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Genomic Evidence for Phototrophic Oxidation of Small Alkanes 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:
Recent genomic and microcosm based studies revealed a wide diversity of previously unknown
microbial processes involved in alkane and methane metabolism. Here we described a new
bacterial genome from a member of the Chloroflexi phylum—termed here
Candidatus
Chlorolinea photoalkanotrophicum—with cooccurring pathways for phototrophy and the
oxidation of methane and/or other small alkanes. Recovered as a metagenome-assembled
genome from microbial mats in an iron-rich hot spring in Japan,
Ca.
‘C. photoalkanotrophicum’
forms a new lineage within the Chloroflexi phylum and expands the known metabolic diversity
of this already diverse clade.
Ca.
‘C. photoalkanotrophicum’ appears to be metabolically
versatile, capable of phototrophy (via a Type 2 reaction center), aerobic respiration, nitrite
reduction, oxidation of carbon monoxide, oxidation and incorporation of carbon from methane
and/or other short-chain alkanes such as propane, and potentially carbon fixation via a novel
pathway composed of hybridized components of the serine cycle and the 3-hydroxypropionate
bi-cycle. 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.
Key words: lateral gene transfer, metagenomics, photosynthesis, greenhouse gas,
propanotrophy, photoalkanotrophy
Introduction
Microbial oxidation of alkanes facilitates the breakdown of environmental hydrocarbons
including methane from geological, biological, and anthropogenic sources, and is therefore an
important component of the global carbon cycle. Microbial fluxes of methane production and
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oxidation alone are on the order of one billion tons of methane per year (Reeburgh 2007).
Understanding the diversity and activity of microbes that are involved in cycling of methane and
other alkanes thus represents an important challenge to developing accurate climate models that
are applicable both today for understanding global warming (e.g. Thauer 2010), and in the past,
when methane may have played an important role in maintaining habitable conditions under a
fainter sun deep in Earth history (e.g. Pavlov et al. 2000). Recently, awareness of archaeal
metabolism of methane and other small alkanes has expanded dramatically, particularly through
genome-centric metagenomics as well as enrichment-based approaches (e.g. Evans et al. 2015,
Laso-Pérez
et al. 2016, Vanwonterghem et al. 2016 Borrel et al. 2019, Chen et al. 2019); this
spate of discoveries strongly suggests an incomplete understanding of the microbial cycling of
small hydrocarbons that may be improved with further sampling of novel microbial diversity.
One hypothetical microbial metabolism is that of methane oxidation coupled to
photosynthesis. This metabolism has previously been proposed (e.g. Vishniac 1960), 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, e.g. Oswald et al.
2015). A report exists of methane utilization by a phototrophic strain tentatively classified as
Rhodopseudomonas gelatinosa
(Wertlieb and Vishniac 1967), 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. Ratering 1996, Frantz 2009). The capacity for
photomethanotrophy has therefore remained elusive. Here we describe a microbe with
cooccurring pathways for phototrophy and the consumption of methane and/or other small
alkanes such as propane, 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 based on presence/absence and redundancy of single-copy
marker genes.
Multiple phylogenetic analyses 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 (Yoon et al. 2017) was calculated to be ~71%, while the
average amino acid identity (AAI) (Rodriguez and Konstantinidis 2014) was ~66%. 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 (Parks et al. 2018). The
OHK40 genome is somewhat larger than that of its close relatives (~5.7 Mb for
Ca.
Chloroploca
asiatica and
Ca.
Viridilinea mediisalina). 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
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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 (quinone-reducing) reaction center, an alternative complex III, and
synthesis of bacteriochlorophylls
a
and
c
(e.g. BchH, BchD, BchI, BchM, AcsF, BchL, BchN,
BchB, BchX, BchY, BchZ, BchF, BchG, BchQ, BchU, and BchK). 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 (hydroxylase), component C
(reductase), and regulatory protein B—but lacks the gamma chain of the A subunit typical of
sMMO complexes, Rosenzweig et al. 1997). These proteins were recovered on a fairly long
(~68kb) contig together with many hypothetical and annotated metabolic genes (e.g. sulfate
permease, mannonate dehydratase, the starvation sensing protein RspA, and multiple peptide
ABC transporters) that were determined via similarity and phylogenetic analyses to be most
closely related to those from members of the Chloroflexaceae and therefore to have been
vertically inherited genes that belong in the OHK40 genome, strengthening interpretations that
this contig belongs in the OHK40 genome and was not recovered due to contamination of the
MAG.A cytochrome
c
552
may enable nitrite reduction to ammonium, and a Form I CO
oxidoreductase suggests a capacity to oxidize CO. 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 bi-cycle (3HP) while
glyoxylate produced as a byproduct of the 3HP cycle and methane- or propane-derived carbon
are incorporated via the serine cycle (Figure 3, and described in detail below).
