The complete genome sequence of Eubacterium limosum SA11, a metabolically versatile rumen acetogen
Kelly et al. Standards in Genomic Sciences
The complete genome sequence of Eubacterium limosum SA11, a metabolically versatile rumen acetogen
William J. Kelly 0
Gemma Henderson 0
Diana M. Pacheco 0
Dong Li 0
Kerri Reilly 0
Graham E. Naylor 0
Peter H. Janssen 0
Graeme T. Attwood 0
Eric Altermann 0
Sinead C. Leahy 0
0 Rumen Microbiology, Animal Science, AgResearch Limited, Grasslands Research Centre , Tennent Drive, Private Bag 11008, Palmerston North 4442 , New Zealand
Acetogens are a specialized group of anaerobic bacteria able to produce acetate from CO2 and H2 via the Wood-Ljungdahl pathway. In some gut environments acetogens can compete with methanogens for H2, and as a result rumen acetogens are of interest in the development of microbial approaches for methane mitigation. The acetogen Eubacterium limosum SA11 was isolated from the rumen of a New Zealand sheep and its genome has been sequenced to examine its potential application in methane mitigation strategies, particularly in situations where hydrogenotrophic methanogens are inhibited resulting in increased H2 levels in the rumen. The 4.15 Mb chromosome of SA11 has an average G + C content of 47 %, and encodes 3805 protein-coding genes. There is a single prophage inserted in the chromosome, and several other gene clusters appear to have been acquired by horizontal transfer. These include genes for cell wall glycopolymers, a type VII secretion system, cell surface proteins and chemotaxis. SA11 is able to use a variety of organic substrates in addition to H2/CO2, with acetate and butyrate as the principal fermentation end-products, and genes involved in these metabolic pathways have been identified. An unusual feature is the presence of 39 genes encoding trimethylamine methyltransferase family proteins, more than any other bacterial genome. Overall, SA11 is a metabolically versatile organism, but its ability to grow on such a wide range of substrates suggests it may not be a suitable candidate to take the place of hydrogen-utilizing methanogens in the rumen.
Acetogen; Methane mitigation; Rumen; Eubacterium limosum; Wood-Ljungdahl pathway; Butyrate
Methane produced by methanogenic archaea during the
fermentation of plant material in the rumen is widely
regarded as a significant contributor to anthropogenic
greenhouse gas emissions from ruminant livestock.
Several approaches to reduce CH4 emissions from farmed
animals are currently being investigated, and the genomes of
several rumen methanogens have been sequenced to
support strategies designed to reduce the number or
metabolic activity of methanogens in the rumen [
is necessary for methanogenesis and this has led to
proposals that organisms which compete with methanogens
for H2 could be used to reduce CH4 production [
Anaerobic bacteria capable of reductive acetogenesis are
of particular interest as these organisms use the
Wood–Ljungdahl pathway to synthesize acetyl-CoA
by the reduction of CO or CO2 and H2 with the
resulting acetate available to the animal . Thus an
additional strategy proposed is the use of acetogens in
conjunction with methanogen inhibition so that hydrogen
does not accumulate and inhibit fermentation.
In some gut environments acetogens can compete with
methanogens for H2, although the process is not
energetically favoured by conditions found in the mature rumen
]. Nevertheless, reductive acetogenesis has been shown
to occur in batch cultures when methanogenesis is
inhibited and acetogens are added [
]. Acetogenic bacteria
are thought to be the dominant hydrogenotrophs in early
rumen microbiota [
], and understanding their ecology
in the developing digestive tract of ruminants may reveal
key features that lead to the prevalence of methanogens
and the restriction of homoacetogens in the adult rumen.
Consequently, rumen acetogens are of interest in the
development of microbial approaches to methane mitigation.
Several acetogens have been isolated from the rumen [
and analyses of sequences of formyltetrahydrofolate
synthetase, a key enzyme of the Wood–Ljungdahl pathway,
indicate that additional species remain uncultured [
Here we present the genome sequence of E. limosum strain
SA11 isolated from the rumen of a sheep .
