The complete genome sequence of the methanogenic archaeon ISO4-H5 provides insights into the methylotrophic lifestyle of a ruminal representative of the Methanomassiliicoccales
Li et al. Standards in Genomic Sciences
The complete genome sequence of the methanogenic archaeon ISO4-H5 provides insights into the methylotrophic lifestyle of a ruminal representative of the Methanomassiliicoccales
Yang Li 0 1 2
Sinead C. Leahy 0 1
Jeyamalar Jeyanathan 0 1
Gemma Henderson 0 1
Faith Cox 0 1
Eric Altermann 0 1
William J. Kelly 0 1
Suzanne C. Lambie 0 1
Peter H. Janssen 0 1
Jasna Rakonjac 1 2
Graeme T. Attwood 0 1
0 AgResearch Limited, Grasslands Research Centre , Palmerston North , New Zealand
1 Abbreviations: bp, Base pair; Cdc, Cell Division Control Protein; COG, Cluster of Orthologous Groups; CoM, Coenzyme M; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat; Fpo , F
2 Institute of Fundamental Sciences, Massey University , Palmerston North , New Zealand
Methane emissions from agriculture represent around 9 % of global anthropogenic greenhouse emissions. The single largest source of this methane is animal enteric fermentation, predominantly from ruminant livestock where it is produced mainly in their fermentative forestomach (or reticulo-rumen) by a group of archaea known as methanogens. In order to reduce methane emissions from ruminants, it is necessary to understand the role of methanogenic archaea in the rumen, and to identify their distinguishing characteristics that can be used to develop methane mitigation technologies. To gain insights into the role of methylotrophic methanogens in the rumen environment, the genome of a methanogenic archaeon has been sequenced. This isolate, strain ISO4-H5, was isolated from the ovine rumen and belongs to the order Methanomassiliicoccales. Genomic analysis suggests ISO4-H5 is an obligate hydrogen-dependent methylotrophic methanogen, able to use methanol and methylamines as substrates for methanogenesis. Like other organisms within this order, ISO4-H5 does not possess genes required for the first six steps of hydrogenotrophic methanogenesis. Comparison between the genomes of different members of the order Methanomassiliicoccales revealed strong conservation in energy metabolism, particularly in genes of the methylotrophic methanogenesis pathway, as well as in the biosynthesis and use of pyrrolysine. Unlike members of Methanomassiliicoccales from human sources, ISO4-H5 does not contain the genes required for production of coenzyme M, and so likely requires external coenzyme M to survive.
Methanogen; Methane; Ruminant; Methanomassiliicoccales; Pyrrolysine
Ruminant animals have evolved a digestive system in
which microbes in their rumen break down plant fiber
and provide fermentation end-products and other
nutrients for growth and development of the animal [
rumen is densely populated with bacteria, archaea, ciliate
protozoa, anaerobic fungi and viruses which participate
in complex interactions to bring about the digestion of
forage material. The archaeal community is made up
almost exclusively of methanogens, which use simple
energy sources such as hydrogen, formate and methyl
compounds and produce methane. Rumen
methanogens play an important role in preventing the
accumulation of hydrogen derived from microbial fermentation
of plant polysaccharides. This allows reduced cofactors,
generated during microbial fermentation, to be
reoxidised so that the main fiber-degrading function of
the rumen can continue. The methane formed from
this process is belched from the animal to the
atmosphere, where it contributes a global warming potential
(over 100 years, GWP100) of around 34× that of carbon
]. The production of methane represents a
loss of energy from the ruminant, and depending on
the diet, this loss can represent 3.8 to 12.8 % of energy
contained in the diet [
Methanogens are classified into three broad categories
based on the compounds they use for methanogenesis:
hydrogenotrophic, methylotrophic and acetoclastic [
In the rumen, methane is formed mainly via the
hydrogenotrophic and methylotrophic pathways. Members of
the new order of methanogenic archaea,
Methanomassiliicoccales, are hydrogen-dependent methylotrophic
methanogens and have been detected in various
habitats, including landfills, rice fields, marine thermal
vents, fresh water, and in the digestive tracts of termites,
millipedes, chickens, ruminants and humans [
Methanomassiliicoccales are considered to be an
important group in the rumen environment and were originally
referred to as Rumen Cluster C methanogens [
Their abundance in the rumen is highly variable,
according to 16S ribosomal RNA gene surveys [
], but on
average, they are the second most abundant order of
rumen methanogens and constitute around 16 % of the
rumen archaeal community based on clone library
analyses , and 13 % of rumen archaeal community
based on pyrosequencing [
]. Representatives of these
organisms have only recently been isolated in culture,
and genomic information on members of the
Methanomassiliicoccales are available only for isolates from
human, bovine [
] and termite sources (NCBI
Reference Sequence: NC_020892.1). This study reports
the complete genome sequence of an ovine rumen
member of Methanomassiliicoccales, designated
methanogenic archaeon ISO4-H5.
