Metagenomic Approaches Unearth Methanotroph Phylogenetic and Metabolic Diversity

Garrett J. Smith1* and Kelly C. Wrighton1,2*

1Department of Microbiology, Te Ohio State University, Columbus, OH, USA. 2Department of Soil and Crop Science, Colorado State University, Fort Collins, CO, USA. *Correspondence: [email protected] and [email protected] htps://doi.org/10.21775/cimb.033.057

Abstract gases from a variety of ecosystems, metagenom- Methanotrophic microorganisms utilize methane ics will continue to be an important component as an electron donor and a carbon source. To in the arsenal of tools needed for understanding date, the capacity to oxidize methane is restricted methanotroph diversity and metabolism in both to microorganisms from three bacterial and engineered and natural systems. one archaeal phyla. Most of our knowledge of methanotrophic metabolism has been obtained using highly enriched or pure cultures grown in Introduction the laboratory. However, many methanotrophs Ferdinand Cohn described a flamentous microor- currently evade cultivation, thus metagenomics ganism that quickly earned infamy for fourishing provides a complementary approach for gaining in European waterworks and obstructing the insight into currently unisolated microorganisms. fow of water in the late 1800s (Cohn, 1870). Here we synthesize the studies using metagenom- More than 100 years afer the initial discovery ics to glean information about methanotrophs. of Crenothrix polyspora, this organism still evades We complement this summary with an analysis cultivation. In the past 40 years, ultrastructure of methanotroph marker genes from 235 publicly evidence (Völker et al., 1977) and the presence available metagenomic datasets. We analyse the of key functional genes (Stoecker et al., 2006) phylogenetic and environmental distribution of hinted that these pervasive flamentous cells methanotrophs sampled by metagenomics. We grew using methane as a carbon and energy also highlight metabolic insights that methano- source. In 2017, metagenomic sequencing, or troph genomes assembled from metagenomes the untargeted sequencing of DNA directly from are illuminating. In summary, metagenomics a microbial community, led to the frst insight has increased methanotrophic foliage within the into Crenothrix genomic contents (Oswald et al., tree of life, as well as provided new insights into 2017). In combination with other chemical and methanotroph metabolism, which collectively can imaging technologies, here metagenomics con- guide new cultivation eforts. Lastly, given the frmed that these mysterious, flamentous cells importance of methanotrophs for biotechnological were methanotrophs. Tis is only one example applications and their capacity to flter greenhouse of how metagenomics, especially when combined

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with other methodologies, has demystifed the Overview of methanotroph physiology of uncultivated microorganisms phylogenetic and metabolic (Brown et al., 2015; Solden et al., 2016). diversity Methanotrophic microorganisms play Methanotrophs are currently assigned to three bac- immense roles in mediating biogeochemical terial and one archaeal phyla (Fig. 3.1). Today all cycles. Tese organisms can flter 50–97% of known Bacterial methanotrophs encode methane methane from ecosystems (Segarra et al., 2015), monooxygenase (MMO), the enzyme complex and therefore have a large impact on the release that oxidizes a C–H bond in methane using oxygen. of this potent greenhouse gas to the atmos- Tere are two forms of this enzyme, particulate phere. In addition to the carbon cycle, it has (pMMO) and soluble (sMMO), and at least one of also been shown that methanotrophs can aid in these is required for aerobic oxidation of methane the removal of 90–99% of the nitrogen during (Dalton, 1983). Functional marker genes for the wastewater treatment (Jewell et al., 1992; He et particulate (pmoA) and soluble (mmoX) forms are al., 2015). More recently, methanotrophic strains commonly used to probe for bacterial methano- have been harnessed to degrade organic pollut- trophs (McDonald et al., 2008; Knief, 2015). ants, produce a variety of industrial precursors, Te most well studied bacterial methanotrophs and scavenge metals such as copper (Sullivan belong to the phylum Proteobacteria specifcally et al., 1998; Semrau et al., 2010; Puri et al., within the Gammaproteobacteria and Alphapro- 2015; Strong et al., 2015, 2016). Given their teobacteria classes. Te Gammaproteobacteria important biogeochemical and biotechnological methanotrophs belong to the order Methylococ- applications, metagenomics is a critical research cales, and are ofen referred to as ‘Type I’ and ‘Type tool for improved understanding of methano- X’ methanotrophs. Within the Alphaproteobacte- troph diversity and physiology in natural and ria, methanotrophs are associated with the order manmade setings. Rhizobiales, referred to as ‘Type II’ methano- Today, with the power of recovering near- trophs (Dalton, 1983). Tese two Proteobacteria complete to closed genomes directly from methanotroph classes are distinguished not only by microbial communities, axenic cultivation is no phylogenetic assignment, but also have diferences longer a requirement for investigating a micro- in carbon assimilation pathways and intra-cytoplas- organism’s metabolic capabilities (Solden et al., mic membrane ultrastructure (Dalton, 1983; Knief, 2016). Metagenomics, which provides an inven- 2015). tory of the metabolic potential of a community, Te second bacterial phylum containing is especially powerful when utilized in parallel methanotrophs is the Verrucomicrobia. Here with metatranscriptomics (community RNA members of the family Methylacidiphilales, have sequencing) or metaproteomics (community core methane oxidation pathways resembling Pro- protein analyses). Tese later technologies pro- teobacteria methanotrophs. However, unlike the vide information on gene and protein expression Proteobacteria methanotrophs, these taxa may also in a microbial community, indicating metabolic utilize hydrogen as an alternative electron donor processes and organisms that are responding and require lanthanide metals for growth (Pol et al., to specifc environmental conditions. Here our 2007, 2014; Mohammadi et al., 2017). Te third objective is to describe how metagenomics and bacterial phylum of methanotrophs is the Methy- enabled ‘omic’ technologies (multi-omics) have lomirabilota, which contains the genus ‘Candidatus altered perspectives on methanotroph environ- Methylomirabilis’ and were previously classifed as mental distribution, phylogenetic diversity, and members of the candidate NC10 phylum (Etwig et metabolism. Leveraging recent metagenomic al., 2010; Glöckner et al., 2017; Parks et al., 2018). studies, we (i) summarize key fndings from a Tese methanotrophs are proposed to generate variety of ecosystems, (ii) describe the metha- molecular oxygen for methane oxidation by reduc- notroph genomic foliage added to the tree ing nitrite, followed by dismutation of nitric oxide of life, and (iii) discuss how metagenomic into dinitrogen and dioxygen (Etwig et al., 2008, analyses of methanotrophs has impacted other 2010; Wu et al., 2015). Tus, these bacteria are pro- scientifc disciplines. posed to couple aerobic methane oxidation with

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(a ) (b ) Proteobacteria Methylacidiphilales Bacteria

- CH4 O2 NOx O2 2e- pmoA cyt c narG cyt c mmoX cyt bd nirK/S cyt bd CH3OH 2e- mxaF

Alpha xoxF CH2O

CH O

Gamma 2

norB Biomass Energy 4e- H O RuMP Serine H2ON2O 2 CBB CO2 Candidatus ANME ANME Methylomirabilis -2 -1 - - CH4 none - NO2 NO3 2e mcrA nirS narG Methyl-S-CoM Fe3+/Mn3+

MHC Methyl-H4MPT e- Biomass Energy

Sulfate Reducing 6e- nod Partner nrfH Reverse - Anammox O2+N2 NO2 Partner CO2 Methanogenesis Figure 3.1. Overview of energy generation and methane oxidation by Figure 3.1 Overviewknown of methanotrophic energy generation clades. and methane (a) Energy oxidation generation by known pathway(s) methanotrophic and clades. (a) Energy generation pathway(s) and (b) methane metabolism. Typical morphologies for a model in each clade are shown. Electron(b) methane acceptors metabolism. for respiration Typical (blue) and morphologies marker genes (red)for a are model labelled in for each each pathway. Anaerobic metabolismsclade are are shown.identifed Electron by the grey acceptors background. for respirationAcronyms are (blue) alphabetically and marker as follows: CBB, Calvin-Benson-Basshamgenes (red) cycle; are cyt labeled c, cytochrome for each c oxidase; pathway, cyt andbd, cytochrometransformations bd ubiquinol:oxidreductase; that are mcrA, methyl coenzymevariable reductase among thesubunit groups A; mmoX are, soluble depicted methane as broken monooxygenase lines. Anaerobic component A; mxaF, methanol dehydrogenasemetabolisms alpha are subunit; identified narG, respiratory by the gray nitrate background. reductase subunit Acronyms alpha; arenirK /S, copper- containing (K) or cytochrome (S) nitrite reductase; norB; nitric oxide reductase; nod, nitric oxide dismutase; nrfH, tetraheme alphabeticallyNapC/NirT-type cytochromeas follows: c; CBB, pmoA Calvin-Benson-Bassham, particulate methane monooxygenase cycle; cyt subunit c, A; RuMP, Ribulose MonoPhoshatecytochrome cycle; c xoxFoxidase;, PQQ-dependent cyt bd, cytochrome ethanol/methanol bd ubiquinol:oxidreductase; dehydrogenase. mcrA, methyl coenzyme reductase subunit A; mmoX, soluble methane monooxygenase component A; mxaF, methanol dehydrogenase alpha denitrifcation subunit; through annarG intra-aerobic, respiratory pathway. nitrate acids, reductase iron or subunit manganese. alpha; Tese nirK/S organisms, thrive Across all the bacterialcopper-containing methanotrophs (K) sampled or cytochrome to in (S) anoxic nitrite environments, reductase; and,norB like; nitric methanogens, date, while muchoxide of the reductase; enzymatic machinerynod, nitric for oxideare subjects dismutase; to oxygen nrfH toxicity, tetraheme (Haroon et al., 2013; aerobic methaneNapC/NirT-type oxidation is strongly cytochrome conserved c; SchellerpmoA et, al., particulate2016; Cai et al., methane 2018). T e variation (e.g. MMO), energymonooxygenase generation and subunitother pathways A; RuMP, in Ribuloseelectron acceptors, MonoPhoshate methane inputs, cycle; or biomass difer between thexoxF phylum, PQQ-dependent (Fig. 3.1) or even ethanol/methanol strains diferences dehydrogenase. may partially explain the highly variable (Hoefman et al., 2014; Heylen et al., 2016). rates of anaerobic methane oxidation for these taxa Unlike the bacterial methanotrophs, the archaeal across ecosystems (Knitel and Boetius, 2009). methanotrophs do not employ MMO. Archaeal Te genes for the enzyme responsible for ANaerobic MEthanotrophs (ANME) instead anaerobic oxidation of methane by ANME, reverse the pathway used by the methanogens to methyl co-enzyme reductase (MCR), can be used produce methane, hereby oxidizing methane in the in conjunction with 16S rRNA genes to phylo- absence of molecular oxygen. Archaeal methane genetically identify these methanotrophs, and oxidation can be coupled to numerous electron can diferentiate them from related methanogens acceptors including sulfate, nitrate, nitrite, humic (Hallam et al., 2003). Using these marker genes,