Classification and features
Eubacterium limosum SA11 was isolated from the
rumen of a New Zealand sheep grazing fresh forage [
and was originally described as sheep acetogen SA11 but
not characterized further. Cells of SA11 are Gram
positive non-motile rods occurring singly and in pairs
(Fig. 1). The 16S rRNA from SA11 is 97 % similar to the
E. limosum type strain ATCC 8486T which was isolated
from human faeces, and as such SA11 can be considered
as a rumen strain of E. limosum (Fig. 2). Strains of E.
limosum have been isolated from various anaerobic
environments including the gastrointestinal tract of various
animals, sewage and mud [
]. E. limosum was the
first rumen acetogen to be isolated , and this strain
(RF) was characterized [
] and used in co-culture
studies with the pectin-degrading rumen bacterium
Lachnospira multipara . These studies showed E.
limosum to be a metabolically versatile bacterium able
to grow on a wide variety of compounds including CO,
CO2/H2, hexoses, pentoses, alcohols, methyl-containing
compounds, formate, lactate, and some amino acids.
Acetate and butyrate are the main fermentation
endproducts, although butyrate production is low when
grown on CO2/H2 [
]. Additional characteristics of
strain SA11 are shown in Table 1.
Genome sequencing information
Genome project history
Eubacterium limosum SA11 was selected for genome
sequencing as an example of a rumen acetogen isolated in
New Zealand with potential application in methane
mitigation strategies. A summary of the genome project
information is shown in Table 2 and Additional file 1: Table S1 .
Growth conditions and genomic DNA preparation
Strain SA11 was able to grow in CO2-containing media
with the following energy sources (all tested at 10 mM):
hydrogen, formate, D-glucose, D-fructose, D-xylose,
Dribose, maltose, pyruvate, L-lactate, methanol, vanillate,
syringate, and 3,4,5-trimethoxybenzoate. Growth was assessed
as an increase in culture density compared to cultures that
contained none of the added energy sources. The following
did not support growth: D-mannose, D-galactose,
Larabinose, L-rhamnose, D-cellobiose, sucrose, lactose,
melibiose, raffinose, D-mannitol, D-sorbitol, glycerol, succinate,
ethanol, ethylene glycol, 2-methoxyethanol, gallate, ferulate,
aesculin, glycine, L-glutamate, and betaine. Glucose and
methanol are the best substrates and support the growth of
SA11 to a high cell density. Strain SA11 grew most rapidly
at pH values of 6.5 to 7.0 (Fig. 3a) and at a temperature of
about 40 °C (Fig. 3b). These are typical of its rumen
Cells of SA11 grown with hydrogen or glucose were
resuspended in fresh medium and 5000 Pa hydrogen
was added to the culture headspace. Cells grown with
both substrates were able to used gaseous hydrogen to a
threshold concentration of 347 to 375 Pa (Fig. 4), at
which point hydrogen use stopped. These concentrations
are equivalent to 2.10 to 2.25 μM dissolved hydrogen.
Normal ruminal hydrogen concentrations can exceed
this directly after feeding, but are also below this over
the animal feeding cycle [
], meaning that strain SA11
probably can grow as a hydrogen-dependent
homoacetogen at times when hydrogen concentrations are high in
SA11 cells for genome sequencing were grown in
RM02 medium [
] with 10 mM glucose and 0.1 % yeast
extract but without rumen fluid. Culture purity was
confirmed by Gram stain and sequencing of the 16S rRNA
gene. Genomic DNA was extracted from freshly grown
cells by standard cell lysis methods using lysozyme,
proteinase K and sodium dodecyl sulphate, followed by
phenol-chloroform extraction, and purified using the
Qiagen Genomic-Tip 500 Maxi kit (Qiagen, Hilden,
Germany). Genomic DNA was precipitated by the
addition of 0.7 vol isopropanol, and collected by
centrifugation at 12,000 × g for 10 min at room temperature.
The supernatant was removed, and the DNA pellet
was washed in 70 % ethanol, re-dissolved in TE buffer
(10 mM Tris-HCl, 1 mM EDTA pH 7.5) and stored
at -20 °C until required.
Genome sequencing and assembly
The complete genome sequence of SA11 was determined
using pyrosequencing of a paired-end 454 GS-FLX
sequence library with Titanium chemistry (Macrogen, Korea).
Pyrosequencing reads provided 43× coverage of the
genome and were assembled using the Newbler assembler
version 2.0 (Roche 454 Life Sciences, USA). The assembly
process resulted in 39 contigs across 1 scaffold. Gap closure
was managed using the Staden package [
] and gaps were
closed using additional Sanger sequencing by standard and
inverse PCR based techniques.
Genome annotation of the SA11 genome was managed
as described previously [
]. The genome sequence was
prepared for NCBI submission using Sequin [
the adenine residue of the start codon of the
chromosomal replication initiator protein DnaA (ACH52_0001)
gene was chosen as the first base for the genome.
