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Phototrophic Methane Oxidation in a Member of the Chloroflexi Phylum
Lewis M. Ward
1,2
, Patrick M. Shih
3,4
, James Hemp
5
, Takeshi Kakegawa
6
, Woodward W.
Fischer
7
, Shawn E. McGlynn
2,8,9
1. Department of Earth & Planetary Sciences, Harvard University, Cambridge, MA USA.
2. Earth-Life Science Institute, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo,
Japan.
3. Department of Plant Biology, University of California, Davis, Davis, CA USA.
4. Department of Energy, Joint BioEnergy Institute, Emeryville, CA USA.
5. School of Medicine, University of Utah, Salt Lake City, UT USA.
6. Department of Geosciences, Tohoku University, Sendai City, Japan
7. Division of Geological & Planetary Sciences, California Institute of Technology, Pasadena,
CA USA.
8. Biofunctional Catalyst Research Team, RIKEN Center for Sustainable Resource Science,
Wako-shi Japan
9. Blue Marble Space Institute of Science, Seattle, WA, USA
Abstract:
Biological methane cycling plays an important role in Earth’s climate and the global carbon
cycle, with biological methane oxidation (methanotrophy) modulating methane release from
numerous environments including soils, sediments, and water columns. Methanotrophy is
typically coupled to aerobic respiration or anaerobically via the reduction of sulfate, nitrate, or
metal oxides, and while the possibility of coupling methane oxidation to phototrophy
(photomethanotrophy) has been proposed, no organism has ever been described that is capable of
this metabolism. Here we described a new bacterial genome from a member of the Chloroflexi
phylum—termed here
Candidatus
Chlorolinea photomethanotrophicum—with cooccurring
methanotrophy and phototrophy pathways, suggesting a novel link between these two
metabolisms. Recovered as a metagenome-assembled genome from microbial mats in an iron-
rich hot spring in Japan,
Ca.
‘C. photomethanotrophicum’ forms a new lineage within the
Chloroflexi phylum and expands the known metabolic diversity of this already diverse clade.
Ca.
‘C. photomethanotrophicum’ appears to be metabolically versatile, capable of phototrophy (via a
Type 2 reaction center), aerobic respiration, nitrite reduction, oxidation of methane and carbon
monoxide, and potentially carbon fixation via a novel pathway composed of hybridized
components of the serine cycle and the 3-hydroxypropionate bicycle. The biochemical network
of this organism is constructed from components from multiple organisms and pathways, further
demonstrating the modular nature of metabolic machinery and the ecological and evolutionary
importance of horizontal gene transfer in the establishment of novel pathways.
Significance:
Methane is a major greenhouse gas, and the production and consumption of methane is largely
driven by the metabolism of microorganisms. Although it has been hypothesized for decades that
some bacteria may be capable of growth by eating methane and conserving energy from sunlight
(photomethanotrophy), this metabolism has never been discovered in nature. Here, we describe
the first genetic evidence for a bacterium capable of photomethanotrophy, adding a new pathway
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to the known diversity of how microbes can make a living. This discovery also adds a new link
to the global carbon cycle, and may provide new opportunities for designing biotechnological
tools for processing methane.
Key words: lateral gene transfer, metagenomics, photosynthesis, greenhouse gas
Introduction
Methane is a critical component of the global carbon cycle, and fluxes of microbial
methane production and oxidation are on the order of one billion tons of methane per year (1).
Understanding the diversity and activity of microbes that are involved in methane cycling thus
represents an important challenge to developing accurate climate models that are applicable both
today for understanding global warming (e.g. 2), and in the past, where it may have played an
important role in maintaining habitable conditions under a fainter sun deep in Earth history (e.g.