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 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 (e.g. Klatt et al.
2013). 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,
CO dehydrogenase, and methyl acetate hydroxylase) appear to have been acquired via HGT from
more distantly related taxa (Figure 2, Supplemental Figures 5-10). 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
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Proteobacteria and a clade that include
s the propane monooxygenase (PrMO) of
Methylocella
sylvestris
(Crombie and Murrell 2014) (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
(Colby et al. 1977,
Coleman et al. 2006, Hakemian and Rosenzweig 2007). 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. Vishniac 1960), but neither this capacity nor the
more general phototrophic oxidation of small alkanes (photoalkanotrophy) has never previously
been confirmed to exist in a single organism. The genome described here, OHK40, provides the
first genomic description of the 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
photoalkanotrophicum, 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 (Parks et al. 2018).
The coupling of methanotrophy or propanotrophy to phototrophy would be enabled by
the modular nature of high-potential electron transfer pathways (Ward et al. 2018a, Fischer et al.
2016, Shih et al. 2017). Electrons derived from oxidation of single-carbon compounds or short
alkanes 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 could also be directly
incorporated into biomass from methane or propane via methanol and 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).
In nonphototrophic aerobic methanotrophs, electrons from methane are run through
electron transport chains and ultimately donated to O
2
(or, rarely, oxidized nitrogen species, e.g.
Skennerton et al. 2015) at complex IV; this respiration of methane-derived electrons results in a
small, finite number of protons pumped across the membrane per methane molecule oxidized, in
contrast to phototrophs which can conserve energy from light via cyclic electron transfer without
net consumption of electrons. As a result, the energetic yield per methane molecule of
photomethanotrophy could be much higher than purely respiratory methanotrophy.
The 3-hydroxypropionate bi-cycle is the characteristic carbon fixation pathway found in
most phototrophic members of the Chloroflexaceae (Ward et al. 2018a, Shih et al. 2017).
Ca.
C.
photoalkanotrophicum encodes portions of the 3-hydroxypropionate bi-cycle, but is lacking
some key proteins (Figure 3). The canonical 3HP pathways involves two cycles for
carboxylation and the subsequent incorporation of glyoxylate.
Ca.
C. photoalkanotrophicum
does not encode the second (right) cycle of 3HP which is responsible for conversion of
glyoxylate into pyruvate. This suggests that
Ca.
C. photoalkanotrophicum may primarily
function as a photoheterotroph. It is possible, however, that
Ca.
C. photoalkanotrophicum may
encode an alternative mechanism of glyoxylate incorporation via conversion to glycerate by way
of components of the serine cycle. This proposed carbon metabolism is expected to function
similarly whether
Ca.
C. photoalkanotrophicum is consuming methane or propane, as these
compounds share an uptake pathway via methanol as an early intermediate (Figure 3).
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Ca.
C. photoalkanotrophicum 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). Although not recovered in the MAG, the presence in the
genome of a gene encoding serine-glyoxylate aminotransferase
would enable a complete
hybridized 3HP/serine cycle for autotrophic carbon fixation through either or both bicarbonate
and alkanes such as methane and propane. Its absence would result in a cycle which could
incorporate methane or other short alkanes 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 this case, methane and propane would only be directly incorporated into
serine-derived biomass, including cysteine, glycine, threonine, and porphyrins such as
bacteriochlorophylls (themselves derived primarily from glycine). The remainder of biomass
could be produced through the 3HP variant present in
Ca.
C. photoalkanotrophicum, or could be
derived from exogenous organic carbon leading to a mixotrophic lifestyle (which is common
among phototrophic Chloroflexi, e.g. Klatt et al. 2013). 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.
photoalkanotrophicum may encode a complete 3HP/serine cycle. There is already precedence for
the plasticity of Chloroflexi to transition between photoautotrophy and photoheterotrophy and 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
with some simultaneously acquiring other carbon fixation pathways (i.e., the Calvin cycle via
RuBisCO and phosphoribulokinase) (Ward et al. 2018a, Shih et al. 2017).
Additionally, it appears that
Ca.