The genome of E. limosum SA11 consists of a single
4,150,332 basepair (bp) circular chromosome with an
average G + C content of 47.4 %. A total of 3902 genes
were predicted, of which 3805 were protein-coding
genes. The properties and statistics of the SA11 genome
are summarized in Tables 3 and 4, and the nucleotide
sequence has been deposited in Genbank under
accession number CP011914. The genome atlas for E.
limosum SA11 is shown in Fig. 5. Three other E. limosum
strains have had their genome sequences determined.
These are the closed genome of strain KIST612
(4,276,902 bp) isolated from an anaerobic digester [
the draft genome of the type strain ATCC 8486T
(4,370,113 bp) isolated from human faeces [
], and the
draft genome of strain 32_A2 isolated from a deep
subsurface shale carbon reservoir (Project ID: Gp0114934).
Insights from the genome sequence
Chemical analysis of the cell wall of the type strain of E.
limosum (ATCC 8486T) shows the presence of the
amino sugars N-acetylmuramic acid (2.9 % dry weight),
N-acetylglucosamine (2.1 %) and N-acetylgalactosamine
(3.9 %) together with larger amounts of rhamnose
(20.4 %), glucose and galactose (together 14.9 %). Amino
Family: Eubacteriaceae TAS [
14, 54, 57
acids identified as present in peptidoglycan were alanine
(3.6 %), glutamic acid (8.0 %), lysine (9.0 %), ornithine
(12.1 %) and serine (3.4 %) and a putative structure of
the peptidoglycan was proposed [
]. In strain SA11 the
genes for peptidoglycan biosynthesis are similar to those
from other Gram positive bacteria but without the
mreBCD genes predicted to control cell shape. The
SA11 genome contains a large number of genes
predicted to be involved in the synthesis of cell wall
glycopolymers. These are ordered in six clusters,
(ACH52_0663-687 which contains rhamnose biosynthesis
genes, ACH52_1029-1040*, ACH52_1350-1371* which
contains sialic acid biosynthesis genes, ACH52_1470-1484,
ACH52_1620-1630* and ACH52_2094-2105*). Four of
these clusters (marked *) are located next to transposase
genes. There are also numerous cell surface proteins
which contain a variety of domains. SA11 has one
cluster of genes (ACH52_2223-2229) predicted to be
involved in the biosynthesis and export of a
nonribosomally synthesised peptide of unknown
function. The non-ribosomal peptide synthetase gene
(ACH52_2225) encodes a 2442 amino acid protein
which shows 90 % identity with a similar protein
(also 2442 amino acids) from E. limosum KIST612.
The genomic location of the non-ribosomal peptide
synthetase gene differs in the two strains.
SA11 has a 55 kb prophage (Fig. 6) integrated into the
genome (ACH52_1707-1805) adjacent to a serine tRNA.
Strain KIST612 does not have a prophage at this
location but has three prophages at other sites on the
chromosome. In terms of phage defense systems the
SA11 chromosome has one cluster of CRISPR genes and
two spacer regions at the same locations as found in
strain KIST612, but does not contain genes for
components of restriction/modification systems. However,
there is a gene for a restriction alleviation protein
(ACH52_1751) located in the prophage. In addition to
the prophage several other gene clusters appear to have
been acquired by horizontal transfer. These include all
six of the cell wall glycopolymer gene clusters as well as
genes for a type VII secretion system (ACH52_0209-0234),
cell surface proteins (ACH52_0843-0846), and genes of
unknown function ACH52_1057-1076, ACH52_1256-1271,
and ACH52_3658-3696). SA11 also has chemotaxis
% of total
genes (ACH52_0307-0324 and ACH52_3642-3645) which
are not present in strain KIST612, but the function of
these is unknown as no flagella genes are found in either
SA11 has a large repertoire of genes involved in central
metabolism and grew with hydrogen, formate, some
sugars, some compounds containing methoxyl-groups
such as methanol and methoxylated benzoates, lactate
and pyruvate. These are all typical energy sources for
The Wood-Ljungdahl pathway and energy conservation
The Wood-Ljungdahl pathway is central to the
metabolism of acetogens and the genes encoding this pathway are
found in three distinct clusters in SA11 (ACH52_291-295,
ACH52_2912-2912, ACH52_3087-3089) as has been
reported for strain KIST612 [
]. SA11 produced only
acetate from hydrogen plus carbon dioxide and from glucose,
consistent with the use of the Wood-Ljungdahl pathway.