3). Biological methane cycling is currently only known to be carried out via production by
members of the Euryarchaeota and potentially some Bathyarchaeota and Verstraetearchaeota (4-
6), incidental production by some bacteria (7-9), and consumption by anaerobic ANME archaea
and aerobic and/or denitrifying bacteria (2, 10, 11). However, additional links in the global
biogeochemical methane cycle are conceivable, and there is a history of discoveries highlighting
microbial energy acquisition strategies which are proposed to be viable and only subsequently
identified to occur in natural organisms, including anammox, comammox, and photoferrotrophy
(12-18). While discovery of many of these missing biogeochemical links have been made by
culture-based approaches, modern genomic sequencing can provide new insights into the
existence of outstanding predicted metabolisms in the absence of readily culturable organisms.
One hypothetical microbial metabolism is that of methane oxidation coupled to
photosynthesis. This metabolism has previously been proposed (e.g. 19), and indirect evidence
exists that is consistent with this metabolism playing a role in natural environments (e.g.
environmental measurements of light-dependent methane oxidation, 20-22). A report exists of
methane utilization by a phototrophic strain tentatively classified as
Rhodopseudomonas
gelatinosa
(23), but to our knowledge was never confirmed; the strain in question is no longer
available, and subsequent attempts to culture photomethanotrophic organisms have failed (e.g.
24). The capacity for photomethanotrophy has therefore not been confirmed in any single
organism. It therefore remains plausible but u
nproven that photosynthetic methanotrophy—an
energetically favorable and biochemically feasible metabolism—should exist in the environment.
Here we describe a microbe with cooccurring methanotrophy and phototrophy, representing the
first genomic evidence for a single organism with the potential for acquiring electrons and
carbon from methane into a photosynthetic electron transport chain and biomass, respectively.
Results
OHK40 was recovered as a metagenome-assembled genome (MAG) from shotgun
metagenome sequencing at Okuoku-hachikurou Onsen (OHK) in Akita, Prefecture, Japan. This
MAG consists of 301 contigs totaling 6.93 Mb and 5980 coding sequences. GC content is 67.8%,
N50 is 36593. 47 RNAs were recovered. The genome was estimated by CheckM to be 98%
complete, with 2.8% contamination.
Phylogenetic analyses using markers including the RpoB protein (a single copy marker
that is typically vertically inherited and more commonly recovered in MAGs than 16S rDNA, 25,
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26), concatenated ribosomal protein sequences (following methods from 27), a partial 16S
sequence, and analysis with GTDB-Tk (28) each independently place OHK40 within the
Chloroflexaceae
clade of phototrophs in the Chloroflexi phylum (Figure 1). These analyses
illustrate that OHK40 is most closely related to
Ca.
Chloroploca asiatica and
Ca.
Viridilinea
mediisalina
(Figure 1). Comparison of OHK40 to
Ca.
Chloroploca asiatica and
Ca
. Viridilinea
mediisalina via OrthoANI identity was calculated to be ~71% (29), while the average amino acid
identity (AAI) was ~66% (30), while a partial 16S sequence from OHK40 (348 nucleotides long)
was determined to be 93% similarity to
Ca.
Viridilinea mediisalina. These pairwise differences
suggest divergence of OHK from these other taxa to at least the genus level; classification with
the Genome Taxonomy Database supports assignment of OHK40 to a novel genus within the
Chloroflexaceae (28). The OHK40 genome is somewhat larger than that of its close relatives
(~5.7 Mb for
Ca.
Chloroploca asiatica and
Ca.
Viridilinea mediisalina, 31). However, the
genome of OHK40 is still significantly smaller than the largest Chloroflexi genome known to
date (8.7 Mb for
Kouleothrix aurantiaca
, 26). The larger genome size of OHK40 is associated
with 160 annotated coding sequences that were not recovered in the draft genomes of either of
the closely related species
Ca.
Chloroploca asiatica or
Ca
. Viridilinea mediisalina (Supplemental
Table 1-3)—these represent candidates for recent HGT into OHK40. Protein phylogenies were
used to verify HGT into OHK40 of genes coding for metabolically relevant proteins as discussed
below (Figure 2, Supplemental Figures 1-6).