Classification and features
A methane-forming enrichment culture was originally
obtained from a 9-year-old Romney wether sheep in
New Zealand grazing a ryegrass-clover pasture diet [
The enrichment culture contained the methanogenic
archaeon, ISO4-H5, and a Gram-negative bacterium,
subsequently identified as being closely related to
Succinivibrio dextrinosolvens and designated as strain H5.
The methanogenic archaeon ISO4-H5 grows slowly and
requires 3 to 4 days to generate detectable methane in the
culture headspace. The optical density of cultures after
maximal methane formation is very low and ISO4-H5 cells
cannot be visualized via fluorescence microscopy at
420 nm due to the apparent lack of the fluorescent
8hydroxy-5-deazaflavin cofactor, known as F420 [
organism has only a thin bi-layer cell membrane, and no
S-layer or cell wall was observed in electron micrographs of
thin sections of ISO4-H5 cells (Fig. 1). The 16S ribosomal
RNA gene of ISO4-H5 is 96 % identical to “Candidatus
Methanomethylophilus alvus” Mx1201 enriched from
human feces [
], and 95 % identical to Thermoplasmatales
archaeon BRNA1 enriched from bovine rumen (Fig. 2). All
three are members of the order Methanomassiliicoccales,
but potentially each represent different species [
general features of methanogenic archaeon ISO4-H5 are
shown in Table 1 and Additional file 1: Table S1.
Genome sequencing information
Genome project history
To gain insight into the role of methylotrophic
methanogens in the rumen environment, the genome of the
methanogenic archaeon isolate ISO4-H5 was sequenced.
Methanogenic archaeon isolate ISO4-H5 represents the
Fig. 1 Transmission electron micrograph of negatively stained thin
section of the methanogenic archaeon ISO4-H5. The sample was
prepared as previously described [
]. Images were captured using a
Philips CM10 Transmission Electron Microscope, using an Olympus
SIS Morada camera and SIS iTEM software (Germany)
first genome sequence of a member of the order
Methanomassiliicoccales isolated from the ovine rumen.
A summary of the genome project information is
shown in Table 2.
Growth conditions and genomic DNA preparation
The initial enrichment cultures were obtained by
inoculation of sheep rumen contents into BY medium [
upplemented with (final concentrations), SL10 trace
elements solution (1 mL/L) [
], selenite/tungstate solution
(1 mL/L) [
], sodium acetate (20 mM), sodium formate
(60 mM), methanol (20 mM), vitamin 10 solution
(0.1 ml per 10 mL culture tube) [
], and coenzyme M
(CoM) (10 μM) [
]. The last two additives were added
to the sterilized medium from filter-sterilized stock
solutions. Hydrogen (H2) was supplied as the energy source
by pumping the culture vessels to 180 kPa over pressure
with an 80:20 mixture of H2: carbon dioxide (CO2).