Energy conservation in the Wood-Ljungdahl pathway and
in acetogens in general has been the focus of extensive
study but is not yet fully understood [
]. Key elements of
energy conservation systems in E. limosum are the
membrane-bound Na+-translocating Rnf
(ACH52_14101415) and ATP synthase complexes [
]. As reported for
strain KIST612 [
], SA11 has two sets of ATP synthase
genes which show different gene orders
(ACH52_16101617 and ACH52_1920-1928).
In contrast to most rumen bacteria, SA11 has very few
genes encoding glycoside hydrolases. There are two
genes encoding GH3 family proteins, one of which
(ACH52_0577) has a signal peptide and probably also has
a role in cell wall biosynthesis. The gene for a secreted
GH4 family protein is located next to an alpha-glucoside
specific PTS transport system protein (sa1_0874-0875).
SA11 has six genes encoding GH13 family proteins, all of
which are predicted to be intracellular and one of which is
part of a gene cluster involved in glycogen biosynthesis
and degradation (ACH52_0652-0657).
SA11 has a large conserved genetic region
associated with selenium-dependent molybdenum
hydroxylases (ACH52_1581-1608) [
] which ends with the
molybdate ABC transporter genes. The role of these
genes in SA11 is not known but it is likely that
they encode the selenium-containing xanthine
dehydrogenase characterized from the closely related
Eubacterium barkeri [
Unlike most rumen anaerobes, SA11 has several genes that
are either components of, or associated with, PTS
carbohydrate transporters [
]. These include PTS transporters for
glucose (ACH52_2633) and fructose (ACH52_0805-807)
(Fig. 3), as well as glucitol/sorbitol (ACH52_0168-0172,
ACH52_1560-1563) and galactitol (ACH52_0007-0009,
Rhamnose and fucose are common components of plant
cell walls and bacterial exopolysaccharides, and their
degradation in the rumen results in lactaldehyde, which
is reduced by lactaldehyde reductase to 1,2 propanediol
(1,2-PD). There is no literature on the metabolism of
1,2-PD by E. limosum, but the acetogen Acetobacterium
woodii can grow on 1,2-PD producing propionate and
propanol as end products [
]. This process occurs
independently of acetogenesis. The 1,2-PD degradative
pathway has been determined in Salmonella enterica and,
because the propionaldehyde intermediate is highly toxic
to the cell, the process occurs within an organelle called
a bacterial microcompartment (BMC) [
]. The BMC
consists of a thin protein shell made up of several
thousand copies of polypeptides with conserved domains
described by the Pfams PF00936 (found in 7 proteins in
SA11) and PF03319 (1 protein in SA11). SA11 has a
cluster of 19 pdu genes encoding degradative enzymes
and BMC production (ACH52_0472-490). The gene
arrangement is identical to A. woodii [
], except the
pduO’ gene (Awo_c25780) is not present.
Pectins make up a significant proportion of plant cell
walls and their complex structures are often highly
methylated so that action of the enzyme pectin methyl
esterase produces methanol in the rumen [
limosum grows well on methanol [
] and has a
methanol:corrinoid methyltransferase (ACH52_2073) as part of a
larger gene cluster. Phenyl methyl ethers are degradation
products of lignin, and their methyl groups can be
utilized as carbon and energy sources by acetogens
including E. limosum [
] and the closely related E.
]. The ether cleavage is mediated by the
Odemethylases, which consist of four different proteins:
two methyltransferases, a corrinoid protein, and an
activating enzyme. SA11 has several genes similar to those
described from other bacteria [
] and one gene cluster
(ACH52_0344-0347), which is not present in the KIST612
strain, may be involved in the metabolism of these
compounds. An unusual feature of the SA11 genome is the
presence of multiple copies of genes encoding
trimethylamine methyltransferase family proteins (COG05598). SA11
has 39 genes in this category, more than any other bacterial
Fig. 6 Genome organization of the prophage from E. limosum SA11. ORFs are drawn to scale and annotations are shown in vertical text. The
absolute size of the phage genome is indicated as a horizontal bar below the genome map, and the numbers indicate nucleotide position
genome, and this seems to be a characteristic of the species
as the KIST612 strain has 31 examples. These genes are
restricted to the two-thirds of the genome closest to the
origin of replication with none found between ACH52_1572
and ACH52_2975. All of these genes are similar in size and
predicted to encode proteins between 458 and 492 amino
acids. They are usually associated with genes for cobalamin
B12-binding proteins (COG05012), BCCT
(betaine/carnitine/choline transporter, COG01292, [
]) and MFS family
transporters and GntR family transcriptional regulators.
