International Journal of Systematic and Evolutionary Microbiology (2012), 62, 1902–1907
DOI 10.1099/ijs.0.033712-0
Methanomassiliicoccus luminyensis gen. nov., sp.
nov., a methanogenic archaeon isolated from
human faeces
Bédis Dridi,1 Marie-Laure Fardeau,2 Bernard Ollivier,2 Didier Raoult1
and Michel Drancourt1
Correspondence
Michel Drancourt
[email protected]
1
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR CNRS 6236
IDR 198, Faculté de Médecine, IFR48, Aix-Marseille-Université, Marseille, France
2
Laboratoire de Microbiologie IRD, UMR D180, Microbiologie et Biotechnologie des
Environnements Chauds, Aix-Provence Université, École Supérieure d’Ingénieurs de Luminy,
Marseille, France
During attempts to obtain novel, human-associated species of the domain Archaea, a coccoid
micro-organism, designated strain B10T, was isolated in pure culture from a sample of human
faeces collected in Marseille, France. On the basis of its phenotypic characteristics and 16S
rRNA and mcrA gene sequences, the novel strain was classified as a methanogenic archaeon.
Cells of the strain were non-motile, Gram-staining-positive cocci that were approximately 850 nm
in diameter and showed autofluorescence at 420 nm. Cells were lysed by 0.1 % (w/v) SDS. With
hydrogen as the electron donor, strain B10T produced methane by reducing methanol. The novel
strain was unable to produce methane when hydrogen or methanol was the sole energy source. In
an atmosphere containing CO2, strain B10T could not produce methane from formate, acetate,
trimethylamine, 2-butanol, 2-propanol, cyclopentanol, 2-pentanol, ethanol, 1-propanol or 2,3butanediol. Strain B10T grew optimally with 0.5–1.0 % (w/v) NaCl, at pH 7.6 and at 37 6C. It
required tungstate-selenite for growth. The complete genome of the novel strain was sequenced;
the size of the genome was estimated to be 2.05 Mb and the genomic DNA G+C content was
59.93 mol%. In phylogenetic analyses based on 16S rRNA gene sequences, the highest
sequence similarities (98.0–98.7 %) were seen between strain B10T and several uncultured,
methanogenic Archaea that had been collected from the digestive tracts of a cockroach, a
chicken and mammals. In the same analysis, the non-methanogenic ‘Candidatus
Aciduliprofundum boonei’ DSM 19572 was identified as the cultured micro-organism that was
most closely related to strain B10T (83.0 % 16S rRNA gene sequence similarity). Each of the
three treeing algorithms used in the analysis of 16S rRNA gene sequences indicated that strain
B10T belongs to a novel order that is distinct from the Thermoplasmatales. The novel strain also
appeared to be distinct from Methanosphaera stadtmanae DSM 3091T (72.9 % 16S rRNA gene
sequence similarity), another methanogenic archaeon that was isolated from human faeces and
can use methanol in the presence of hydrogen. Based on the genetic and phenotypic evidence,
strain B10T represents a novel species of a new genus for which the name
Methanomassiliicoccus luminyensis gen. nov., sp. nov. is proposed. The type strain of the type
species is B10T (5DSM 24529T5CSUR P135T).
At the time of writing, only three species in the domain
Archaea have been isolated and cultured from microbiota
associated with human mucosae. Two of these species,
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and
mcrA gene sequences of strain B10T are HQ896499 and HQ896500,
respectively.
Two supplementary figures are available with the online version of this
paper.
1902
Methanobrevibacter smithii (Miller et al., 1982; Samuel
et al., 2007) and Methanosphaera stadtmanae (Miller &
Wolin, 1985), were found in the microbiota of the gut but
the other species, Methanobrevibacter oralis (Ferrari et al.,
1994), was isolated from oral mucosa. Over the last decade,
metagenomic and molecular analyses have broadened the
spectrum of known human-associated Archaea to include
non-methanogenic organisms such as Halobacteria (Oxley
et al., 2010) and Crenarchaeota (Rieu-Lesme et al., 2005).
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Methanomassiliicoccus luminyensis gen. nov., sp. nov.