Like closely related members of the Chloroflexaceae, the OHK40 genome encodes
pathways for aerobic respiration (via an A-family and a B-family heme copper oxidoreductase),
phototrophy via a Type 2 reaction center, an alternative complex III, and synthesis of
bacteriochlorophylls
a
and
c
. Additionally, OHK40 encodes a soluble methane monooxygenase
(sMMO) (including all subunits found in closely related sequences—the alpha and beta chains of
the A subunit, component C, and regulatory protein B—but lacking the gamma chain of the A
subunit typical of sMMO complexes, 32), a cytochrome
c
552
enabling nitrite reduction to
ammonium, and a Form I CO oxidoreductase. OHK40 does not encode genes for nitrogen
fixation, canonical denitrification (i.e. the stepwise reduction of NO
3
-
to NO
2
-
, NO, N
2
O, and
finally N
2
,), the RuMP pathway for methane incorporation, or known genes for dissimilatory
oxidation or reduction of sulfur- or iron-bearing compounds. The genome encodes a partial 3-
hydroxypropionate cycle that is potentially linked to components of the serine cycle, in which
carboxylation is performed by the left branch of the 3-hydroxypropionate bicycle (3HP) while
glyoxylate produced as a byproduct of the 3HP cycle and methane-derived carbon are
incorporated via the serine cycle (Figure 3, and described in detail below). The OHK40 genome
does not encode the catalytic subunits of an uptake hydrogenase (HypA, HypC-F); however the
genome does have assembly proteins for a NiFe uptake hydrogenase homologous to those from
other phototrophic Chloroflexi. These genes are at the end of a contig and the portion of the
genome corresponding to that of
Chloroflexus aggregans
and
Roseiflexus castenholzii
that
encodes catalytic subunit genes of the hydrogenase are missing in the OHK40 genome,
suggesting that these genes may be encoded in the source genome but were not recovered in the
MAG (despite the low MetaPOAP False Negative estimate ~0.02).
Protein phylogenies for multiple phototrophy, respiration, and carbon fixation genes are
congruent with organismal phylogenies within the Chloroflexaceae (Supplemental Figures 1-3),
consistent with vertical inheritance of these traits from the last common ancestor of the clade. In
contrast, the putative sMMO and numerous other proteins (e.g. cytochrome
c
552
nitrite reductase
and CO dehydrogenase) appear to have been acquired via HGT from more distantly related taxa
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(Figure 2, Supplemental Figures 5-9). The putative sMMO protein sequence in OHK40 is most
closely related to sequences from uncultured organisms including the putatively methanotrophic
gammaproteobacterium UB981 on a branch of the multisubunit monooxgenase tree between
verified sMMO proteins from obligate methanotrophic Proteobacteria and a clade that includes
the propane monooxygenase (PrMO) of
Methylocella sylvestris
(33) (Figure 2). Moreover, this
enzyme family typically oxidizes a broad range of substrates including methane, propane, and
other small hydrocarbons
in vitro
, whether or not this allows growth on a range of substrates
in
vivo
(34-36). Interpretations of the full range and preferred substrate(s) of the monooxygenase in
OHK40 are therefore tentative pending experimental data following isolation or enrichment of
this organism.
Discussion and conclusions
The metabolic coupling of phototrophy and methanotrophy (photomethanotrophy) has
been hypothesized to be viable for decades (e.g. 19), but has never previously been confirmed to
exist in a single organism. The genome described here, OHK40, provides the first description of
the genomic potential for photomethanotrophy. Given the degree of genetic divergence of
OHK40 and its apparent unique metabolic attributes, we propose a new genus and species
designation within the Chloroflexaceae family of Chloroflexi,
Candidatus
Chlorolinea
photomethanotrophicum, pending isolation and further characterization. The designation of
OHK40 as a new genus-level lineage is consistent with recent proposals for standardized genome
sequence-based taxonomy (28).