Their substrate is not known. E. limosum is known to have
the ability to demethylate, and thereby increase the
bioactivity of, a range of plant isoflavonoids [
]. This has
led to it being linked with possible health benefits and
Lactate is used for growth by E. limosum SA11, and the
mechanism of lactate utilization in acetogens has
recently been determined in Acetobacterium woodii [
In this species a stable complex is formed between
lactate dehydrogenase and the two subunits of an
electrontransferring flavoprotein. This complex uses flavin-based
electron bifurcation for energetic coupling. The genes
for this complex have been identified in A. woodii, and a
similar gene cluster is found in E. limosum KIST612 [
and also in SA11 (ACH52_2109-2113).
Butyrate is produced when E. limosum is grown on a
range of substrates and butyrate production by strain
KIST612 grown on CO has been studied [
]. The genes
for the pathway from acetyl-CoA to butyryl-CoA have
been identified and are also found in SA11
(ACH52_34843489). The cluster of butyrate genes also includes the two
subunits of an electron-transferring flavoprotein (EtfAB)
and it is proposed that butyryl-CoA dehydrogenase forms
a complex with EtfAB and also uses flavin-based electron
bifurcation as reported in Clostridium kluyveri [
limosum does not have a butyrate kinase and uses the
alternative pathway that transfers the CoA moiety from
butyryl-CoA onto acetate (butyryl-CoA:acetate
CoAtransferase, ACH52_2647) as the final step in butyrate
formation. The SA11 genome contains three EtfAB pairs
(ACH52_238-239, ACH52_3175-3176 and
ACH52_31783179) additional to the ones involved in lactate and
butyrate metabolism but the function of these is not known,
and is not apparent from their genome context.
The genome sequence of Eubacterium limosum SA11
provides insights into the metabolism of this versatile
rumen acetogen. SA11 can grow autotrophically using
CO2/H2 or heterotrophically using a diverse range of
substrates with the best growth on glucose or methanol.
If autotrophic growth could be encouraged, and
hydrogenotrphic methanogens inhibited, then SA11 could be
a useful addition to methane mitigation strategies.
However, it is apparent that in the rumen SA11 would have a
number of different substrates to select from and that
autotrophic growth is unlikely to be the norm.
Consequently, it is unlikely to be a suitable candidate to take
the place of hydrogenotrophic methanogens in the
rumen. SA11 does grow well on methanol and it would
be interesting to determine if it is able to compete with
the methylotrophic methanogens such as
Methanosphaera species and members of the order
Methanomassiliicoccales that are present in the rumen.
Additional file 1: Table S1. Associated MIGS record for M. millerae SM9,
which links to the SIGS supplementary content website. (DOC 70 kb)
CH4: methane; CO: carbon monoxide; CO2: carbon dioxide; H2: hydrogen.
The authors declare that they have no competing interests.
WJK, GH, GTA, EA, SCL conceived and designed the experiments. DMP, DL,
KR, SCL performed the sequencing and assembly experiments. GH, GEN, PHJ
performed the bacterial growth studies. WJK, EA, SCL performed the genome
annotation and comparative studies. WJK, GH, SCL wrote the manuscript. All
authors commented on the manuscript before submission. All authors read and
approved the final manuscript.
The SA11 genome sequencing project was funded by the New Zealand
Pastoral Greenhouse Gas Research Consortium (PGgRc) and the New
Zealand Agricultural Greenhouse Gas Research Centre (NZAGRC).
1. Leahy SC , Kelly WJ , Ronimus RS , Wedlock N , Altermann E , Attwood GT . Genome sequencing of rumen bacteria and archaea and its application to methane mitigation strategies . Animal . 2013 ; 7 Suppl 2 : 235 - 43 .
2. Joblin KN . Ruminal acetogens and their potential to lower ruminant methane emissions . Aust J Agric Res . 1999 ; 50 : 1307 - 13 .
3. Wright ADG , Klieve AV . Does the complexity of the rumen microbial ecology preclude methane mitigation? Animal Feed Sci Technol . 2011 ; 166 - 167 : 248 - 53 .
4. Weimer PJ . Redundancy, resilience, and host specificity of the ruminal microbiota: implications for engineering improved ruminal fermentations . Front Microbiol . 2015 ; 6 : 296 .