Such analyses have also indicated the presence in humans of
several phylotypes that would need to be placed in a new
order of Archaea, although, until now, such strains have not
been isolated (Mihajlovski et al., 2008, 2010; Scanlan et al.,
2008). While investigating the diversity of Archaea in the
human gut, by the PCR amplification and sequencing of 16S
rRNA genes, we recently detected one such phylotype in a
sample of human faeces. This micro-organism, a strict
anaerobe that was designated strain B10T, was successfully
isolated and cultured. In the present study, which was
approved by the Ethics Committee of the Institut Fédératif
de Recherche 48 (Marseille, France), the taxonomic
affiliation of strain B10T was assessed by using a polyphasic
approach. The results indicate that the strain represents a
novel species of a new genus of methanogenic Archaea.
produced colonies that appeared identical and cells that
were undistinguishable by microscopic examination, were
found to be phylogenetically similar (16S rRNA gene
sequence similarities of 99.8–100 %) and all five could use
H2 as an electron donor in the presence of methanol as
electron acceptor. Strain B10T was selected for further
investigation. The purity of strain B10T was verified by
incubating it in medium containing yeast extract (1 g l21),
bio-trypticase (1 g l21) and glucose (20 mM), to check that
no fermentative microbial contaminants were present.
Purity of the isolate was also confirmed when DNA extracted
from it (Dridi et al., 2009) failed to yield any amplicons
when run in a PCR based on the universal bacterial 16S
rRNA gene primers fD1 and rp2 (Weisburg et al., 1991),
which do not amplify archaeal 16S rRNA genes.
A faecal specimen collected from an 86-year-old healthy
man was submitted to the clinical microbiology laboratory,
Aix-Marseille-Université, Marseille, France. Although the
specimen tested negative for known enteric pathogens, it
gave positive results, in real-time PCR for the detection of
Archaea (Dridi et al., 2009), for both Methanobrevibacter
smithii [cycle threshold (Ct)535] and a novel phylotype
(Ct528). Enrichment and isolation of the methanogenic
archaea in the sample were then attempted using Methanobrevibacter medium (MM) containing (l21) 0.5 g
KH2PO4, 0.4 g MgSO4 . 7H2O, 5 g NaCl, 1 g NH4Cl,
0.05 g CaCl2 . 2H2O, 1.6 g sodium acetate, 0.5 g cysteineHCl, 1 g yeast extract, 1 g trypticase peptone, 0.001 g
resazurin, 1 ml Widdel trace elements solution and 2 ml
tungstate-selenite solution. The tungstate-selenite solution
contained (l21) 0.4 g NaOH, 2 mg Na2SeO3 and 4 mg
Na2WO4 . 5H2O. The pH of the medium was adjusted to
7.5 with 10 M KOH before the medium was autoclaved,
boiled under a stream of O2-free N2 gas and then allowed
to cool to room temperature. The medium was then
dispensed into Hungate tubes (5 ml medium per tube) or
serum bottles (20 ml per bottle) under a stream of N2/CO2
(80 : 20, v/v) at atmospheric pressure. The tubes and bottles
were autoclaved for 20 min at 120 uC before the medium in each vessel was supplemented with sterile NaHCO3
(4 g l21), Na2S . 9H2O (0.5 g l21), sodium formate (2 g l21)
and vitamin solution (10 ml l21; Balch et al., 1979); finally,
methanol (40 mM) and H2/CO2 [80 : 20, v/v; at 1 bar
(100 kPa) pressure] were added as growth substrates. After
inoculation with the faecal specimen (at 10 %, v/v), each
vessel was incubated at 37 uC with shaking. Positive enrichment cultures were obtained after 1 week. Methane production was detectable 3 days after inoculation. Pure strains
were obtained by repeated use of the Hungate roll-tube
method (Hungate & Macy, 1973) with H2/CO2 (80 : 20, v/v;
at 1 bar pressure) and basal growth medium that was
solidified with 1.5 % (w/v) Noble agar (Difco) and supplemented with vancomycin (25 mg l21) and methanol
(40 mM). At 37 uC, regular circular, yellow-blue colonies
approximately 0.5–1.0 mm in diameter developed in the
agar roll tubes after 1 month. Five strains, designated B10T,
B11, B12, B13 and B14, were isolated. These strains, which
Cells of strain B10T are non-motile, Gram-stain-positive,
regular cocci that show autofluorescence at 420 nm (Fig.