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all currently known ANME lineages are members inserts of DNA cloned into plasmids (Hallam et al., within the Methanomicrobia class of the Euryar- 2004; Meyerdierks et al., 2005; Ricke et al., 2005; chaeota phylum. Within the Methanomicrobia, the Dumont et al., 2006; Chen and Murrell, 2010). ANME-1 appear to be a distinct order, several line- Contemporary metagenomics leverage next-gener- ages of ANME-2 form a monophyletic clade within ation sequencing advancements, typically resulting the Methanosarcinales order, while the ANME-3 in fragments (reads) with increased sequencing likely represent a specifc genus within the Metha- depth per sample (Escobar-Zepeda et al., 2015). nosarcinaceae family (Knitel and Boetius, 2009; However, as sequencing technology continues to Glöckner et al., 2017; Timmers et al., 2017). improve, metagenomics show increasing promise Single marker gene sequencing of the 16S rRNA in complete genome recovery from complex envi- genes along with bacterial MMO (pmoA, mmoX) ronmental samples. and archaeal MCR (mcrA) genes have sampled Metagenomic data can be analysed using two methanotrophs from a wide range of environments. main approaches: gene-centric or genome-centric From these studies it is inferred that a minor frac- (Fig. 3.2). In one gene-centric approach, sequenc- tion of methanotrophic phylogenetic diversity ing reads are aligned (mapped) to a database of is represented by axenic cultures. In fact, entire microbial genomes or reference genes to assign clades of methanotrophs still lack a single isolated reads to closely related genes, pathways or genomes representative, including ANME and Ca. Methy- contained in a database. While both time- and cost- lomirabilis. By comparing small subunit ribosomal efective, this approach may miss novel genomic protein S3 (rpS3) genes recovered in metagenomes information, owing to insufcient representation in with known reference isolate genomic sequences, databases. In an alternative gene-centric approach, we highlight that metagenomics continues to sequencing reads can be de novo assembled into recover potentially novel species, i.e. 251 out of larger genome fragments, which are then com- 641 recovered genes shared < 95% amino acid iden- pared to nearest neighbour genomes. Here the tity to methanotroph isolate genomes (see Web advantage is that novel functional and phylogenetic resources). Furthermore, this includes the identif- markers can be reconstructed from an environ- cation of numerous potentially novel species, using mental sample. Te limitation of both gene-centric the same criteria, even among the well-sampled approaches is the lack of a holistic genome content phylogenetic groups such as the Gammaproteobac- and organization, hindering the precise under- teria, e.g. 170 out of 539 rpS3 genes recovered from standing of the metabolic pathways or phylogenetic metagenomes (see Web resources). Recent meta- signals essential in description of novel taxa and omic studies continue to uncover and defne new pathways. lineages of methanotrophs, as well as provide nec- In contrast, in the genome-centric approach, essary metabolic and ecological context, thereby the assembled genomic fragments (contiguous playing a critical role in advancing our under- sequences, or contigs) are clustered together to standing of methanotrophy from single genes, to form distinct, population-level genome bins. In microbial communities, to entire ecosystems. the past 15 years, these Metagenome-Assembled Genome bins (MAGs) have yielded near-complete or essentially closed genomes from a myriad Metagenomic approaches used of environmental samples (Tyson et al., 2004; to study methanotrophs in situ Sharon and Banfeld, 2013; Parks et al., 2017). Tis and in vitro approach allows for genome wide comparisons, A ‘metagenome’ is the theoretical collection of which provide expanded phylogenetic resolution genomes or genomic information from members of through the use of conserved housekeeping genes, a given microbial community. While metagenomic residing at various locations in the genome (Rinke methods and sequencing technologies have changed et al., 2013; Hug et al., 2016; Yeoh et al., 2016; Parks over time, the core principle remains: the untargeted et al., 2017), as well as for the extended analysis of sequencing of DNA from a microbial community. the metabolic pathway content (Anantharaman et Te earliest metagenomic methanotroph studies al., 2016), virus–host linkages via CRISPR-Cas relied on sequencing libraries containing large loci, etc. (Edwards et al., 2016). A combination

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Environmental Sample

Approach Gene-centric Genome-centric Functional

CH 4 smo mcr 13 O2 mmo Fe(III) + CH4 +/- e- accept mcr CH OH 3 Fe(III) Time pmo mcr mdh Abundance NO - Fe(III) 2 13C-DNA CH2OH

Gene specific analyses Genomes of strains or Data and Isotopically labeled DNA of reads or assembed closely related (or RNA) Analysis fragments populations

Estimate known Capture new Enrich for low diversity and abundance phylogenetic diversity abundance members Purpose Reconstruct known Identify novel Examine community pathways metabolisms dynamics

Arctic permafrosts Lake waters Lake Washington Examples Deepwater Horizon Marine OMZ Wastewater treatment Rice paddies Wetland soils Natural methane seeps

Figure 3.2 Schematic overview of metagenomics approaches. There are two primary methods of metagenomic analysis: gene-centric that maps reads, genes, or contigs (represented by coloured dashes) to a reference database (boxed genes), and genome-centric that reconstructs genomes (broken circles) independent of reference databases. Functional metagenomics precedes these other methods using an initial enrichment, with or without a label, to more directly target methanotrophs, e.g. 13C-methane. The utility of these methods and example ecosystems are outlined for each approach. Acronyms are alphabetically as follows: mmo, methane monooxygenase genes; mdh, methanol dehydrogenase genes; mcr, methyl coenzyme M reductase genes; pmo, particulate methane monooxygenase genes; smo, soluble methane monooxygenase genes. of the established bioinformatics platforms and sample with 13C-methane, followed by the analysis the afordable sequencing technologies now allow of 13C-DNA (Friedrich, 2006). Sequencing of the for prevalent use of the genome-centric approach. labelled DNA, referred to as functional metagenom- Given sufcient depth of sequencing, this method ics, followed by gene- or genome-centric analysis, can recover genomic bins of the quality comparable directly links methane consumption to specifc taxa to the quality of draf genomes from isolate cultures (McDonald et al., 2005; Kalyuzhnaya et al., 2008; (Bowers et al., 2017), and thus this method repre- Chen and Murrell, 2010). Functional metagenom- sents an important avenue for discovering novel ics, with or without SIP, have been proven to be methanotrophs. Here we catalogue the methano- exceptionally useful for examining slow-growing or troph MAGs from multiple databases, to provide consortia-dependent methanotroph taxa, provid- facile access to these genomic data, and summa- ing a majority of genomic information to date for rize their diversity and ecosystem and high-level lineages such as ANME and Ca. Methylomirabilis genomic information (see Web resources). that lack pure culture representatives (discussed Given that all methanotrophs, by defnition, are below). Te difculties facing both methanotroph able to consume methane, metagenomic methods isolation and metagenomic sequencing of complex have been successfully combined with stable isotope samples can be simplifed or overcome by the use of probing (SIP), or incubation of an environmental enrichment-based metagenomics.