5. Schiel-Bengelsdorf B , Dürre P . Pathway engineering and synthetic biology using acetogens . FEBS Lett . 2012 ; 586 : 2191 - 8 .
6. Jeyanathan J , Martin C , Morgavi DP . The use of direct-fed microbials for mitigation of ruminant methane emissions: a review . Animal . 2014 ; 8 : 250 - 61 .
7. Nollet L , Demeyer D , Verstraete W. Effect of 2-bromoethanesulfonic acid and Peptostreptococcus productus ATCC 35244 addition on stimulation of reductive acetogenesis in the ruminal ecosystem by selective inhibition of methanogenesis . Appl Environ Microbiol . 1997 ; 63 : 194 - 200 .
8. Le Van TD , Robinson JA , Ralph J , Greening RC , Smolenski WJ , Leedle JA , et al. Assessment of reductive acetogenesis with indigenous ruminal bacterium populations and Acetitomaculum ruminis . Appl Environ Microbiol . 1998 ; 64 : 3429 - 36 .
9. Fonty G , Joblin K , Chavarot M , Roux R , Naylor G , Michallon F . Establishment and development of ruminal hydrogenotrophs in methanogen free lambs . Appl Environ Microbiol . 2007 ; 73 : 6391 - 403 .
10. Gagen EJ , Mosoni P , Denman SE , Al Jassim R , McSweeney CS , Forano E. Methanogen colonization does not significantly alter acetogen diversity in lambs isolated 17h after birth and raised aseptically . Microb Ecol . 2012 ; 64 : 628 - 40 .
11. Henderson G , Naylor GE , Leahy SC , Janssen PH . Presence of novel, potentially homoacetogenic bacteria in the rumen as determined by analysis of formyltetrahydrofolate synthetase sequences from ruminants . Appl Environ Microb . 2010 ; 76 : 2058 - 66 .
12. Gagen EJ , Padmanabha J , Denman SE , McSweeney CS . Hydrogenotrophic culture enrichment reveals rumen Lachnospiraceae and Ruminococcaceae acetogens and hydrogen-responsive Bacteroidetes from pasture-fed cattle . FEMS Microbiol Lett . 2015 ; 362 : fnv104 .
13. Genthner BRS , Davis CL , Bryant MP . Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H2-CO2-utilizing species . Appl Environ Microbiol . 1981 ; 42 : 12 - 9 .
14. Wade WG , Genus I . Eubacterium Prévot 1938 , 294AL . In: De Vos P , Garrity GM , Jones D , Krieg NR , Ludwig W , Rainey FA , Schleifer K-H , Whitman WB , editors. Bergey's Manual of Systematic Bacteriology , Volume 3 . 2nd ed. New York: Springer; 2009 . p. 865 - 91 .
15. Genthner BRS , Bryant MP . Growth of Eubacterium limosum with carbon monoxide as the energy source . Appl Environ Microbiol . 1982 ; 43 : 70 - 4 .
16. Genthner BRS , Bryant MP . Additional characteristics of one-carboncompound utilization by Eubacterium limosum and Acetobacterium woodii . Appl Environ Microbiol . 1987 ; 53 : 471 - 6 .
17. Rode LM , Genthner BRS , Bryant MP . Syntrophic association by cocultures of the methanol- and CO2-H2-utilizing species Eubacterium limosum and pectin-fermenting Lachnospira multiparus during growth in a pectin medium . Appl Environ Microbiol . 1981 ; 42 : 20 - 2 .
18. Janssen PH . Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics . Anim Feed Sci Technol . 2010 ; 160 : 1 - 22 .
19. Kenters N , Henderson G , Jeyanathan J , Kittelmann S , Janssen PH . Isolation of previously uncultured rumen bacteria by dilution to extinction using a new liquid culture medium . J Microbiol Methods . 2011 ; 84 : 52 - 60 .
20. Staden R , Beal KF , Bonfield JK . The Staden package, 1998 . Methods Mol Biol . 2000 ; 132 : 115 - 30 .
21. Kelly WJ , Leahy SC , Li D , Perry R , Lambie SC , Attwood GT , et al. The complete genome sequence of the rumen methanogen Methanobacterium formicicum BRM9 . Stand Genomic Sci . 2014 ; 9 : 15 .
22. Benson DA , Cavanaugh M , Clark K , Karsch-Mizrachi I , Lipman DJ , Ostell J , et al. GenBank. Nucleic Acids Res . 2013 ; 41 : D36 - 42 .