C. photoalkanotrophicum 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. To our knowledge, this is the first evidence for phototrophic CO metabolism
in the Chloroflexi. Previous reports of CO metabolism in Chloroflexi have been restricted to
nonphototrophic lineages (e.g. Islam et al. 2019). Phototrophic CO oxidation has previously been
proposed for the anoxygenic phototrophic proteobacterium
Rhodopseudomonas palustris
(Larimer et al. 2004). A separate route for 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 discovery of alkanotrophy generally and perhaps methanotrophy specifically in a
member of the Chloroflexi phylum expands the known metabolic diversity of this phylum and
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 (Kawaichi et al. 2013), complete denitrification (Hemp et al. 2015c),
sulfate reduction (Anantharaman et al. 2018), nitrite oxidation (Sorokin et al. 2012), and
lithoautotrophic hydrogen oxidation (Ward et al. 2018f). The discovery of putative
photomethanotrophy helps to fill a gap in thermodynamically favorable metabolisms
hypothesized to exist long before their discovery in the environment, alongside metabolisms
such as anammox, comammox, and photoferrotrophy (Kuenen 2008, Daims et al. 2015, van
Kessel et al. 2015, Widdel et al. 1993, Vishniac 1960).
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While
Ca.
C. photoalkanotrophicum 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. photoalkanotrophicum, 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 (e.g. Oswald et al. 2015). 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).
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 (Takashima et al. 2011, Ward et
al. 2017a). 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 (Ward et al.
2017a). 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+
(Ward et al. 2017a).
Samples for shotgun metagenomic sequencing were collected in September 2016 from
the “Shallow Source” and “Canal” sites described in (Ward et al. 2017a). Thin biofilms (<1 mm)
were scraped from mineral precipitates using sterile forceps and spatulas (~0.25 cm
3
of material).
Cells were 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:
Metagenomic sequencing and analysis, including genome binning, followed methods
described previously (Ward et al. 2018a, Ward et al. 2018f) and described in the SI.
Presence of metabolic pathways of interest was predicted with MetaPOAP (Ward et al.
2018b) 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
Stamatakis 2014) on the Cipres science gateway (Miller et al. 2010). Transfer bootstrap support
values were calculated by BOOSTER (Lemoine et al. 2018), and trees were visualized with the
Interactive Tree of Life viewer (Letunic and Bork 2016). Taxonomic assignment of the OHK40
genome was determined using markers including placement in reference phylogenies built using
the RpoB protein (a single copy marker that is typically vertically inherited and more commonly
recovered in MAGs than 16S rDNA, e.g. Ward et al. 2018a) and concatenated ribosomal protein
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sequences (following methods from Hug et al. 2016) and using a partial 16S rDNA sequence
recovered in the genome. Taxonomic assignment was further confirmed with GTDB-Tk (Parks
et al. 2018).
Protein structural modeling of the methane monooxygenase was done with the SWISS-
MODEL workspace (Bienert et al. 2017). 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 Hug et al. 2016. The analysis contains members of
the Chloroflexi phylum previously described and members of the closely related phylum
Armatimonadetes as an outgroup. 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 photoalkanotrophicum 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
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/propane 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; 26) acetone monooxygenase; 27) methyl
acetate hydrolase.
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Figure 4:
Diagram of putative electron transfer in OHK40 in redox potential space.
Electrons sourced from methanol (derived from oxidation of methane or propane), 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. Fischer et al. 2016).
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 Astrobiology Center Program of
the National Institutes of Natural Sciences (grant no. AB311013).
.
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Supplemental Information:
Supplemental Methods:
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 (Ward 2017). Raw sequence reads from the two samples were
coassembled with MegaHit v. 1.02 (Li et l. 2016). Genome bins were constructed using
MetaBAT (Kang et al. 2015), CONCOCT (Alneberg et al. 2013), and MaxBin (Wu et al. 2014)
before being dereplicated and refined with DASTool (Sieber et al. 2018). Genome bins were
assessed for completeness and contamination using CheckM (Parks et al. 2015) and
contamination reduced with RefineM (Parks et al. 2017). The OHK40 genome was uploaded to
RAST for preliminary annotation and characterization (Aziz et al. 2008). Sequences of ribosomal
and metabolic proteins used in analyses (see below) were identified locally with the
tblastn
function of BLAST+ (Camacho et al. 2008), aligned with MUSCLE (68), and manually curated
in Jalview (Waterhouse et al. 2009). 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 confirmed to be located on large, well-assembled (i.e. 10s of kb) contigs
and screened against outlier (e.g. likely contaminant) contigs as determined by CheckM (Parks et
al. 2015) and RefineM (Parks et al. 2017) using tetranucleotide, GC, and coding density content.
.
CC-BY-NC-ND 4.0 International license
It is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/531582
doi:
bioRxiv preprint first posted online Jan. 26, 2019;