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Metagenomic sampling of in depth, the ANME types were the most abun- methanotroph phylogeny and dant methanotroph taxa. Here, it was inferred physiology across diverse that the electron acceptor for methane oxidation habitats was ferric hydroxides or sulfate, the later reduced We queried 741 metagenomes and metatranscrip- through a partnership with sulfate-reducing bacte- tomes associated with methane cycling from the ria (SRB). Tese results are consistent with much Integrated Microbial Genomes database (IMG, of our understanding of extremely diverse ANME 4th June 2018), provided by the Joint Genome metabolism derived primarily from naturally and Institute. We used ribosomal protein S3 (rpS3) artifcially enriched vent samples (Knitel and genes from know methanotroph genomes to mine Boetius, 2009), discussed in more detail in sections a total of 566 methanotroph rpS3 proteins, from below. Beyond anaerobic methane oxidation, some 235 metagenomic samples, spanning a variety of additional evidence was provided that aerobic taxa and ecosystems (see Web resources). In addi- methanotrophs may also be important in marine tion to this gene-specifc search, we also recovered methane seep systems, as reads mapping to aerobic the rpS3 genes from 75 methanotroph MAGs (37 methanotroph genomes from the Proteobacteria inferred methanotroph MAGs lacked this gene) (Methylococcales and Rhizobiales), Verrucomicro- (see Web resources). Tese two metagenome- bia (Methylacidiphilales), and Methylomirabilota derived rpS3 data sets, along with reference rpS3 (Ca. Methylomirabilis) were also present in many proteins from the known methanotroph isolate of the metagenomes (Håvelsrud et al., 2011, lineages, were used to construct a phylogenetic 2012; Tu et al., 2017). Additionally, gene-centric tree visualizing the methanotroph phylogenetic metagenomics performed on a hydrocarbon seep diversity captured by both isolation and metagen- in the Red Sea reconstructed functional genes for omic sampling (Fig. 3.3). Auxiliary rings on the methane oxidation not detected in clone libraries, phylogenetic tree provide additional information and was one of the frst studies showing that sMMO for class level assignment, the type of sequencing genes were more abundant than pMMO genes in a performed, and the ecosystem from which the natural ecosystem (Abdallah et al., 2014). sequence was recovered. Te dissimilarity between In addition to the naturally occurring methane reference isolate genomes and MAGs’ rpS3 genes seeps, anthropogenic sources of hydrocarbons shows that many MAGs are not well represented also occur in marine and terrestrial systems, and by isolates – in only 22 out of 112 MAGs did the represent another focus for the methanotrophy rpS3 gene peptides share > 95% amino acid identity research. Following the Deepwater Horizon spill with reference genomes. MAGs continue to play an in 2010, several metagenomic studies revealed that important role in expanding the methanotroph foli- aerobic Gammaproteobacteria methanotrophs of age on the tree of life. Using our meta-analysis as a the order Methylococcales must have consumed guide, below we highlight some of the prominent nearly all the methane in the water column, mitigat- metagenomic studies performed across a range of ing methane release to the atmosphere (Mason et diferent habitats, and indicate the insights gleaned al., 2014; King et al., 2015; Yergeau et al., 2015). about methanotrophs from these studies. Te methane consumption activity also caused an oxygen anomaly as well as depleted certain metals Marine and terrestrial hydrocarbon in the water column (Kessler et al., 2011; King et and methane seeps al., 2015; Shiller et al., 2017). Taken together, these Gene-centric metagenomics have provided studies demonstrate both the positive and poten- genomic insights into the anaerobic methane- tially the negative impacts methanotroph activity oxidizing taxa from natural hydrocarbon seeps may have on an ecosystem scale. at Coal Oil Point (California) (Håvelsrud et al., An additional hydrocarbon source is oil sand 2011), Troll petroleum reservoir (Norway) (Håv- tailing ponds, which are small natural freshwater elsrud et al., 2012), Lei-Gong-Hou mud volcano bodies contaminated with hydrocarbon waste afer (Taiwan) (Tu et al., 2017), and the Mississippi industrial petroleum extraction from bituminous Canyon (Gulf of Mexico) (Vigneron et al., 2013). sands. SIP-enabled gene-centric metagenomics In these ecosystems, reaching up to 1100 metres of Canadian tailings pond sediments recovered

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Sample Treatment Native Enrichment Isolate ANME−1 ANME−2

Methylomirabilis Unknown

Methylacidiphilae Sample Treatment Ca. Sequencing Type Sequencing Type Ecosystem Metatranscriptome Metagenome

Alphaproteobacteria Single-amplified genome Fosmid

Ecosystem Agriculture Alkaline/Hypersaline Contaminated/Wastewater 1 amino acid substitutions per site Endosymbiont Freshwater Bootstraps >70% Forest Geothermal Marine Natural Seep Peat Permafrost Wetland Gammaprotebacteria Unknown

Figure 3.3 Phylogeny of methanotroph ribosomal protein S3 (rpS3) genes identifed in metagenomic datasets and isolates. A total 566 recovered and 152 reference – 77 isolates and 75 Metagenome-Assembled Genomes (MAGs) – rpS3 genes were used for analysis if the rpS3 gene was greater than 180 amino acids, approximately 50% of the maximum reference sequence length. Protein sequences were aligned using MUSCLE and modifed to remove predominantly gapped, clade-specifc positions resulting in 183 residues. The maximum- likelihood phylogenetic tree was generated using RAxML with the GAMMA model of rate heterogeneity and WAG substitution matrix, with 100 bootstrap replicates. Auxiliary layers around the tree are colour-coded for metadata accompanying the genomic information and indicate cultivation status (Sample Treatment), the type of sequencing performed (Sequencing Type), and the environmental source (Ecosystem) of the samples and genomic information. No information is provided for non-methanotrophic out-group sequences.

pMMO genes most similar to the Gammaproteo- and artifcially hydrocarbon- or methane-enriched bacteria Methylococcales member of the genus environments support diverse communities of Methylocaldum, inferred to cross-feed with non- methanotrophs that mitigate methane release into methanotrophic methylotrophic taxa via methanol the atmosphere. (Saidi-Mehrabad et al., 2013). Methanol-mediated communal metabolism of methane has been dem- Atmospheric methane sinks in dry onstrated in other ecosystems (Beck et al., 2013; soils Krause et al., 2017), suggesting that, while methano- Forest soils have long been of interest for the study troph and methylotroph genera may difer between of methanotrophs, due to their ability to act as ecosystems, the metabolic exchanges may be con- net methane sinks by consuming trace (< 2 parts served. Although generally in low quantities in the per million) quantities of methane present in the most marine and terrestrial environments, naturally atmosphere. Several studies have inferred that

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this phenomenon, named high-afnity methane permafrosts, suggests the importance of HAMO on oxidation (HAMO), likely occurred through a global scale. the activity of uncultivated Alphaproteobacteria In addition to the USCα methanotrophs, the methanotrophs (Dunfeld et al., 1999; Holmes et frst nearly complete genome of the divergent Gam- al., 1999; Henckel et al., 2000). One of the early maproteobacterial Upland Soil Cluster (USCγ) SIP-enabled metagenomics projects generated methanotroph has recently been recovered from sequences of Bacterial Artifcial Chromosomes cold, arid soils. Tis genome was shown to be sig- (BACs) from forest soil, recovering fragments of nifcantly divergent from the genomes of known genomes most closely related to members of the Methylococcales (Edwards et al., 2017) (Fig. 3.3; Alphaproteobacteria genus Methylocystis (Dumont see Web resources), while encoding typical methy- et al., 2006). Tis demonstrated the application of lotrophy metabolic pathways. Since their initial this technique for recovering the genomic contents discovery in forest soils, the recovery of USCγ of methanotrophs that are generally present at from Taylor Dry Valley in Antarctica demonstrates low abundance in complex soil communities. In a their distribution beyond forested environments separate study, sequencing BACs from forest soils (Edwards et al., 2017). Importantly, for both USCα provided the frst genomic information for the and USCγ lineages, these MAGs alone cannot Alphaproteobacterial Upland Soil Cluster (USCα) explain the specifc metabolic requirements or the methanotrophs, a prevalent lineage in forest soils enzymatic diferences that enable HAMO. Given implicated in oxidizing methane at atmospheric the broad environmental distribution of these pressure levels (Ricke et al., 2005). Te gene-cen- organisms (northern latitude forests, Antarctic tric approach revealed that, based on the pMMO deserts, and permafrost ecosystems), there is a need and neighbouring genes, the proposed USCα for cultivation and physiological studies targeting methanotroph was most similar to the known these lineages. Alphaproteobacteria methanotrophs (Ricke et al., 2005). Freshwater lake sediments Recently, Pratscher et al. (2018) provided the One of the best-characterized ecosystems for

frst morphological information for the USCα, microbial C1 cycling is freshwater lake sediments. and recovered the frst draf genome of USCα Tese systems have anoxic conditions in the methanotroph, by combining targeted cell sorting deeper sediments, resulting in methanogenesis, and metagenomic sequencing. Te resulting MAG with methane difusing to oxic sediment–water lacked sMMO genes, but contained several pMMO interface, where aerobic methanotrophy occurs. gene variants (Pratscher et al., 2011, 2018). It was One of the earliest uses of SIP in combination proposed that these diferent pMMO variants may with metagenomics investigated the microbial function in a manner similar to variants previously taxa involved in methylotrophy in Lake Washing- identifed in Methylocystis isolates, showing difer- ton sediments (Kalyuzhnaya et al., 2008). Here ent substrate afnities towards methane (Baani sediments incubated with 13C-methane largely and Liesack, 2008; Dam et al., 2013). Additionally, recaptured the data from the cultivation eforts, the USCα genome encoded pathways for acetate showing that Methylobacter, of the Gammaproteo- utilization, signifying the signifcance of the capa- bacteria Methylococcales clade, were the dominant bility to utilize alternative carbon substrates as a methane-oxidizing taxa (Auman et al., 2000). means of adaptation for HAMO (Pratscher et al., Additionally, these methane-fed communities were 2011, 2018). Mining of 16S rRNA gene-databases enriched for Betaproteobacteria species Methyloten- posited that USCα species are not restricted to era, which were likely labelled through consuming forest soils, but are also present in lava cave bio- methanol, a by-product of methane oxidation. Tis flms and arctic permafrost (Pratscher et al., 2018). metabolic cross-feeding spurred a series of experi- Similar to these fndings, Singleton et al. (2018) ments showing that at lower oxygen:methane recovered a partial USCα methanotroph genome mixing ratios, Methylobacter, consistently outcom- from partially thawed permafrost soils in Sweden. peted other methanotrophs, and also produced Te much wider distribution of USCα methano- methanol to cross-feed Methylotenera (Beck et al., trophs, including non-arid environments such as 2011; Oshkin et al., 2015; Krause et al., 2017).