ISO4-H5 was enriched in tubes receiving sheep rumen
contents diluted by a factor of 16,384,000 [
approaches were used to reduce the bacteria in the
enrichment culture, including a 10-fold dilution, the
addition of antibiotics (combinations of streptomycin,
ampicillin, bacitracin at 10 μg/mL each, and vancomycin
at 86.7 μg/mL), heat treatment of the enrichment culture
at 50 °C for 10 to 30 min, and application of lysozyme
(2.5 mg/mL). These approaches produced a limited
diversity enrichment culture containing ISO4-H5 and S.
dextrinosolvens H5, which was verified by phase contrast
epifluorescence microscopy and bacterial and archaeal
16S rRNA gene sequencing. Genomic DNA was
extracted from cells harvested from a freshly grown (7 d
incubation time) 2 L enrichment culture using a
modified version of a liquid N2 freezing and grinding method
], in which treatment with 2.5 mg lysozyme/mL and
0.8 mg proteinase K/mL replaced the 1 % w/v sodium
dodecyl sulfate step, before a Genomic-tip 500/G
(Qiagen, Germany) was used, following the manufacturer’s
instructions, in place of the phenol/chloroform
Genome sequencing and assembly
The DNA extracted from the ISO4-H5 enrichment
culture was sequenced via pyrosequencing of a 3 kb mate
aEvidence codes – TAS Traceable Author Statement (i.e., a direct report exists in the literature), IDA Inferred from Direct Assay, NAS Non-traceable Author Statement
(i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes
are from the Gene Ontology project [
paired-end sequence library using the 454 GS FLX
platform with Titanium chemistry (Macrogen, Korea).
Pyrosequencing reads provided 43.8× coverage of the
combined ISO4-H5 and Succinivibrio dextrinosolvens
H5 genomes, and were assembled using the Newbler
assembler version 2.7 (Roche 454 Life Sciences, USA). The
Newbler assembly resulted in 176 Succinivibrio
dextrinosolvens H5 contigs across 28 scaffolds and 47 ISO4-H5
contigs in a single scaffold. The assignment of
scaffolds to genomes was based on G + C content
analysis and identification of the methanogenesis marker
gene, methyl coenzyme M reductase (mrtA).
Sequence gap closure was managed using the Staden
] and gaps were closed using standard
PCR techniques with Sanger sequencing. A total of
163 additional sequencing reactions were used to
close gaps and to improve the quality of the genome
sequence, ensuring correct assembly and to resolve
Genome annotation was carried out as previously
] and the ISO4-H5 genome sequence
was prepared for NCBI submission using Sequin .
The guanosine residue of the start codon of the Cdc6-1
replication initiation protein gene (AR505_0001) was
High-quality, closed genome
454 3 kb mate paired-end library
% of totala
chosen as the first base for the ISO4-H5 genome. The
nucleotide sequence of the ISO4-H5 chromosome has
been deposited in Genbank under accession number
The genome of ISO4-H5 consists of a single,
1,937,882 bp, circular chromosome with a G + C
content of 54 %. A total of 1,817 protein-coding genes
were predicted, representing 90.2 % of the total
genome sequence. A Cluster of Orthologous Groups
category was assigned to 1,434 of the protein-coding
genes, and the properties of the genome are
summarized in Tables 3 and 4.
ISO4-H5 is predicted to contain two Cdc6 genes.
Cdc6.1 (AR505_0001) is adjacent to two origin
recognition box (ORB) motifs downstream [
], while Cdc6.2
(AR505_1205) is located 661 kb away from the Cdc6.1
gene and is not associated with any ORB motif.
Therefore, Cdc6.1 is predicted to be the origin of replication
for ISO4-H5 (Fig. 3). The presence of multiple origins of
replications is a feature also observed in the genome
sequences of other members of Methanomassiliicoccales,
including BRNA1 (TALC00001, TALC00716, 645 kb
apart), Mx1201 (MMALV_00010, MMALV_10400, 637 kb
apart), Mx1-Issoire (H729_00005, H729_08750, 90 kb
apart), and B10 (WP_019178385, WP_019178317). The
ISO4-H5 genome contains genes predicted to be
integrases (AR505_0313, 0669, 0931, 1543, 1570, 1640, 1697),
as well as several Clustered Regularly Interspaced Short
Palindromic Repeat (CRISPR) genes (AR505_1089 –
1095) associated with a CRISPR region containing 35
repeats (bases 1,153,894 to 1,155,995). There is evidence of
Genome size (bp)
DNA coding (bp)
G + C content (bp)
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
aTotal is based on either the size of the genome in base pairs, or the total
number of protein coding genes in the annotated genome
a mobile element in the ISO4-H5 genome (AR505_0313–
AR505_0358) which excised and segregated from the
chromosome over several passages between the
sequencing of the genome and subsequent analyses of the
annotated locus. The 32 kb mobile element harbors 37
hypothetical protein genes, three adhesin-like protein
genes, three DNA-cytosine methyltransferase genes,
one phage integrase gene, one DNA mismatch
endonuclease gene and one Membrane Occupation and
Recognition Nexus (MORN) repeat-containing protein
]. No plasmids were identified in the ISO4-H5
genome. The genome contains a predicted
toxin/antitoxin module (AR505_0857, 0858) and a
death-oncuring family protein (AR505_1566), although the
latter lacks an identifiable gene encoding a partner
Insights from the genome
The genomes of several members of
Methanomassiliicoccales are publically available, including M.
