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Metagenomic Approaches Unearth Methanotroph Phylogenetic and Metabolic Diversity 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 Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb 58 | Smith and Wrighton 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 Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Metagenomics Unearthing Methanotroph Diversity | 59 (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 foreach 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, solubledepicted methane as broken monooxygenase lines. Anaerobic component A; mxaF, methanol dehydrogenasemetabolisms alpha are subunit; identified narG, respiratoryby 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
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