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Alternatively, under high oxygen:methane mixing single genus of methanotrophic bacteria (Tveit et ratios, members of the same family but diferent al., 2015). genus (Methylosarcina) cross-fed diferent Betapro- Other studies have implicated Alphaproteo- teobacteria microorganisms (Methylophilus) bacteria as key methanotrophs to respond to (Hernandez et al., 2015). Tese fndings highlight permafrost thaw. Mackelprang et al. (2011) used the role of cooperative metabolic exchanges fuelled changes in the relative abundance of 16S rRNA by methanotrophy occurring across ecosystems, and pMMO genes during in vitro thaw simulation and the value of both laboratory and feld based experiment, to infer members of the genus Methy- studies. locystis as responsive species. A large-scale study at Stordalen Mire in Sweden found both classes Thawing arctic permafrost of Proteobacteria methanotrophs to be present, Tere is growing interest in the metabolic activity but these were diferentially distributed across the of methanotrophs in arctic peat and permafrost thaw gradient (Singleton et al., 2018). Metagen- soils. Tese environments store approximately omes from partially thawed and intact permafrosts 1300–1600 petagrams of carbon that is suscep- contained mostly reads mapping to Alphaproteo- tible to release under predicted climate warming bacteria methanotrophs, particularly Methylocystis scenarios (Dean et al., 2015). Warming tempera- and USCα MAGs, while fully thawed permafrost ture trends threaten to thaw permafrost, making metagenomes harboured reads that mapped mostly the previously inaccessible carbon newly avail- to Gammaproteobacteria Methylococcales MAGs. able. Te newly accessible organic carbon can Tis trend was matched by the distribution and be degraded to yield methane, which may be diversity of pMMO and sMMO genes identifed consumed by the methanotrophs or be emited in the metagenomes, as well as metatranscriptomic into the atmosphere, the later with future climatic reads mapping to these genes (Singleton et al., feedbacks (MacKelprang et al., 2011). 2018). Several studies by Tveit et al. (2013, 2014) Two other studies in the Canadian High Arctic clearly articulated the importance of aerobic Gam- also used metagenomics technologies to investigate maproteobacteria Methylococcales in warming methanotroph diversity and activity in perma- permafrost. In the frst two studies, metatranscrip- frost soils. One gene-centric metagenomics study tomic data were collected from permafrost soils concluded that Gammaproteobacterial Methy- in Svalbard, Norway. Here 14 nearly full-length lococcales were the dominant methanotrophs in assembled 16S rRNA genes (> 1330 nucleotides) the active layer and up to 2 m depth into the arctic in the metatranscriptome were assigned to the permafrost (Yergeau et al., 2010). In contrast, a genus Methylobacter. Metatranscriptomic reads paired metagenomic, metatranscriptomic, and were mapped to pMMO, methanol dehydroge- metaproteomic investigation of another perma- nase, and formaldehyde oxidation pathways from frost site noted that Alphaproteobacterial USCα reference genomes, further demonstrating that methanotrophs recruited most reads and peptide members of Methylobacter were primarily respon- fragments in all datasets (Lau et al., 2015). It was sible for methane oxidation in these permafrost inferred that the relative aridity and atmospheric soils. Notably, genes from other methanotrophs methane consumption in this site, compared to were absent, or present at negligible counts (Tveit other permafrost feld locations, may have contrib- et al., 2013, 2014), demonstrating the metabolic uted to the prevalence of USCα. Similarly, Siberian prevalence of this one genus in these soils. Labora- permafrosts were also dominated by Alphaproteo- tory mesocosm incubations designed to examine bacteria members of the genus Methylocystis, but the efects of thaw gradient (temperatures ranging Gammaproteobacteria methanotrophs could also from 1 to 30 °C) on these soils also demonstrated be detected (Rivkina et al., 2016). that Methylobacter remained the dominant metha- In summary, while the active methane-oxidizing notroph (Tveit et al., 2015). Taking these studies communities across several diferent permafrost together, it is possible that in these permafrost habitats were dominated by Proteobacteria metha- soils, active methanotrophy today and under notrophs, it appeared that the thaw stage was a major future warming scenarios may be mediated by a controlling factor for the distribution and activity

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of diferent clades of methanotrophs in these per- primarily by Alphaproteobacteria Methylocystis mafrost ecosystems. Terefore, understanding and Methylosinus. Data collected from grasslands, the physiological constraints, e.g. temperature, forests, mangroves, and systems replete in plants hydrology, methane oxidation rates, etc. of Proteo- corroborated the fnding that Methylocystis types bacteria methanotrophs will be critically important were the most prevalent methanotrophs in soils for improving predictions of methane release from within the rhizosphere (Xu et al., 2014). Tere- Northern thawing permafrost ecosystems. Under- fore, further understanding of the physiology standing these dynamics is important for predicting and metabolism of Methylocystis is important for the efcacy of methanotrophs in reducing methane improving models for methane cycling in vegetated release from ecosystems with large amounts of environments. stored carbon. Te Great Lakes region on the border between the United States and Canada stores large amounts Temperate hydric soils and of carbon (Nahlik and Fennessy, 2016), and sediments (peats or wetlands) wetlands adjacent to Lake Erie are known to be Temperate wetlands can be defned as fooded or net methane emiters, with seasonal variations in seasonally fooded soils located in regions with rela- methane output (Angle et al., 2017; Rey-Sanchez tively mild climates, and these include freshwater, et al., 2018). Gene-centric approaches, 16S rRNA coastal, and peatland soils. Despite their small land gene abundances and pMMO gene transcript coverage, on a global scale, these systems account inventories, have shown that Methylobacter species for a disproportionate fraction of the total methane dominated the methanotroph community (Smith budget (Nazaries et al., 2013; Dean et al., 2018). et al., 2018). Furthermore, pMMO transcription Below we summarize some of the recent metagen- was season-dependent, and found to be increased omic investigations of these habitats. in autumn relative to summer. In contrast, metha- In a boreal peat bog in Minnesota, SIP- nogen gene expression was relatively stable over the enabled metagenomes revealed enrichment for sampling period, suggesting that the greater meth- Alphaproteobacteria Methylocystis and Gammapro- ane emissions observed in the summer months teobacterial Methylomonas pMMO genes, at a ratio might be due to the reduced methanotroph activ- of about 3:1 (Esson et al., 2016). At increasing ity and not to increased methanogenesis activity depths, the Methylomonas signal was eventually (Smith et al., 2018). A biogeographical survey con- below detection, consistent with qPCR and micro- ducted by Smith et al. (Smith et al., 2018) detected array data from the same study site (Esson et al., pMMO genes similar to the those of Methylobacter 2016). Other studies also found that pMMO genes lineages found in many methane-emiting Northern afliated with Methylocystis and other Alphapro- latitude environments that include Lake Washing- teobacteria methanotrophs tended to be more ton (Chistoserdova, 2011a), the restored Twitchell abundant, possibly owing to the acidic pH or to Island peatlands (He, S. et al., 2015), and the Prairie copper limitation (Chen et al., 2008; Kolb and Potholes region of North Dakota (Dalcin Martins Horn, 2012; Lin et al., 2014; Graham et al., 2017). et al., 2017). Despite major diferences in the Corroborating decades of interest in peatland latitude, the prevalence of Methylobacter genotypes systems, gene-centric analyses have reafrmed the and their genomic conservation across geographi- importance of Alphaproteobacteria methanotrophs cally distinct wetland and permafrost habitats in many of these acidic environments as natural fl- suggests that further studies of the representatives ters of methane release. of this genus are relevant for global climate change In a restored peat ecosystem in California, research (Tveit et al., 2013). metagenomes from samples collected up to 25 cm into the soil column revealed methanotroph rela- Rice paddies and agricultural soils tive abundances ranging from ≈ 1% to 3% of total Rice paddies represent inundated agricultural soils microbial community (S. He et al., 2015). Both the that contribute 4–20% to the atmospheric methane number of pMMO genes and the reads mapped to budget (Nazaries et al., 2013; Dean et al., 2018), components of methane oxidation pathways were despite most of the methane generated (up to 90%) enriched in plant-associated samples, dominated being consumed by the methanotrophs (Bao et al.,