luminyensis B10 isolated from a human source, “Candidatus
Methanomethylophilus alvus” Mx1201 and “Candidatus
Methanomassiliicoccus intestinalis” Mx1-Issoire enriched
from human sources, “Candidatus Methanoplasma
termitum” MpT1 enriched from termite gut, and
Thermoplasmatales archaeon BRNA1 enriched from the bovine
rumen. These genomes were compared with ISO4-H5
(Table 5). ISO4-H5 is very similar in genome size to the
other members of Methanomassiliicoccales, with M.
luminyensis B10 being the exception, with a genome
35 % larger than ISO4-H5. The genomic G + C
content of the Methanomassiliicoccales range from 49 to
60 %, with “Candidatus Methanomassiliicoccus
intestinalis” Mx1-Issoire being different to the rest
with a genomic G + C content of 41 %. The
organization of genes within the ISO4-H5 genome
shows best synteny with “Candidatus
Methanomethylophilus alvus” Mx1201 and
Thermoplasmatales archaeon BRNA1 (Fig. 4), its two closest
Members of the order Methanomassiliicoccales rely
solely on hydrogen-dependent methylotrophic
methanogenesis to produce energy. However, they use only part
of the pathway reported for other methylotrophic
methanogens (Fig. 5), such as members of the genera
Methanosarcina and Methanosphaera [
Methanosarcina spp. disproportionate methanol by electron
bifurcation, oxidizing one mole to produce CO2 while
generating reducing potential to reduce three further
moles to methane. The methanogenesis pathway in
ISO4-H5 lacks the genes encoding the enzymes required
to oxidize methanol to CO2, and is predicted to only
reduce methylated compounds directly to methane.
Functionally, this is similar to Methanosphaera stadtmanae
MCB-3, which encodes all the genes for the enzymes
needed to oxidize methanol to CO2 but does not use this
pathway due to the lack of genes encoding synthesis of
molybdopterin, a cofactor required for formation of an
active formylmethanofuran dehydrogenase .
ISO4H5 is predicted to use a heterodisulfide reductase
(HdrABC) and a methyl-viologen hydrogenase (MvhADG)
to recycle CoM, using reducing equivalents generated
from the hydrogenase. However, unlike M. stadtmanae,
the Hdr and Mvh complexes in ISO4-H5 are not predicted
to be coupled to an energy-converting-hydrogenase
], but rather are coupled to a F420-dehydrogenase
Fpo-like complex to generate the membrane potential
necessary for energy formation via ATP synthase [
The energy converting-hydrogenase complex identified
in M. luminyensis B10 and “Candidatus
Methanomassiliicoccus intestinalis” Mx1-Issoire could possibly
have an anaplerotic role . Based on the lack of
the corresponding genes, the ISO4-H5 Fpo-like
complex lacks the FpoF and FpoO subunits, which in
other methanogens contain the iron-sulfur centers
likely responsible for interacting with coenzyme F420
and methanophenazine, respectively [
]. This is
expected, as ISO4-H5 cells do not fluoresce when
illuminated at 420 nm, suggesting that coenzyme F420 is
not present in this organism. Furthermore, the
genome does not contain genes for cytochrome
biosynthesis, which suggests that methanophenazine is also
absent. A hypothetical protein (AR505_1626) in the
Fpo operon, between fpoK (AR505_1625) and fpoJ
(AR505_1627) genes, is predicted to be a
transmembrane protein and shares 49.5, 54.4 and 45.9 % amino
acid identity to MMALV_02020 of Mx1201,
TALC_00216 of BRNA1 and Mpt1_c12590 of MpT1
respectively. In addition, this gene is also located in
an operon whose organization is similar to those
encoding BRNA1, Mx1201, and MpT1, and is possibly a
subunit of the Fpo-like complex.