The coupling of methanotrophy to phototrophy would be enabled by the modular nature
of high-potential electron transfer pathways (26, 37, 38). Electrons derived from oxidation of
single-carbon compounds can be fed into the phototrophic reaction center to drive cyclic electron
flow for energy conservation and subsequently used for carbon fixation (Figure 4). Carbon can
also be directly incorporated into biomass from methane via formaldehyde in order to
supplement or replace the more energetically costly fixation of dissolved inorganic carbon (DIC,
e.g. CO
2
and HCO
3
-
) (Figure 3) (see below). The initial activation of methane via methane
monooxygenase has an obligate requirement for O
2
, so photomethanotrophy is likely only
possible in aerotolerant Type 2 phototrophs, and not in Type 1 phototrophic lineages such as
Chlorobi and Heliobacteria which are typically more O
2
-sensitive (though the discovery of
aerobic or microaerobic members of Chlorobi and Chloracidobacteria suggests the potential for
previously recognized aerotolerance in Type 1 phototrophs, 39, 40). In nonphototrophic aerobic
methanotrophs, electrons from methane are run through electron transport chains and ultimately
donated to O
2
(or, rarely, oxidized nitrogen species, 41, 42) at complex IV; this respiration of
methane-derived electrons results in a small, finite number of protons pumped across the
membrane, in contrast to phototrophically cycled electrons, which can be cycled indefinitely
through the reaction center and electron transport chain to conserve energy from light. As a
result, the energetic yield per methane molecule of photomethanotrophy could be much higher
than purely respiratory methanotrophy.
Additionally, it appears that
Ca.
C. photomethanotrophicum could be capable of
harvesting electrons from carbon monoxide via a Form 1 CO dehydrogenase to feed into the
phototrophy pathway. During carbon monoxide metabolism, CO is oxidized completely to CO
2
;
CO-derived carbon cannot be directly incorporated into biomass, but electrons from CO could be
used to fix DIC into biomass. Phototrophic CO oxidation metabolism has previously been
proposed for the anoxygenic phototroph
Rhodopseudomonas palustris
(43). A separate route for
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electron intake could occur by the activity of a [NiFe] hydrogenase, which could feed electrons
to the phototrophic reaction center and ultimately to CO
2
for carbon fixation or onto a respiratory
electron acceptor (Figure 4).
The putative carbon metabolism of
Ca.
C. photomethanotrophicum is detailed in Figure
3. The 3-hydroxypropionate bicycle is the characteristic carbon fixation pathway found in most
phototrophic members of the Chloroflexaceae (26, 38, 44). The canonical 3HP pathways
involves two cycles for carboxylation and the subsequent incorporation of glyoxylate.
Ca.
C.
photomethanotrophicum does not encode the second (right) cycle of 3HP which is responsible
for conversion of glyoxylate into pyruvate. This suggests that
Ca.
C. photomethanotrophicum
may primarily function as a photoheterotroph. It is possible, however, that
Ca.
C.
photomethanotrophicum may encode an alternative mechanism of glyoxylate incorporation via
conversion to glycerate by way of components of the serine cycle.
There is already precedence for the plasticity of Chloroflexi to mix and match various
metabolic pathways within central carbon metabolism, as various members have already been
demonstrated to have lost enzymes involved in the 3HP pathway and simultaneously incorporate
other carbon fixation pathways (i.e., the Calvin cycle via RuBisCO and phosphoribulokinase)
(26, 38).
Ca.
C. photomethanotrophicum recovered nearly the complete set of genes involved in
the proposed hybrid 3HP/serine cycle. The only step that was not recovered was
serine/glyoxylate aminotransferase (step 7 in Figure 2). MetaPOAP analyses show that the
probability of failure to recover this one gene in the MAG is low (~0.02), but considered in the
context of the entire 3HP/serine cycle, the probability of failing to recover at least one gene out
of the ~20 involved in the total pathway is fairly high (~0.33, assuming failure to recover any
one gene is ~0.02). It is therefore difficult to reject the hypothesis that
Ca.
