrate that these communities mostly select for two functional guilds, methanotrophs of the family Methylococcaceae and non-methanotrophic methylotrophs of the family Methylophilaceae, these taxonomic guilds outcompeting all other species included in the synthetic mix. The metatranscriptomics analysis uncovered that in both Methylococcaceae and Methylophilaceae, some of the most highly transcribed genes were the ones encoding methanol dehydrogenases (MDH). Remarkably, expression of alternative MDH genes (mxaFI versus xoxF), previously shown to be subjects to the rare Earth element switch, was found to depend on environmental conditions such as nitrogen source and methane and O2 partial pressures, and also to be species-specific. Along with the xoxF genes, genes encoding divergent cytochromes were highly expressed in both Methylophilaceae and Methylococcaceae, suggesting their function in methanol metabolism, likely encoding proteins serving as electron acceptors from XoxF enzymes. The research presented tested a synthetic community model that is much simplified compared to natural communities consuming methane, but more complex than the previously utilized two-species model. The performance of this model identifies prominent species for future synthetic ecology experiments and highlights both advantages of this approach and the challenges that it presents.

Application of Omics Approaches to Studying Methylotrophs and Methylotroph Comunities.

This review covers some recent advances in application of omics technologies to studying methylotrophs, with special reference to their activities in natural environments. Some of the developments highlighted in this review are the new outlook at the role of the XoxF-type, lanthanum-dependent methanol dehydrogenase in natural habitats, new mechanistic details of methane oxidation through the reverse methanogenesis pathway, propensity of 'aerobic' methanotrophs to thrive in hypoxic environments and potential connection of this process to denitrification, and a novel outlook at methane oxidation as a community function.

Communal metabolism of methane and the rare Earth element switch.

Metabolism of methane is an important part of biogeochemical cycling of carbon. Methane is also a major contributor to climate change. A specialized group of microbes that consume methane, the methanotrophs, represent a natural filter preventing an even faster accumulation of methane in the atmosphere. Methanotrophy can proceed via both anaerobic and aerobic modes. The anaerobic methanotrophs, represented by both archaea and bacteria, all appear to be engaged in syntrophic interdependencies with other species, to overcome the energetic barriers of methane metabolism in the absence of oxygen. In contrast, aerobic methanotrophy can be carried out by pure cultures of bacteria. Nevertheless, a concept of communal function in aerobic methane oxidation has been gaining momentum, based on data from natural cooccurrence of specific functional guilds, and based on results from laboratory manipulations. The mechanistic details are still sparse on how and why the methanotrophs share their carbon with other species, and whether and what they gain in return. In this minireview we highlight recent studies that led to this new concept of community function in aerobic methane oxidation. We first describe the stable isotope probing experiments employing heavy carbon-labeled methane, tracing methane carbon consumption. We then follow up with analysis of data from microcosm community dynamics. We further discuss the role of a synthetic community approach in unraveling the principles of carbon flow and species cooperation in methane consumption. Finally, we touch on the role of lanthanides, which are rare Earth elements, previously thought to be biologically inert, in bacterial metabolism of methane.

Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interactions.

The utilization of methane, a potent greenhouse gas, is an important component of local and global carbon cycles that is characterized by tight linkages between methane-utilizing (methanotrophic) and nonmethanotrophic bacteria. It has been suggested that the methanotroph sustains these nonmethanotrophs by cross-feeding, because subsequent products of the methane oxidation pathway, such as methanol, represent alternative carbon sources. We established cocultures in a microcosm model system to determine the mechanism and substrate that underlay the observed cross-feeding in the environment. Lanthanum, a rare earth element, was applied because of its increasing importance in methylotrophy. We used co-occurring strains isolated from Lake Washington sediment that are involved in methane utilization: a methanotroph and two nonmethanotrophic methylotrophs. Gene-expression profiles and mutant analyses suggest that methanol is the dominant carbon and energy source the methanotroph provides to support growth of the nonmethanotrophs. However, in the presence of the nonmethanotroph, gene expression of the dominant methanol dehydrogenase (MDH) shifts from the lanthanide-dependent MDH (XoxF)-type, to the calcium-dependent MDH (MxaF)-type. Correspondingly, methanol is released into the medium only when the methanotroph expresses the MxaF-type MDH. These results suggest a cross-feeding mechanism in which the nonmethanotrophic partner induces a change in expression of methanotroph MDHs, resulting in release of methanol for its growth. This partner-induced change in gene expression that benefits the partner is a paradigm for microbial interactions that cannot be observed in studies of pure cultures, underscoring the importance of synthetic microbial community approaches to understand environmental microbiomes.