ISO4-H5 is predicted to have essentially the same
methane formation pathway as “Candidatus
Methanoplasma termitum” [
] and likely pumps only one ion
across the cell membrane for every two methanes
formed, to generate a membrane gradient. This is in
contrast to M. stadtmanae, which has the same general
metabolic stoichiometry but pumps two ions per
methane formed [
]. Since ATP synthesis in all of these
methanogens is via a membrane-bound ATP synthase,
ISO4-H5 is predicted to have a have a much lower ATP
(and growth) yield than Methanosphaera spp. which is
consistent with the very low culture densities observed
when the isolate is grown in the laboratory. However, it
can be expected to have a lower threshold for hydrogen,
using the same rationale proposed by Lang et al. (2015)
for “Candidatus Methanoplasma termitum”. This
therefore differentiates it ecologically from Methanosphaera,
and suggests that Methanosphaera spp. and members of
Methanomassiliiococcales, both of which occur in the
], occupy different niches.
Interestingly, the cysteate synthase, cysteate
aminotransferase (serC) and sulfopyruvate decarboxylase (comDE)
genes required for the synthesis of CoM [
] are absent
from the ISO4-H5 genome. This suggests that ISO4-H5
cannot synthesize CoM, and requires an external supply
of CoM to survive within the rumen, similar to
Methanobrevibacter ruminantium M1 [
] and MpT1 [
explains the requirement for CoM supplementation in the
initial enrichments of ISO4-H5 [
]. ISO4-H5 also
possesses only a subset of methanogenesis marker genes: 1-8,
11, 13, 15-17 (AR505_1391, 0786, 1390, 1417, 1388, 1389,
1385, 1203, 1637, 0362, 1387, 0724, and 1386
respectively). This suggests that the remaining
methanogenesis marker genes (mmp 9, 10, 12 and 14) are not
required for the truncated methyl-reducing pathway
used by ISO4-H5.
ISO4-H5 possesses a complete operon predicted to
encode the genes required for the biosynthesis of pyrrolysine
and for aminoacylation of a transfer RNA (tRNA) to
pyrrolysine (Fig. 6) [
], enabling read-through of the
amber stop codon, UAG. Pyrrolysine is produced from
two molecules of lysine by the gene products PylBCD.
Methylornithine synthase (PylB) converts L-lysine to
(3R)3-methyl-D-ornithine, which in turn is ligated with a
second molecule of L-lysine to produce (2R,
3R)-3-methylornithyl-N6 lysine, catalysed by
(2R,3R)-3-methylornithylN6-lysine synthase (PylC); pyrrolysine synthase (PylD)
converts (2R,3R)-3-methylornithyl-N6-lysine to
pyrrolysine . Pyrrolysine-tRNA ligase (PylS) catalyses the
aminocylation of tRNA (CUA) which itself is encoded by pylT
]. The operon organization is conserved across the
Methanomassiliicoccales (Fig. 6), suggesting pyrrolysine
use is important for members of this order. The in-frame
amber codon occurs in 46 ISO4-H5 genes, including the
genes encoding methylamine use;
trimethylamine:corrinoid methyltransferase, mttB (AR505_0772); methanol
corrinoid protein, mtaC (AR505_0952);
monomethylamine methyltransferase, mtmB (AR505_1327, 1328);
and dimethylamine:corrinoid methyltransferase, mtbB
(AR505_1332). The amber codon is also found in the
mmp 8 gene, a predicted nitrogenase gene (AR505_1289),
an adenylate kinase gene (AR505_1784) involved in purine
biosynthesis, a bifunctional
phosphoglucose/phosphomannose isomerase gene (AR505_0560) involved in the last
step of gluconeogenesis, two geranylgeranyl reductase
genes (AR505_1433, AR505_1618) that are likely involved
in cell membrane lipid biosynthesis, and the
CRISPRassociated endonuclease Cas3 gene (AR505_1089) that
is involved in acquired immunity against foreign DNA.