C.
photomethanotrophicum may encode a complete 3HP/serine cycle. The presence of
sga
would
enable a complete hybridized 3HP/serine cycle for autotrophic carbon fixation through either or
both bicarbonate and methane, while its absence would result in a cycle which could incorporate
methane into some, but not all, biomolecules, with the remainder coming from exogenous
organic carbon (mixotrophy) or a modified form of the 3HP cycle (autotrophy). In the absence of
the closed 3HP/serine cycle, methane would only be directly incorporated into serine-derived
biomass, including cysteine, glycine, threonine, and porphyrins such as bacteriochlorophylls
(themselves derived primarily from glycine). While the amino acid composition of bacterial
proteins varies somewhat based on factors such as GC content, serine and serine-derived amino
acids make up approximately 15-20% of residues in cellular proteins (45); given an average
protein content of ~55% of dry weight (46) this equates to ~10% of cellular carbon. The
remainder of biomass could be produced through the 3HP variant present in
Ca.
C.
photomethanotrophicum, or could be derived from exogenous organic carbon leading to a
mixotrophic lifestyle (which is common among phototrophic Chloroflexi, e.g. 47). These
possibilities could be untangled by compound-specific carbon isotope measurements given the
expected substantial difference in
δ
13
C of organic carbon derived from methane compared with
that derived from HCO
3
-
uptake via the 3-hydroxypropoionate cycle.
The discovery of methanotrophy in a member of the Chloroflexi phylum expands the
known metabolic further reinforces interpretations of the Chloroflexi as one of the most
metabolically diverse bacterial phyla, following recent descriptions of Chloroflexi with diverse
metabolic traits including iron reduction (48), complete denitrification (49), sulfate reduction
(50), nitrite oxidation (51), and lithoautotrophic hydrogen oxidation (52).The putative discovery
of photomethanotrophy helps to fill a gap in thermodynamically favorable metabolisms that were
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hypothesized to exist long before their discovery in the environment, alongside metabolisms
such as anammox and photoferrotrophy (12, 13, 17-19). While
Ca.
C. photomethanotrophicum is
the first described genome of a putatively photomethanotrophic organism, this metabolism may
be more broadly distributed. Though to our knowledge it has never been discussed in the
literature, genes for methanotrophy and phototrophy also cooccur in several sequenced members
of the Proteobacteria
including
Methylocella silvestris
(NCBI-WP_012591068.1),
Methylocystis
palsarum
(NCBI-WP_091684863.1), and
Methylocystis rosea
(NCBI-WP_018406831.1), though
their capacity for photomethanotrophy has not yet been demonstrated. These organisms are not
closely related to
Ca.
C. photomethanotrophicum, and so photomethanotrophy may have evolved
convergently in both the Chloroflexi and Proteobacteria phyla. If photomethanotrophy is more
widespread, it may play a previously unrecognized role in modulating methane release in
methane-rich photic environments such as wetlands, stratified lakes, and thawing permafrost.
Photomethanotrophy may even have contributed to previous observations of light-dependent
methane oxidation in diverse environments (20-22). However, the derived phylogenetic
placement of putative photomethanotrophs and the obligate O
2
requirement of aerobic
photomethanotrophy suggest that this metabolism did not play a role early in Earth history,
before the rise of oxygen (supplemental information).
Among remaining undiscovered metabolisms, phototrophic ammonia oxidation
(“photoammox”) is a viable metabolism that could be biochemically wired in a fashion similar to
photomethanotrophy as described here: O
2
-enabled (via ammonia monooxygenase, AMO)
anoxygenic phototrophic ammonia oxidation. AMO is homologous to particulate methane
monooxygenase, and could work similarly to methane monooxygenase to activate ammonia
using O
2
, producing hydroxylamine. Hydroxylamine oxidase would then produce nitrite and
yield biologically useful electrons that could by fed into the phototrophic electron transport chain
in a manner analogous to the photomethanotrophy pathway described here. No organism has yet
been described which encodes both a phototrophic reaction center and genes for ammonia
oxidation, though phototrophic nitrite oxidation by members of the Proteobacteria has recently
been described (53).
Microbial methane metabolisms are critical not only in the biogeochemical carbon cycle
but also for industrial purposes to mitigate the release of methane as a greenhouse gas and for the
54, 55). The methane metabolism described here—involving thermotolerant enzymes, coupling
to light energy, potentially with incorporation of methane into biomass without complete
oxidation to CO
2
—may represent a biotechnologically valuable resource for future investigation.