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2014; Lee et al., 2015). Given the large error in cur- Seasonal dynamics likely play a role in constrain- rent predictions for methane emissions from these ing methanotroph abundance and activity in rice habitats, there is a desire to beter understand how paddies. During the drought season, rice paddies agricultural land management impacts the activity typically serve as net sinks of methane (Harriss et of methane-cycling microorganisms. In particular, al., 1982; Kelley et al., 1995; Melling et al., 2007; rice paddies are characterized by drastic oxyclines Kolb and Horn, 2012). Incubation of rice paddy that occur along the water depth profle and along soils at high versus near-atmospheric methane the distance from the plant roots, conducive of mixing rations found a strong enrichment for Gam- both aerobic and anaerobic methanotrophy (Reim maproteobacteria genus Methylosarcina and mild et al., 2012; Lee et al., 2015). A study on Indian rice enrichment of Alphaproteobacteria Methylocystis paddies with diferent fertilizers by Bhatacharyya (Cai et al., 2016). From a combination of metatran- et al. taxonomically classifed reads, fnding that scriptomics and SIP experiments, it was thought Gammaproteobacteria Methylococcales, par- that Methylosarcina responded to the initial spikes ticularly Methylobacter and Methylococcus, were of methane (≈9000 ppm methane), consuming the most abundant methanotroph taxa, regard- methane to produce metabolites that are necessary less of fertilizer treatment (Philippot et al., 2010; to support HAMO (≈2 ppm methane) by the Alp- Bhatacharyya et al., 2017). Alphaproteobacteria haproteobacteria methanotrophs. Interestingly, this and Verrucomicrobia methanotrophs were also study noted that neither of the USC clades were detected, but were less abundant. Te reconstruc- detected in the metagenomes or metatranscrip- tion of the formaldehyde assimilation pathways tomes, therefore HAMO observed in these samples showed predominance of the serine cycle genes, was dependent on the known methanotrophs and which are present in both Alphaproteobacteria and not on the expected USC-type methanotrophs. Gammaproteobacteria methanotrophs (Ward et al., 2004; Takeuchi et al., 2014). Subsurface ecosystems sustained by Profling active methane metabolism along methane rice paddy soil compartments, including water, Microbial life in caves and subsurface ecosys- soil, phyllo-, rhizo-, and endo-spheres, is a current tems, which typically lack organic carbon input research area. One study that applied metagenom- by plant primary productivity, is dependent on ics in combination with metaproteomics to both chemolithotrophic metabolisms. Methane is a the phyllosphere and the rhizosphere has found relatively rich source of energy and carbon in a that proteins for both methane oxidation and variety of geographically unrelated subsurface methanogenesis were signifcantly enriched in the ecosystems (Hutchens et al., 2004; Brankovits et rhizospheres. Both Alphaproteobacteria and Gam- al., 2017; Karwautz et al., 2018). A model cave maproteobacteria methanotrophs were detected, system in Romania, Movile Cave, undergoes and both sMMO and pMMO were expressed at seasonal extremes of methane concentrations due approximately equal peptide spectra (Knief et to consumption by up to four distinct bacterial al., 2012). In contrast, Bao et al. (2014) failed to clades of methanotrophs – USCα, USCγ, and two recover any peptides mapping to Methylococcales potentially novel lineages (Waring et al., 2017). and found that rice root metaproteomes mainly Metagenomic analysis revealed that aerobic represented methanotrophic Alphaproteobacteria methanotrophic marker genes were present in of the Methylocystaceae family, which carry out both the oxic mat and the anoxic sediment, and both methanotrophy and dinitrogen fxation in the also showed that the predominant MMO types vascular bundles and epidermal cells of rice roots. varied between niches (Kumaresan et al., 2018). A separate metatranscriptomic analysis of rice In another subsurface environment, an artifcial paddy laboratory incubations found Methylococ- mine in a granitic system in Japan, methane oxi- cales to be the predominant methane oxidizer, but dation appeared to entirely rely on the ANME was restricted to the very narrow 2 mm thick oxic type Archaea, and a nearly complete genome of surface layers, and no transcripts afliated with an ANME-2d organism has been reconstructed methanotrophs were detected in the anoxic zone at that difered from the previously reconstructed 6–8 cm (Kim and Liesack, 2015). ANME-2d genomes by lacking any genes for

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denitrifcation or metal reduction (Ino et al., frst near-complete pathway for Archaeal methane 2018). Instead, the genome encoded functions oxidation, inferring the methanogenesis pathway

for a putative respiratory H2 oxidation as well as operating in reverse (Hallam et al., 2003; Meyer- a possible pathway for utilizing zero-valent sulfur dierks et al., 2010). Additionally, they recovered the to exchange electrons with syntrophic partners frst [Fe–Fe] hydrogenases in Archaea, as well as the (Milucka et al., 2012; Ino et al., 2018). Over- multiple multi-haem c type Cytochromes (MHC) all, caves and other subsurface ecosystems are that were proposed to act as the electron shutling relatively understudied environments, but the few intermediates involved in interactions with the examples in the literature suggest that these eco- sulfate-reducing bacteria (SRB) or in metal reduc- systems are home to interesting methanotrophs, tion (Meyerdierks et al., 2010). Tese genomes and that methane oxidation occurs diferentially were critical, providing the frst evidence for the along ecological niches. types of anaerobic energy generation mechanisms encoded by the archaeal methanotrophs. Shortly afer, combined metagenomic and metaprot- Metagenome-derived genomes eomic/metatranscriptomic studies corroborated reveal methanotroph metabolic the genomic potential postulated from the frst variability ANME-1 genome (Stokke et al., 2012; Krukenberg In this review, we compiled the genomic informa- et al., 2018). tion for all 80 isolates and 112 MAGs available via Shortly afer the recovery of the frst ANME-1 the IMG or the National Center for Biotechnology genome, a MAG representative of the ANME-2d lin- Information (NCBI), assigned to methanotroph eage was reconstructed from a methane consuming taxa or taxa described as a putative methanotrophs and denitrifying bioreactor inoculated with a mix- in publications (Fig. 3.4; see Web resources). ture of waste sludge and freshwater lake sediments Reconstruction of MAGs provides genomic infor- (Haroon et al., 2013). Tis near-complete genome mation for major clades entirely lacking a single pure is referred to as Candidatus Methanoperedens culture representative, e.g. ANME, USCα, USCγ nitroreducens, named afer the inferred metabolic or Ca. Methylomirabilis. Tese MAGs provide capacity to couple methane oxidation, via reverse essential information about these microorganisms methanogenesis, to dissimilatory nitrate reduction that are phylogenetically and physiologically diver- (Haroon et al., 2013). In contrast to ANME-1, the gent from the known, isolated methanotrophs. Ca. Methanoperedens genome contained a full Genome-centric metagenomics approaches have reverse methanogenesis pathway. An additional more than doubled methanotroph genomic data- unique feature of this genome was the presence of base sampling in as litle time as a decade. Below, only the frst gene for denitrifcation, nar, which we summarize the major discoveries that analyses would only allow Ca. Methanoperedens nitrore- of MAGs have added to the phylogenetic and meta- ducens to reduce nitrate to nitrite (Haroon et al., bolic understanding of methanotrophs in natural as 2013). Metatranscriptomic coupled to 15N-nitrate well as artifcial setings. SIP analysis applied to the bioreactor microbial community suggested that the nitrite generated MAGs represent the only genomic was likely consumed by the nitrite-utilizing micro- information from some uncultivated organisms, including Ca. Methylomirabilis, or by methanotroph lineages anaerobic ammonia-oxidizing microorganisms To date there is not a single ANME representative in (Raghoebarsing et al., 2006; Haroon et al., 2013). axenic culture. Genomic information for ANME-1 Subsequent ANME-2 MAGs from enriched fresh- was frst obtained in 2005, linking methanogenesis water canal sediments in the Netherlands found and 16S rRNA genes on the same contig (Mey- that other Ca. Methanoperedens nitroreducens erdierks et al., 2005). Tis fnding was followed MAGs encoded the capacity to perform dissimila- by the frst MAG described for ANME-1 lineage, tory nitrate reduction to Ammonia (DNR, nrfH) recovered in 2010 by Meyerdierks et al. (2010) by (Arshad et al., 2015). Taken together, these stud- fosmid sequencing of microbial mats in the Black ies are important for highlighting the coupling of Sea. From this MAG, the authors reconstructed the anaerobic methane oxidation to nitrate reduction.

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Major clades with axenic cultures Major clades without Gammaproteobacteria Alphaproteobacteria axenic cultures Methylococcaceae Beijerinckaceae 60 Methylococcus USCα Methylomirabilota Unknown Methylosinus Methylomirabilaceae Methylomonaceae Methylocystis Ca. Methylomirabilis Endosymbionts Methylocapsa Crenothrix Hyphomicrobiaceae Methanomicrobia Methylovulum Unknown ANME-1 Methylosarcina Unkown Methylomonas Verrucomicrobia ANME-2 40 Methyloglobulus Methylacidiphilaceae Ca. Methanoperedens Methylobacter Unknown Unknown Unknown Unknown USCγ OPU3 Unknown 20

0 Number of Metagenome-Assembled Genomes ANME

MethylacidiphilaeMethylomirabilis Alphaproteobacteria Ca. Gammaproteobacteria

Figure 3.4 Phylogenetic composition of rpS3 genes recovered in putative methanotrophic Metagenome- Assembled Genomes (MAGs). Distribution of MAGs at the genus level, if known, among the major clades of methanotrophs and grouped by family according to SILVA (SSU refNR 132) and the Genome Taxonomy DataBase (GTDB). Yellow stars indicate the lack of axenic, i.e. pure, cultures of specifc methanotrophic genera (Proteobacteria and Verrucomicrobia) and classes (Methylomirabilota and Methanomicrobia).