Additionally, 17 genes encoding hypothetical proteins,
one adhesin-like protein gene, and 10 insertion
sequence elements have amber codons. Similar findings
have been reported in the genomes of members of
Methanomassiliicoccales of human origin and it has
been suggested that pyrrolysine synthesis is a particular
feature of this order and an important marker in the
evolution of methanogenic archaea [
ISO4-H5 has a genome size of approximately 1.9 Mb,
and a genomic G + C content of 54 %, similar to the
genomes of Mx1201, B10 and BRNA1. ISO4-H5 encodes
the key genes and pathways required for
hydrogendependent methylotrophic methanogenesis by reduction
of methyl substrates, without the ability to oxidize
methyl substrates to carbon dioxide. The wide range of
methyl substrates predicted to be used by ISO4-H5
suggests it is more metabolically versatile than other
methylotrophic methanogens within the rumen.
Members of Methanomassiliicoccales co-exist in the
rumen with Methanosphaera spp. [
24, 25, 56
] and share
similar substrate requirements. Methanomassiliicoccales
are probably able to outcompete Methanosphaera in the
rumen at low substrate concentrations, due to the lower
thresholds conferred by the low ATP gain, but are
probably disadvantaged when substrate concentrations are
high and the low ATP yield limits their ability to
proliferate. The variability of fermentation rates in the
rumen associated with periods of feeding or fasting is
therefore expected to give both groups of
methylotrophic methanogens opportunities to grow.
ISO4-H5 appears to be reliant on the Hdr, Mvh and
Fpo-like complexes for electron bifurcation, membrane
potential generation and energy conservation, which is
identical to what has been described in other members
of Methanomassiliicoccales. However, ISO4-H5 is
incapable of producing CoM, which suggests that
ISO4H5 has adapted to the rumen environment, where
CoM produced by other methanogens would be able
to supplement ISO4-H5. ISO4-H5 also lacks the genes
encoding cofactor F420 synthesis, rendering it
nonfluorescent under illumination at 420 nm. This trait
has also been reported amongst other members of
Methanomassiliicoccales, and is likely one of the key
characteristics of this particular order of methanogens.
However, a culture of B10 has been reported to
] and this may be consistent with B10
belonging to the deepest branching group within
The use of pyrrolysine in proteins carrying out various
cellular functions suggests it is important for ISO4-H5.
While pyrrolysine is important in methylamine
utilisation by all members of Methanomassiliicoccales
sequenced thus far, pyrrolysine also appears to play a role
in methanol use by ISO4-H5, as the
methanol:methyltransferase corrinoid protein, MtaC1, is predicted to
contain a pyrrolysine in its full length protein. The use
of pyrrolysine and the Fpo-like complex by ISO4-H5
adds further weight to the hypothesis that the order
Methanomassiliicoccales is evolutionary closer to the
order Methanosarcinales, supporting findings from a
previous phylogenetic study [
]. By analyzing the
genome of ISO4-H5, our knowledge of the order
Methanomassiliicoccales has been expanded, and
together with the genomes of other members of the
Methanomassiliicoccales, will be an important
resource for the development of methane abatement
technologies in ruminants.
Additional file 1: Table S1. Associated MIGS record. (DOC 72 kb)
This work was funded by the New Zealand Agricultural Greenhouse Gas
Research Centre (NZAGRC). We thank the Pastoral Greenhouse Gas Research
Consortium (PGgRc) for providing access to the original enrichment cultures
containing ISO4-H5. We thank Doug Hopcroft and Jordon Taylor (Manawatu
Microscopy and Imaging Centre, Massey University) for technical assistance.
We thank Dong Li and Graham Naylor (AgResearch) for providing specialized
SLe and GA initiated and supervised the study. JJ cultured the original
enrichment containing ISO4-H5. YL, FC conducted the microbial culturing,
SLa extracted genomic DNA, YL performed electron microscopy, assembled
the genome, closed sequence gaps, annotated the genome and drafted the
manuscript. SLe, GA, WK, GH, EA, PJ, JR discussed, analysed the data and revised
the manuscript. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
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