Materials and methods
Geological context and sample collection:
The metagenome-assembled genome described here was derived from shotgun
metagenomic sequencing of microbial communities of Okuoku-hachikurou Onsen (OHK) in
Akita, Prefecture, Japan. OHK is an iron-carbonate hot spring (56, 57). This spring is remarkable
for its unique iron-oxidizing microbial community and the accumulation of iron-rich tufa
(authigenic mineral cement) that has some textural features in common with sedimentary iron
formations deposited during Precambrian time (57). In brief, the geochemistry of OHK derives
from source waters supersaturated in CO
2
, anoxic, pH 6.8, ~45 °C, and containing ~22
μ
m
dissolved NH
3
/NH
4
+
and ~114
μ
m dissolved Fe
2+
(57).
Samples for shotgun metagenomic sequencing were collected in September 2016 from
the “Shallow Source” and “Canal” sites described in (57). Thin biofilms (<1 mm) were scraped
from mineral precipitates using sterile forceps and spatulas (~0.25 cm
3
of material). Cells were
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lysed and DNA preserved in the field using a Zymo Terralyzer BashingBead Matrix and
Xpedition Lysis Buffer. Cells were disrupted immediately by attaching tubes to the blade of a
cordless reciprocating saw, which was run for 60 s.
Sequencing and analysis:
Upon return to the lab, microbial DNA was extracted and purified with a Zymo
Soil/Fecal DNA extraction kit. Following extraction, DNA was quantified with a Qubit 3.0
fluorimeter (Life Technologies, Carlsbad, CA) according to manufacturer’s instructions. Purified
DNA was submitted to SeqMatic LLC (Fremont, CA) for library preparation and 2x100 bp
paired-end sequencing via Illumina HiSeq 4000 technology. The “Shallow Source” and “Canal”
samples were prepared as separate libraries and multiplexed in a single sequencing lane with one
sample from another project (58). Raw sequence reads from the two samples were coassembled
with MegaHit v. 1.02 (59). Genome bins were constructed using MetaBAT (60), CONCOCT
(61), and MaxBin (62) before being dereplicated and refined with DASTool (63). Genome bins
were assessed for completeness and contamination using CheckM (64) and contamination
reduced with RefineM (65). The OHK40 genome was uploaded to RAST for preliminary
annotation and characterization (66). Sequences of ribosomal and metabolic proteins used in
analyses (see below) were identified locally with the
tblastn
function of BLAST+ (67), aligned
with MUSCLE (68), and manually curated in Jalview (69). Positive BLAST hits were considered
to be full length (e.g. >90% the shortest reference sequence from an isolate genome) with
e
-
values greater than 1e-20. Genes of interest were screened against outlier (e.g. likely
contaminant) contigs as determined by CheckM (64) and RefineM (65) using tetranucleotide,
GC, and coding density content. Presence of metabolic pathways of interest was predicted with
MetaPOAP (70) to check for False Positives (contamination) or False Negatives (genes present
in source genome but not recovered in MAG). Phylogenetic trees were calculated using RAxML
(71) on the Cipres science gateway (72). Transfer bootstrap support values were calculated by
BOOSTER (73), and trees were visualized with the Interactive Tree of Life viewer (74).
Taxonomic assignment of the OHK40 genome was confirmed with GTDB-Tk (28) and by
placement in a concatenated ribosomal protein phylogeny following methods from (27) (Figure
1).
Protein structural modeling of the methane monooxygenase was done with the SWISS-
MODEL workspace (75-79). The predicted hydroxylase SMMO subunit was structurally aligned
to the protein data base structure 1MTY within PyMOL.
Figures:
Figure 1:
Phylogeny of the Chloroflexi phylum, built with concatenated ribosomal
protein sequences following methods from (27). The analysis contains members of the
Chloroflexi phylum previously described (26, 31, 49, 51, 80-91), and members of the closely
related phylum Armatimonadetes as an outgroup (92, 93). All nodes recovered TBE support
values greater than 0.7. In cases where reference genomes have a unique strain name or
identifier, this was included; otherwise Genbank WGS genome prefixes were used.