23. Roh H , Ko HJ , Kim D , Choi DG , Park S , Kim S , et al. Complete genome sequence of a carbon monoxide-utilizing acetogen, Eubacterium limosum KIST612 . J Bacteriol . 2011 ; 193 : 307 - 8 .
24. Song Y , Cho BK . Draft genome sequence of chemolithoautotrophic acetogenic butanol-producing Eubacterium limosum ATCC 8486 . Genome Announc . 2015 ; 3 : e01564 - 14 .
25. Zhang X , Rimpiläinen M , Simelyte E , Toivanen P . What determines arthritogenicity of bacterial cell wall? A study on Eubacterium cell wallinduced arthritis . Rheumatology . 2000 ; 39 : 274 - 82 .
26. Jeong J , Bertsch J , Hess V , Choi S , Choi IG , Chang IS , et al. A model for energy conservation based on genomic and experimental analyses in a carbon monoxide-utilizing, butyrate-forming acetogen , Eubacterium limosum KIST612. Appl Environ Microbiol . 2015 ; 81 : 4782 - 90 .
27. Schuchmann K , Müller V . Autotrophy at the thermodynamic limit of life: a model for energy conservation in acetogenic bacteria . Nat Rev Microbiol . 2014 ; 12 : 809 - 21 .
28. Haft DH , Self WT . Orphan SelD proteins and selenium-dependent molybdenum hydroxylases . Biol Direct . 2008 ; 3 : 4 .
29. Schräder T , Rienhöfer A , Andreesen JR . Selenium-containing xanthine dehydrogenase from Eubacterium barkeri . Eur J Biochem . 1999 ; 264 : 862 - 71 .
30. Jiang W , Pinder RS , Patterson JA , Ricke SC . Sugar phosphorylation activity in ruminal acetogens . J Environ Sci Health A Tox Hazard Subst Environ Eng . 2012 ; 47 : 843 - 6 .
31. Schuchmann K , Schmidt S , Martinez Lopez A , Kaberline C , Kuhns M , Lorenzen W , et al. Nonacetogenic growth of the acetogen Acetobacterium woodii on 1,2-propanediol . J Bacteriol . 2015 ; 197 : 382 - 91 .
32. Chowdhury C , Sinha S , Chun S , Yeates TO , Bobik TA . Diverse bacterial microcompartment organelles . Microbiol Mol Biol Rev . 2014 ; 78 : 438 - 68 .
33. Vantcheva ZM , Prodhan K , Hemken RW . Rumen methanol in vivo and in vitro . J Dairy Sci . 1970 ; 53 : 1511 - 4 .
34. Mountfort DO , Grant WD , Clarke R , Asher RA . Eubacterium callanderi sp. nov. that demethoxylates O-methoxylated aromatic acids to volatile fatty acids . Int J Syst Bacteriol . 1988 ; 38 : 254 - 8 .
35. Schilhabel A , Studenik S , Vödisch M , Kreher S , Schlott B , Pierik AJ , et al. The ether-cleaving methyltransferase system of the strict anaerobe Acetobacterium dehalogenans: analysis and expression of the encoding genes . J Bacteriol . 2009 ; 191 : 588 - 99 .
36. Ziegler C , Bremer E , Krämer R . The BCCT family of carriers: from physiology to crystal structure . Mol Microbiol . 2010 ; 78 : 13 - 34 .
37. Hur H-G , Rafii F. Biotransformation of the isoflavonoids biochanin A, formononetin, and glycitein by Eubacterium limosum . FEMS Microbiol Lett . 2000 ; 192 : 21 - 5 .
38. Possemiers S , Rabot S , Espín JC , Bruneau A , Philippe C , González-Sarrías A , et al. Eubacterium limosum activates isoxanthohumol from hops (Humulus lupulus L.) into the potent phytoestrogen 8-prenylnaringenin in vitro and in rat intestine . J Nutr . 2008 ; 138 : 1310 - 6 .
39. Zhang Y , Yang DH , Zhang YT , Chen XM , Li LL , Cai SQ . Biotransformation on the flavonolignan constituents of Silybi Fructus by an intestinal bacterial strain Eubacterium limosum ZL-II . Fitoterapia . 2014 ; 92 : 61 - 71 .
40. Biagi E , Nylund L , Candela M , Ostan R , Bucci L , Pini E , et al. Through ageing, and beyond: gut microbiota and inflammatory status in seniors and centenarians . PLoS One . 2010 ; 5 : e10667 .
41. Weghoff MC , Bertsch J , Müller V. A novel mode of lactate metabolism in strictly anaerobic bacteria . Environ Microbiol . 2015 ; 17 : 670 - 7 .