Since their initial discovery, approximately 20 methane oxidation to iron reduction (Etwig et al., genomes have been reconstructed for the ANME 2016). Furthermore, these analyses also suggested lineages, including 4 ANME-1 and 15 ANME-2 that MHC was the prevailing mode of interaction representatives (Fig. 3.4, see Web resources). between the ANME and the SRB among both MAGs of both ANME-1 and ANME-2 lineages the thermophilic and the psychrotolerant com- contain a surprising number of MHC genes, munities of the, respectively, Guaymas Basin and suggesting that both groups may be capable of the Crimean Peninsula, and the mid-Norwegian coupling methane oxidation to the reduction of margin (Meyerdierks et al., 2010; Stokke et al., metals (Meyerdierks et al., 2010; Wang et al., 2014; 2012; Krukenberg et al., 2018), suggesting that this Cai et al., 2018), or Direct Interspecies Electron function is important across temperature and geo- Transfer (DIET) to bacterial consortia (Wegener et graphic ranges. Genomic inferences suggested that al., 2015). Studies investigating anaerobic methane ANME-2 have the potential for nitrogen fxation, metabolism have shown that ANME-1 increase a fnding confrmed by the cellular incorporation extracellular cytochrome production and form of labelled dinitrogen gas (Pernthaler et al., 2008), cellular structures connecting methane oxidizers and in contrast to the other ANME-2 MAGs to their partnering SRBs (Wegener et al., 2015). reconstructed from subsurface ecosystems (Ino et Others have shown that ANME-2, possessing al., 2018). Taken together, metagenomics, ofen in MHC with 41 haem-binding motifs, coupled combination with other techniques, have provided

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a wealth of information on the metabolic diversity using oxygen-dependent pMMO in anoxic condi- maintained among ANME lineages. tions (Etwig et al., 2010). Tese reconstructed ANME MAGs have also enabled improved detection of the ANME-type MAGs demonstrate the breadth of methanotrophs from a wide range of ecosystems. phylogenetic diversity within the For instance, sequences have been recruited to Proteobacteria methanotrophs ANME genomes from deep marine ecosystems and Beyond the lineages comprised exclusively of wastewater treatment reactors (Raghoebarsing et al., uncultivated methanotrophs, MAGs have also 2006; Håvelsrud et al., 2011, 2012; Lee et al., 2016; sampled lineages represented by or closely related Welte et al., 2016; Cai et al., 2018), but also from to the cultivated methanotrophs (Fig. 3.4). rice paddies, wetlands, and peatlands soils, as well Multiple MAGs have been recovered related to as from subsurface locations (Takeuchi et al., 2011; the members of Methylobacter, Methylomonas, Vaksmaa et al., 2017; Ino et al., 2018). Te impact of and Methylocystis (Fig. 3.4; see Web resources). ANME in ecosystems exposed to dynamic oxygen Additionally, MAGs reconstructed from marine fuctuations is poorly understood relative to marine oxygen minimum zone and marine endosymbiont and wastewater treatment environments, but more samples have sampled novel lineages within the genomic representatives in databases may result in Gammaproteobacteria. In this section, we high- promising new areas for future research. light the metabolic information derived MAG In addition to ANME, methanotrophic repre- representatives of uncultivated Proteobacteria sentatives of the Methylomirabilota, formerly of methanotroph lineages. the candidate NC10 phylum, the Ca. Methylomi- In marine systems, endosymbiotic methano- rabilis genus also lack any pure culture isolates. trophs have been identifed, forming thus far a However, genomic insights into the metabolism monophyletic lineage within Gammaproteobac- of this group are enabled by at least eight MAGs. teria Methylococcales (Petersen and Dubilier, Tese genomes were reconstructed from denitrify- 2009). One of the most well-known marine ani- ing wastewater treatment reactors (Raghoebarsing mals to host the methanotrophs in its gill tissues et al., 2006). Tese anoxic reactor f uids, when is Bathymodiolus, a mussel found at the botom of supplemented with nitrate and methane, became the ocean (Fisher et al., 1987; Nix et al., 1995). enriched with two anaerobic methanotroph Because endosymbiotic bacteria are difcult to populations: ANME-2, described above, and Ca. isolate, knowledge on their metabolism relies on Methylomirabilis (Etwig et al., 2010). Te exact these culture-independent methods. Phylogenetic mechanism of nitrogen transformation in this analyses using 16S rRNA and pMMO genes system was largely unknown until the recovery of and ultrastructure afliated the endosymbiotic MAGs from the bioreactor. Analysis of the Ca. methanotrophs with the Methylococcales genus Methylomirabilis MAGs identifed a paradoxical Methyloprofundus (Spiridonova et al., 2006; lack of the nitrous oxide reductase (nos) genes Tavormina et al., 2015; Sun et al., 2017). that were expected to be responsible for the stoi- Two recent publications independently chiometric conversions of nitrite to dinitrogen gas, reconstructed MAGs from distinct species of observed in previous studies (Etwig et al., 2010, Bathymodiolus (Ponnudurai et al., 2017; Tak- 2012). To explain this phenomenon, Etwig et al. ishita et al., 2017). Te rpS3 genes recovered (2010) hypothesized that an unknown enzyme, a from these genomes were most closely related nitric oxide dismutase (NOD), was responsible for to each other and to Methyloprofundus (Fig. 3.3; disproportionation of two nitric oxide molecules see Web resources). Tese MAGs were not only into one molecular nitrogen and one molecular enlightening for resolving the identity of the oxygen. While the biochemistry of this hypoth- endosymbionts, but also for their function(s) esized enzyme remains to be fully elucidated within the host. Ponnundurai et al. (2017) con- (Reimann et al., 2015), intracellularly generated cluded that the methanotroph supplied carbon oxygen could explain the production dioxygen in dioxide for assimilation by other non-methano- cultures with no other exogenous sources, and trophic endosymbionts, as well as synthesizing enable the traditional aerobic methane oxidation amino acids and necessary cofactors for the host.

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Takishita et al. (2017) found that the metha- genus Methylocapsa (Ricke et al., 2005; Pratscher notrophic endosymbiont synthesized unique et al., 2018; Singleton et al., 2018), but evolution- cholesterol intermediates that the mussel could ary history of the USCγ is less clear (Edwards et not produce on its own. Further investigations al., 2017). While the pMMO marker genes are into these relationships using metagenomics will similar to Methylococcales genus Methylocaldum, likely uncover additional critical functions these the 16S rRNA gene recovered in the MAG was endosymbiotic methanotrophs perform for the most similar to microorganisms within the Gam- host, as well as further insights into the evolution maproteobacteria class Chromatiales (Edwards of methanotroph-animal endosymbioses. et al., 2017), similar to its placement upon the Recovery of MAGs from low-oxygen marine fringe of the Methylococcales clade in the rpS3 ecosystems have enabled the identifcation of gene phylogeny (Fig. 3.3). To our knowledge, multiple uncultivated, novel Gammaproteobacte- there are no other close isolated relatives to the ria clades. From deep-sea marine vents at the USCγ MAG, therefore more cultivation-based Lau Basin, Methylothermaceae B42 is the only sampling is needed to make inferences about genomic representative of a likely novel genus metabolic or phylogenetic relationships for this in the Gammaproteobacteria Methylococcales genome (Edwards et al., 2017). (Skennerton et al., 2015). Tis genome shares Interestingly, the recovery of USCα MAGs in only 94% 16S rRNA sequence identity with thawing permafrost by Singleton et al. (2018) its closest isolated relative in the genus Methy- uncovered more phylogenetic novelty within the lohalobius. Similarly, the frst genome from the Alphaproteobacteria. Tis includes the presence uncultivated Operational PmoA Unit 3 (OPU3) of pMMO and sMMO genes in a novel genus lineage was recovered from Oxygen Minimum of the non-methanotrophic but methylotrophic Zone (OMZ) of Gulfo Dulce, sharing < 93% 16S Alphaproteobacteria family Hyphomicrobiaceae rRNA sequence identity with any Methylococ- (Singleton et al., 2018). Tis is not the frst occur- cales isolates (Hayashi et al., 2007; Padilla et rence of MMO detected in non-methanotrophic al., 2017). OPU3 appear to be cosmopolitan in methylotrophic clade; a species of non-metha- marine systems and are enriched in some OMZ notrophic isolate genus Methyloceanibacter was ecosystems and during the Deepwater Horizon isolated that encoded pMMO genes and could oil spill (Tavormina et al., 2010; Lesniewski et al., utilize methane (Vekeman et al., 2016a). Tese 2012; Li et al., 2014). Both Methylothermaceae genome-centric studies in polar regions have not B42 and OPU3 MAGs were sampled from oxy- only improved the phylogenetic resolution of the gen-limited environments, and other 16S rRNA USC methanotrophs, but have additionally pos- gene surveys indicate that the OPU3 are found ited a novel taxa capable of oxidizing methane. in deep waters (100–2,000 m depth), suggesting Despite being studied for decades, novel line- tolerance to low dissolved oxygen concentrations ages of methanotroph within the Proteobacteria (Tavormina et al., 2010), as discussed below. are constantly being discovered. Tis is true of Tese two studies have both recovered genomic ecosystems ranging from the botom and deep information for uncultivated lineages in marine waters of the ocean to terrestrial ecosystems across systems, which likely signify the beginning of the globe. Interestingly, much of the new diver- future discoveries of novel methanotrophic Gam- sity is found within the existing methanotroph maproteobacteria taxa in marine environments families, e.g. Beijerinckaceae, Methylocystaceae, through metagenomics. and Methylococcaceae. Further analyses of these Although most information regarding the genomes may provide information on the strain- distribution and ecology of USC methanotrophs level variations enabling adaptation to diferent comes from forest soils, MAGs representing both environmental conditions across the globe. It is USCα and USCγ have been reconstructed from apparent that natural ecosystems host many novel polar latitudes (Edwards et al., 2017; Pratscher et methanotrophs, and the employment of metagen- al., 2018; Singleton et al., 2018). Tere is increas- omics will continue to resolve the vast diversity ingly strong evidence that the USCα are a divergent of methanotrophs by reconstructing MAG repre- lineage closely related to Alphaproteobacteria sentatives of uncultivated lineages.