Figure 2:
Phylogeny and structure of the methane monooxygenase hydroxylase
(MMOH) from the Ca. C photomethanotrophicum genome. A) Phylogeny of the protein
sequence of the alpha chain of the A subunit of soluble methane monooxygenase (SmmoA),
showing position of OHK40 relative to other sequences available from NCBI Genbank and WGS
databases, on a branch near members of the genus
Sulfobacillus
and the uncultured
gammaproteobacterial lineage UBA981
,
suggesting that OHK40 acquired this enzyme via
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horizontal gene transfer from a donor outside the Chloroflexi phylum. B) Structural overlay of
the modeled structure (yellow) with pdb entry 1MTY (MMOH) chain D (MmoX) from
Methylococcus capsulatus
(Bath) (blue). C) Expanded view of the di-iron active site of the pdb
derived structure 1MTY with the model (yellow). Nitrogen appears as a blue ball, oxygen in red,
iron in rust.
Figure 3:
Diagram of putative carbon metabolism in OHK40, including incorporation of
methane and bicarbonate into organic carbon via components of the serine and 3HP cycles,
respectively. Dotted arrows indicate steps which were not recovered in the MAG, but which
would enable a more complete hybridized pathway as discussed in the text. Red arrows indicate
steps that appear to have been acquired in OHK via HGT since divergence from
Ca.
Chloroploca
asiatica and
Ca
. Virdilinea mediisalina. A dotted blue arrows indicate a step which is not
encoded by proteins annotated to perform this function, but for which close homologs that could
potentially perform this step are encoded (e.g. acetolactate synthase for tartronate-semialdehyde
synthase). Stoichiometry is 1:1 products to reactants for all steps, with the exception of step 15,
which takes 2 glyoxylate as input and produces one tartronic semialdehyde and one CO
2
.
1) methane monooxygenase; 2) alcohol dehydrogenase; 3) formaldehyde dehydrogenase; 4)
formate dehydrogenase; 5) serine hydroxymethyltransferase; 6) serine deaminase; 7) serine
glyoxylate aminotransferase; 8) hydroxypyruvate reductase; 9) glycerate kinase; 10) enolase; 11)
phosphoenolpyruvate carboxylase; 12) malate dehydrogenase; 13) 2-hydroxy-3-oxopropionate
reductase; 14) hydroxypyruvate isomerase; 15) tartronate-semialdehyde synthase; 16) malyl-
CoA lyase; 17) acetyl-CoA carboxylase; 18) malonyl-CoA reductase; 19) propionyl-CoA
synthase; 20) propionyl-CoA carboxylase; 21) methylmalonyl-CoA epimerase; 22)
methylmalonyl-CoA mutase; 23) succinyl-CoA:(S)-malate-CoA transferase; 24) succinate
dehydrogenase; 25) fumarate hydratase
Figure 4:
Diagram of putative electron transfer in OHK40 in redox potential space.
Electrons sourced from methanol, carbon monoxide, or other donors are siphoned into the
phototrophic electron transfer chain for conservation of energy (i.e. buildup of proton motive
force) before being transferred uphill to reduced electron carriers such as NAD(P)H for carbon
fixation. This is in contrast to more oxidized electron donors for photosynthesis such as H
2
O or
NO
2
-
which must be fed directly into the reaction center (e.g. 37).
Acknowledgements
LMW acknowledges support from NASA NESSF (#
NNX16AP39H), NSF (#OISE 1639454),
NSF GROW (#DGE 1144469), the Earth-Life Science Institute Origins Network (EON), and the
Agouron Institute. P.M.S. was supported by The Branco Weiss Fellowship - Society in Science
from ETH Zurich. WWF acknowledges the generous support of the Caltech Center for
Environment Microbe Interactions, NASA Exobiology (#NNX16AJ57G), and the Simons
Foundation Collaboration on the Origins of Life (SCOL). SEM is supported by NSF Award
1724300, JSPS KAKENHI Grant Number 18H01325, and the Research Foundation for Opto-
Science and Technology.
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