42. Li F , Hinderberger J , Seedorf H , Zhang J , Buckel W , Thauer RK . Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri . J Bacteriol . 2008 ; 190 : 843 - 50 .
43. Saitou N , Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees . Mol Biol Evol . 1987 ; 4 : 406 - 25 .
44. Felsenstein J . Confidence limits on phylogenies: An approach using the bootstrap . Evolution . 1985 ; 39 : 783 - 91 .
45. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences . J Mol Evol . 1980 ; 16 : 111 - 20 .
46. Tamura K , Stecher G , Peterson D , Filipski A , Kumar S . MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol . 2013 ; 30 : 2725 - 9 .
47. Reddy TBK , Thomas A , Stamatis D , Bertsch J , Isbandi M , Jansson J , et al. The Genomes OnLine Database (GOLD) v. 5: a metadata management system based on a four level (meta)genome project classification . Nucleic Acids Res . 2015 ; 43 : D1099 - 106 .
48. Field D , Garrity G , Gray T , Morrison N , Selengut J , Sterk P , et al. Towards a richer description of our complete collection of genomes and metagenomes “Minimum Information about a Genome Sequence” (MIGS) specification . Nat Biotechnol . 2008 ; 26 : 541 - 7 .
49. Woese CR , Kandler O , Wheelis ML . Towards a natural system of organisms: proposal for the do-mains Archaea, Bacteria, and Eucarya . Proc Natl Acad Sci USA . 1990 ; 87 : 4576 - 9 .
50. Phylum SK-H, XIII. Firmicutes Gibbons and Murray 1978 , 5 (Fimacutes [sic] Gibbons and Murray 1978 , 5) . In: De Vos P , Garrity GM , Jones D , Krieg NR , Ludwig W , Rainey FA , Schleifer K-H , Whitman WB , editors. Bergey's Manual of Systematic Bacteriology , Volume 3 . 2nd ed. New York: Springer; 2009 . p. 19 - 1317 .
51. Ludwig W , Schleifer K-H , Whitman WB . Revised road map to the phylum Firmicutes . In: De Vos P , Garrity GM , Jones D , Krieg NR , Ludwig W , Rainey FA , Schleifer K-H , Whitman WB , editors. Bergey's Manual of Systematic Bacteriology , Volume 3 . 2nd ed. New York: Springer; 2009 . p. 1 - 13 .
52. Rainey FA . Class II. Clostridia class. nov . In: De Vos P , Garrity GM , Jones D , Krieg NR , Ludwig W , Rainey FA , Schleifer K-H , Whitman WB , editors. Bergey's Manual of Systematic Bacteriology , vol. 3 . Secondth ed. New York: Springer; 2009 . p. 763 .
53. List Editor. List of new names and new combinations previously effectively, but not validly, published . List no. 132. Int J Syst Evol Microbiol 2010 ; 60 : 469 - 472 . doi: 10 .1099/ijs.0. 022855 - 0 .
54. Skerman VBD , McGowan V , Sneath PHA . Approved Lists of Bacterial Names . Int J Syst Bacteriol . 1980 ; 30 : 225 - 420 .
55. Prévot AR . In: Hauderoy P, Ehringer G , Guillot G , Magrou J , Prévot AR , Rosset D , Urbain A , editors. Dictionnaire des Bactéries Pathogènes . 2nd ed. Paris: Masson et Cie; 1953 . p. 1 - 692 .
56. Ludwig W , Schleifer K-H , Whitman WB . Family II. Eubacteriaceae fam. nov . In: De Vos P , Garrity GM , Jones D , Krieg NR , Ludwig W , Rainey FA , Schleifer K-H , Whitman WB , editors. Bergey's Manual of Systematic Bacteriology , Volume 3 . 2nd ed. New York: Springer; 2009 . p. 865 - 909 .
57. Prévot AR . Études de systématique bactérienne. Ann Inst Pasteur (Paris). 1938 ; 60 : 285 - 307 .
58. Judicial opinion 57. Designation of Eubacterium limosum (Eggerth) Prévot 1938 as the type species of Eubacterium . Int J Syst Bacteriol . 1983 ; 33 : 434 .
59. Ashburner M , Ball CA , Blake JA , Botstein D , Butler H , Cherry JM , et al. Gene ontology: tool for the unification of biology . The Gene Ontology Consortium. Nat Genet . 2000 ; 25 : 25 - 9 .