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Methylococcales MAGs widely metatranscriptomes from these soils (Smith et al., encode the denitrification potential, 2018). potentially enabling methane Te denitrifcation metabolic potential of Meth- oxidation under low oxygen ylococcales isolates and MAGs has been recently conditions inventoried several times (Padilla et al., 2017; Smith Using pure cultures, Stein and colleagues provided et al., 2018). Te most recent analysis by Smith et evidence that Gammaproteobacteria methano- al. (2018) recovered at least partial denitrifcation trophs may conserve oxygen for methane oxidation (nar, nap, or nir) encoded in over 45 out of 57 and use nitrate, rather than oxygen, to generate a combined MAG and isolate genomes. Moreover, proton motive force for energy generation (Kits 77% of the 18 reconstructed MAGs contained the et al., 2015). Tis metabolic potential has been capacity for reducing nitrate (Smith et al., 2018). inferred from multiple, diverse MAGs, located Notably, both analyses failed to identify genes for across a range of ecosystems. It is thought that the fnal step in denitrifcation, the reduction of this capacity may enable methane oxidation in nitrous oxide to dinitrogen gas by nitrous oxide environments with low or below detectable oxygen reductase (nos), in any Methylococcales genomes concentrations. (Padilla et al., 2017; Smith et al., 2018). Terefore, In marine oxygen minimum zones, both the it remains unclear whether the greenhouse gas Methylothermaceae B42 and OPU3 MAGs mitigation benefts from methane consumption encode genes for denitrifcation. T is includes activity across wider oxygen gradients or whether it the capacity to reduce nitrate to nitrous oxide might be ofset by the production of a more potent (nar, nap, nir, nor). Both Methylothermaceae B42 greenhouse gas nitrous oxide. Te prevalence and and OPU3 MAGs contained nar genes that are conservation of this metabolism among Methy- divergent from the genes in other Methylococcales lococcales suggests it is likely a valuable metabolic methanotrophs, suggesting a recent horizontal strategy, however more environmental context and transfer from non-methanotrophs (Skennerton expression data are required to truly understand the et al., 2015; Padilla et al., 2017). It was theorized impacts of methanotroph-mediated denitrifcation. that denitrifcation can support methane oxida- Beyond denitrifcation, methanotrophs may also tion in marine waters where dissolved oxygen is be able to tolerate much lower oxygen concentra- commonly below detection limits. Interestingly, tions than previously thought. Genome analysis of transcripts for one of the nar genes, encoded by MAGs and isolate genomes has identifed the pres- the OPU3 MAG, were detected in anoxic marine ence of high-afnity cytochromes, e.g. cytochrome waters (Padilla et al., 2017). Tis is so far the only bd ubiquinol–oxidoreductase, in addition to the environmental evidence that these denitrifcation traditional low-afnity cytochrome c oxidase. Te pathways, encoded by aerobic Gammaproteobac- use of these high-afnity oxidases would enable teria methanotrophs, are transcribed or expressed methane oxidation coupled to aerobic respiration in situ (Padilla et al., 2017). in nanomolar range of oxygen (Skennerton et al., Beyond marine systems, the genomic potential 2015; Oswald et al., 2017; Smith et al., 2018). Tus, for denitrifcation has been widely reported in meth- it is possible that methane oxidation by aerobic Pro- anotroph MAGs from terrestrial environments. For teobacteria may be occurring in environments with example, Dumont et al. (2011) observed expres- lower dissolved oxygen than currently assumed in sion of Methylobacter-like denitrifcation genes in global models (Riley et al., 2011; Xu et al., 2016). oxic incubations of lake sediments. Additionally, Another possible adaptation for maintaining the frst genome representative of the mysterious methane consumption in low oxygen conditions Crenothrix clade encoded the metabolic capacity is ‘micro-aerobic fermentation’, which has been for denitrifcation, possibly explaining the meth- reported in pure cultures of Methylomicrobium ane consumption activity in anoxic incubations of buryatense (Kalyuzhnaya et al., 2013; Gilman et al., freshwater lake waters (Oswald et al., 2017). Lastly, 2015). Oswald et al. (2017) and Smith et al. (2018) Methylobacter MAGs from freshwater wetland soils both reconstructed pathways for fermentation in, also possessed the potential for denitrifcation, respectively, Crenothrix and Methylobacter MAGs. but the transcripts for this pathway was absent in Tese fermentation pathways would enable the

Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Metagenomics Unearthing Methanotroph Diversity | 73 re-oxidation of NAD(P), and would ultimately lead lanthanide series metal (known as Rare Earth Ele- to the secretion of mixed fermentation products ments, REEs) (Keltjens et al., 2014; Pol et al., 2014; including acetate, lactate, succinate, ethanol, and/ Chistoserdova, 2015, 2016). Low concentrations or hydrogen. However, discerning the likelihood of of REEs have been reported to strongly repress this fermentative metabolism from metagenomic expression of the mxa genes (Chu and Lidstrom, or metatranscriptomic data alone is difcult, and 2016; Chu et al., 2016), suggesting that XoxF may therefore future physiological investigations in be the ‘preferred’ MDH. Microorganisms using these and other lineages are warranted to beter XoxF-type enzymes are now of current interest not understand micro-aerobic fermentation by metha- only for the ability to transform multi-carbon com- notrophs. pounds (Good et al., 2016; Wehrmann et al., 2017), A fnal possible mechanism for consuming or alter trophic interactions (Krause et al., 2017), methane when oxygen is relatively depleted is by but also of for their potential in extracting REEs scavenging oxygen via a bacteriohemerythrin. from electronics (Martinez-Gomez et al., 2016),. Hemerythrins are able to bind molecular oxygen, Verrucomicrobia methanotrophs rely on the and they have been shown to interact with the XoxF MDH as all genomes thus far lack MxaF genes pMMO enzyme, suggesting a role in directly pro- and isolate grow poorly in the absence of lanthanide viding oxygen to the methane oxidation machinery metals. So far, Verrucomicrobia methanotrophs (Karlsen et al., 2005; Kao et al., 2008; Chen et appear to be nearly absent in metagenomes assem- al., 2012). Bacteriohemerythrin genes have been bled from mesophilic grassland and freshwater identifed in 80% of the methanotroph genomes, environments (Kielak et al., 2010; Cabello-Yeves including at least four Methylococcales MAGs, et al., 2017; He et al., 2017) and are primarily 18 Methylococcales, six Alphaproteobacteria, and detected in thermo- and acidophilic geothermal one Verrucomicrobia methanotroph genomes ecosystems (Op den Camp et al., 2009; Sharp et al., (Rahalkar and Bahulikar, 2018). Teir wide dis- 2014; Knief, 2015). One MAG putatively assigned tribution strongly suggests that they provide an to a Methylacidiphilaceae (Methylacidiphilaceae important and conserved mechanism for sustaining UBA1321) was reconstructed by Parks et al. (2017) methane consumption during oxygen limitation from the Twitchell Island restored wetland. How- across the aerobic methanotrophic phyla. In fact, ever, both the MAG and the metagenome lack the up-regulated transcription of this gene has been MMO genes (S. He et al., 2015), suggesting this reported under oxygen-limited conditions in Meth- might not be a bona fde Verrucomicrobia metha- ylomicrobium (Gilman et al., 2017). From these notroph. Tis clade is not well understood given studies, it is clear that MAGs play an important role the few number of isolates and limited metagen- in elucidating the repertoire of mechanisms sup- ome-derived genomic information, however their porting methane oxidation under a variety of redox reliance on XoxF type MDH, hydrogen metabolism conditions, across a range of environments. (Carere et al., 2017; Mohammadi et al., 2017), and heat and acid tolerances make them physiologically unique and interesting. Methanotroph MAGs and Early investigations of xoxF distribution found isolate genomes indicate the the gene present in all the Proteobacteria methy- importance of novel metal lotrophs, ofen in addition to the mxaFI-encoded biochemistry MDH (Chistoserdova, 2011b). Environmental Methanol DeHydrogenases (MDHs) catalyse surveys suggested that xoxF might be more preva- the oxidation of methanol to formaldehyde, an lent and widespread among uncultivated lineages essential step in aerobic methane oxidation. Tere in natural ecosystems than mxaF (Ramachandran are two known MDH enzymes, the canonical and and Walsh, 2015; Taubert et al., 2015). Metagen- long-studied MxaF type and the novel type, XoxF omic analysis of enrichment cultures of two closely (Chistoserdova et al., 2009). Despite similar func- related Methylococcales and native samples from tions, these two diferent MDH require diferent marine ecosystems found MAGs containing only metal cofactors: the MxaFI complex requiring xoxF MDH (Vekeman et al., 2016b; Padilla et calcium, while the XoxF enzyme requiring a light al., 2017). However, as is the case when making