1). They lyse when placed in 0.1 % (w/v) SDS or hypotonic
distilled water (Boone & Whitman, 1988). When ultrathin
sections were prepared and examined in a transmission
electron microscope (Morgagni M 268 D; FEI), following
the methods described by Merhej et al. (2008), the cells of
strain B10T were found to have mean diameters of about
850 nm. The cell wall consisted of one thin electron-dense
layer and one thick transparent layer (140±20 nm) that
contained granular material (Fig. 1). Growth of strain B10T
was assessed at various pH values, temperatures and salt
concentrations in Hungate tubes with basal growth medium
containing 40 mM methanol and with H2/CO2 in the gas
phase (at 1 bar). All of the analytical assays were performed
in duplicate. Growth was measured by directly inserting
the tubes into a UV-160A spectrophotometer (Shimadzu)
and evaluating OD at 580 nm. Methane production was
quantified as described previously (Cord-Ruwisch et al.,
1986). The pH was adjusted to the desired value by the
addition of anaerobic, sterile solutions containing 10 % (w/
v) NaHCO3 or 10 % (w/v) Na2CO3. During growth, the pH
of the medium increased by about 0.1 unit. Growth occurred
at pH 7.2–8.4 (optimum pH 7.6). Subsequently, growth of
strain B10T was tested at temperatures between 25 and 45 uC
in basal growth medium at pH 7.6 (measurements indicated
that pH was stable over the temperature range 25–45uC).
Optimal growth was observed at 37 uC; no growth occurred
at 25 uC or 45 uC. To investigate salt tolerance, NaCl was
directly weighed into Hungate tubes to give 0.1–1.5 % (w/v).
Strain B10T grew in the basal medium in the presence of 0.1–
1.5 % (w/v) NaCl (optimum 1.0 %). Strain B10T did not
grow in oxidized medium (indicated by the pink colour of
resazurin). To determine its optimal physico-chemical
growth conditions and substrate utilization, the novel strain
was always subcultured once under the same experimental
conditions.
http://ijs.sgmjournals.org
In order to determine the pattern of substrate utilization, a
sterile stock solution of each substrate was added to basal
medium to give a final concentration of 10 mM (trimethylamine, 2-butanol, 2-propanol, cyclopentanol, 2-pentanol,
ethanol, 1-propanol and 2,3-butanediol; all with an
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B. Dridi and others
with the BLAST program (blast.ncbi.nlm.nih.gov). The 16S
rRNA and mcrA gene sequences of the novel strain were
aligned with those of various archaea using the CLUSTAL W
program. 16S rRNA gene sequence-based phylogenetic trees
were reconstructed by the neighbour-joining and maximum-likelihood methods using version 4.2 of the MEGA
software package (Kumar et al., 2008), and the MrBayes
algorithm.
Fig. 1. A transmission electron micrograph showing the cell-wall
structure of strain B10T, with one thin electron-dense layer (white
arrow) and one thick transparent layer (black arrow). Bar, 0.3 mm.
atmosphere of N2/CO2), 20 mM (acetate), 40 mM (formate and methanol) or 40 mM (methanol) with an
atmosphere of H2/CO2. Hydrogen oxidation was tested
using H2/CO2 (80 : 20, v/v; at 1 bar) in the gas phase with
and without 2 mM acetate as a carbon source. Formate
oxidation was also tested with and without 2 mM acetate.
These tests indicated that strain B10T oxidized H2 as the
only energy source by reducing methanol to methane.
Strain B10T did not use acetate, trimethylamine, 2-butanol,
2-propanol, cyclopentanol, 2-pentanol, ethanol, 1-propanol or 2,3-butanediol under an atmosphere of N2/CO2.
Furthermore, formate could not be used as energy source.
Yeast extract and tungstate-selenite were required for
growth with H2 and methanol.
The genomic DNA G+C content of strain B10T,
59.93 mol%, was evaluated using the strain’s complete
genome sequence, which was obtained by pyrosequencing
on a GSFLX system (Roche 454) as previously described
(Margulies et al., 2005). The estimated size of the strain’s
complete genome was 2.05 Mb. Two of the genes of strain
B10T were amplified in PCR: 16S rRNA by using the
Met86F (59-GCTCAGTAACACGTGG-39) and Met1340R
(59-CGGTGTGTGCAAGGAG-39) primers (Wright & Pimm,
2003) and mcrA by using the mcrA-F (59-GGTGGTGTMGGATTCACACARTAYGCWACAGC-39) and mcrA-R
(59-TTCATTGCRTAGTTWGGRTAGTT-39) primers (Luton
et al., 2002). Sterile distilled water was used as a negative
control in each PCR. Although mcrA-F and mcrA-R were also
used for the mcrA gene sequencing, an additional internal
primer pair – B10-dir (59-AGCCGCCGCGGTAAYAC-39)
and B10-rev2 (59-CYGGCGTTGAVTCCAATT-39) – was
used for sequencing of the 16S rRNA gene. The PCR
products were purified and sequenced by using a BigDye
Terminator 1.1 Cycle Sequencing kit (Applied Biosystems)
and a 3130 genetic analyzer (Applied Biosystems). The
sequences were analysed with the Seqscape program
(Applied Biosystems) and similarity values were determined
1904
The phylogenetic analysis based on the 16S rRNA gene
sequence of strain B10T (1434 bp) indicated that the novel
strain belonged to the Archaea but its closest, cultivated
phylogenetic relative, ‘Candidatus Aciduliprofundum boonei’, which has been placed in the order Thermoplasmatales,
showed only 83.0 % 16S rRNA gene sequence similarity.