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inferences from non-closed genomes, caution fuctuating redox conditions. Moreover, metagen- must be used when inferring absence of genes from omic studies from marine, freshwater, and polar MAGs. Interestingly, metagenomes from fresh- systems have found surprisingly low numbers of water and terrestrial systems have failed to detect mxaF genes, compared with the number of xoxF, mxaF in the metagenomes using both gene- and potentially further signifying the importance of genome-centric approaches (Oswald et al., 2017; this enzyme and informing cultivation eforts. It Pratscher et al., 2018; Singleton et al., 2018; Smith is clear that metagenomics will continue to play et al., 2018). Tis evidence, composed of both iso- an important role in furthering understanding late and metagenomic information, suggests that of methanotroph physiology and phylogenetic the xoxF is an environmentally relevant MDH, and diversity. therefore cultivation strategies for novel methano- trophs should be modifed to include lanthanides in enrichment medium. Future trends While we acknowledge that our analysis was not comprehensive of all current metagenomic Conclusions/discussion sequencing projects (741 available in IMG before Metagenomics has greatly advanced the feld of 1 June 2016 queried), geographic bias was obvious methane oxidation by providing an unbiased in our meta-analysis (Fig. 3.5). For example, far sequencing approach to successfully capture gene- more methanotroph genomes have been recovered and genomic-level information for novel lineages, from metagenomes from the northern hemisphere, improving our understanding of both the phylo- mostly in the United States and Europe, than genetic and metabolic diversity encompassed by anywhere else. Te limitation in knowledge about methanotrophs. In the past decade metagenomics methanotrophs in more equatorial latitudes may has more than doubled methanotroph genomic have important global ramifcations, as tropical databases by reconstructing MAGs, which now regions account for ≈50–60% of the emissions includes representatives of clades that contain from wetland systems (Dean et al., 2018). Sub- few or no isolated representatives. Specifcally, stantially increased metagenomic sequencing of these enigmatic lineages include members of the more equatorial regions is likely to uncover novel Proteobacteria (e.g. USCα, USCγ, and Creno- methanotrophs involved in mediating the balance thrix), members of the anaerobic methanotrophs between methane source and sinks. (ANME-1, ANME-2), and of the genus Ca. We also note that metagenomic sampling of Methylomirabilis formerly of the candidate NC10 methanotrophs in Eastern and Southern regions phylum. Additionally, MAGs have been recovered were similarly sparse. Tese include major rice that are closely related to the well characterized cultivation areas such as China and India. Rice pad- methanotrophs (e.g. Methylobacter), which open dies are important ecosystems that provide food for up the possibility of investigating fnely resolved a large global population, but also represent major diferences in genotypes that allow for specifc envi- sources of methane to the atmosphere. It is impera- ronmental adaption. tive that we continue to study the distribution Most importantly, metagenomic surveys have and activity of methanotrophs in these and other been critical to furthering our understanding of agricultural systems, to beter understand methane the methanotroph distribution across ecosystems. oxidation with diferent agricultural practices and Tese studies have highlighted roles for methane climatic changes. As metagenomic sequencing oxidation from the depths of the ocean, to the tis- becomes more cost efcient and commonplace, it sues of endosymbionts, to agricultural soils. From is anticipated that methanotroph genomes will be MAGs assembled across these ecosystems, it is beter sampled across the globe in the near future. now possible to extend physiological insights from Technological developments that continue cultivation studies to the feld scale. For instance, to improve ease and efciency of metagenomic recent appreciation for the versatility of methano- sequencing and analysis will enable beter gene- troph energy generation pathways, has highlighted and genome-centric approaches. Sequencing the capacity of these organisms to withstand technologies that generate long reads, for example

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Phylogenetic affiliation metagenomic genes and MAGs Alphaproteobacteria Gammaproteobacteria Ca. Methylomirabilis Methylacidiphilae ANME Number of metagenomic genes and MAGs in unique BioProjects 1 5 10 20

Figure 3.5 Geographic distribution of methanotrophs detected in metagenomic and metatranscriptomic datasets across the globe. Each point represents one of 327 unique BioProjects associated with a metagenome containing a methanotroph rpS3 gene or a putative methanotrophic Metagenome-Assembled Genome (MAG). Phylogenetic afliation of rpS3 genes not in MAGs were assigned to the class of the most similar reference sequence determined by BLASTp and confrmed by topological coherence with reference genes. The colours indicate the phylogenetic afliation of the rpS3 gene, and the size of the point represents the number of recovered sequences per unique BioProject. Sixteen MAGs lacked identifable location information.

Nanopore (Feng et al., 2015) and PacBio (Rhoads et al., 2014), and will likely continue to prove to be and Au, 2015), are already showing great promise exceptionally powerful for exploring methanotroph for improved metagenomic assemblies, yielding ecology and function. greater recovery of genes, pathways, and MAGs According to metabolic reconstructions of among complex, diverse samples. In addition, we ANME and Ca. Methylomirabilis MAGs to date, are hopeful that pairing metagenomics with other members of both lineages should be capable tools (e.g. metatranscriptomics, metaproteomics, of coupling methane oxidation to respiration SIP, Nano-Sims, fuorescent in situ microscopy, independently of consortia, although no isolated or exometabolomics) will further elucidate the representatives currently exist. Increased sampling physical and metabolic interactions between of these lineages both in the laboratory and the feld methanotrophs and other community members. may ofer new physiological variants that beter Coupling metagenomics with measurements of withstand axenic growth. For instance, MAGs gene or protein expression have already proven from ANME and Ca. Methylomirabilis have been invaluable for frmly confrming methane oxidation sampled from wastewater treatment and natural by the Crenothrix flaments (Oswald et al., 2017), methane seep ecosystems (Welte et al., 2016); nuances of trace metal metabolism in ANME (Glass however, these same taxa are also detected and

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ofen prevalent in wetlands, rice paddies, and fresh- methane, as well as the metabolites secreted by water lakes (Figs. 3.3 and 3.5) (Haroon et al., 2013; these methanotrophs. Welte et al., 2016; Narrowe et al., 2017; in ‘t Zandt Additionally, there is increasing evidence that et al., 2018; Graf et al., 2018). Tese later habitats the metabolic versatility of methanotrophs is represent an untapped potential for examination greater than was historically assumed. For exam- of the metabolic versatility of these uncultivated ple, the utilization of hydrogen has been observed methanotroph lineages. Additionally, further in Verrucomicrobia, and may be possible in some genomic investigations of stable consortia may also Proteobacteria methanotrophs (Carere et al., 2017; ofer insights into factors that hinder cultivation Mohammadi et al., 2017; Singleton et al., 2018; of these strains. Isolation of these microorganisms Smith et al., 2018). While the role of hydrogen may be inhibited by their extremely slow growth metabolisms, evident in Verrucomicrobia metha- rates, but this is unlikely to be the sole cause. notrophs, is still unclear, hydrogen production Additional physiological-genomic studies may elu- or consumption by methanotrophs may expand cidate dependencies on other microorganisms, for the niches occupied by methanotrophs as well as example amino acid or vitamin auxotrophies, or the their function in microbial communities. Tese detoxifcation of toxic metabolites. proposed metabolisms require verifcation using MAGs have highlighted the metabolic versatility axenic cultures, however metagenomic sequenc- that may enable methane oxidation along broader ing coupled with activity quantifcation of specifc redox gradients, however much of this fexibility genes or pathways may continue to elucidate the is currently only theoretical or poorly understood. distribution and impact of these genes or pathways An example of a currently only hypothetical under in situ conditions. metabolism is the reverse methanogenesis by Bath- yarchaeota, encoding a divergent MCR gene and Web resources using pathways similar to the ones in ANME (Evans We would like to acknowledge of the Organization et al., 2015; Zhou et al., 2018). While there is no of Methanotroph Genome Analysis (OMeGA) direct evidence for this metabolism, it is expected for their invaluable contributions to the genomic to yield acetate that could be exchanged with SRB. databases of methane oxidizing microorganisms. Further understanding of these exchanges may help Methanotroph genomes sequenced and analysed explain isotopic anomalies already observed in by OMeGA are available online at IMG/MER some habitats (Zhou et al., 2018). (https://img.jgi.doe.gov/cgi-bin/mer/main. Te role of dissimilatory nitrate reduction in the cgi) or at the National Center for Biotechnology aerobic Methylococcales requires further investi- (htps://www.ncbi.nlm.nih.gov/). We would like gation. Leveraging the large number of cultivated to thank IMG/MER for hosting the repository representatives within this clade to beter under- of metagenomic information that can be readily stand oxygen responses in the Methylococcales mined, and Simon Roux for assistance and guid- seems warranted, given the broad distribution and ance for the data mining. activity of these taxa across the globe. For instance, Online supplemental sequences, metadata, tree some of these genomes encode up to four difer- generation commands, and R scripts used for the ent, but possibly overlapping energy generation analyses presented here are available at the Wrighton mechanisms: low-afnity cytochromes for oxic laboratory’s GitHub website: htps://github.com/ aerobic respiration, high-afnity cytochromes for TheWrightonLab/Methanotroph_rpS3Analy- suboxic aerobic respiration, respiratory nitrate ses_SmithWrighton2018. Tis repository includes reduction, and micro-aerobic fermentation (Kits minimally modifed rpS3 amino acids sequences et al., 2015; Skennerton et al., 2015; Padilla et al., for the methanotroph reference and metagenomic 2017; Smith et al., 2018). It is currently almost sequences, the alignment used for phylogenetic completely unknown how these metabolic path- tree generation, the phylogenetic tree displayed in ways are regulated, or what efects they have on Fig. 3.3, and the metadata tables used as inputs for methane oxidation rates. New discoveries could information displayed in Figs 3.3, 3.4, and 3.5, and drastically alter our perception of the redox gradi- fnally the R script for recreation of the analyses and ents across which Methylococcales can consume fgures.

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