However, strain B10T showed higher 16S rRNA gene
sequence similarities (98.0–98.7 %) with several uncultured,
methanogenic archaea previously retrieved from the digestive tracts of various animals, including wallabies (Evans
et al., 2009), humans (Mihajlovski et al., 2008, 2010), pigs
(Nehmé et al., 2009), sheep (Saengkerdsub et al., 2007) and
chickens (Wright et al., 2006). In the phylogenetic trees
based on 16S rRNA gene sequences, i.e. those reconstructed
with the neighbour-joining (Fig. 2), maximum-likelihood
(Fig. S1, available in IJSEM Online) or MrBayes algorithms
(Fig. S2), strain B10T was clustered with these uncultured
archaea in a group that was distinct from members of the
order Thermoplasmatales. Members of the order Thermoplasmatales could also be differentiated from strain B10T by
several phenotypic characteristics (Table 1). The current
members of the order Thermoplasmatales are sulfur-reducing,
thermoacidophilic archaea that have been isolated from
extreme environments (Reysenbach et al., 2006), whereas
strain B10T is a methanol-reducing, mesophilic methanogen
isolated from a clinical sample. Strain B10T was able to produce
methane under anaerobic conditions. This observation is in
agreement with the detection of an mcrA gene (GenBank
accession no. HQ896500) in the strain, as this gene encodes the
alpha subunit of the methyl-coenzyme M reductase that plays a
crucial role in methanogenesis (Hallam et al., 2003). It appears
that strain B10T and its uncultured phylogenetic relatives
belong to a new order within the methanogenic Archaea that is
different from the order Thermoplasmatales.
Strain B10T shows 75.65 % 16S rRNA gene sequence
similarity with the type strain of Methanobrevibacter
smithii, its most closely related methanogen within the
order Methanobacteriales. Two major differences distinguish
strain B10T from the other three cultured archaea that were
isolated from humans (i.e. Methanobrevibacter smithii,
Methanosphaera stadtmanae and Methanobrevibacter oralis):
it requires both a slightly more alkaline pH and tungstateselenite for its growth. Tungstate-selenite has been found
essential for the growth of several other methanogenic archaea
but none of these were isolated from humans (Belay et al.,
1986; Jones & Stadtman, 1977, 1981). Strain B10T and
Methanosphaera stadtmanae share the ability to oxidize H2
and reduce methanol to methane. This ability is not seen in
members of the order Methanosarcinales (e.g. Methanosarcina
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Methanomassiliicoccus luminyensis gen. nov., sp. nov.
Fig. 2. A maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain
B10T and 36 members of the domain Archaea. Bootstrap values ¢90% (based on 1000 replications) are shown at branching
points. Bar, 0.05 substitutions per nucleotide position.
species), which can, however, still use methanol and other
methylotrophic compounds as energy sources (Kendall &
Boone, 2006). In contrast to Methanosphaera stadtmanae,
which only has a thick electron-dense layer as its cell wall
(Miller & Wolin, 1985), the cell wall of strain B10T consists of
one thin electron-dense layer and one thick transparent layer.
Moreover, the genomic DNA G+C content reported for
Methanosphaera stadtmanae (25.8 mol%) is much lower than
that of strain B10T (59.93 mol%) (Table 1).
Based on the phenotypic, molecular and phylogenetic data
presented here, strain B10T represents a novel species of a
new genus within the methanogenic Archaea for which the
name Methanomassiliicoccus luminyensis gen. nov., sp. nov.
is proposed.
kokkos grain, seed) coccus; N.L. masc. n. Methanomassiliicoccus a methane-forming coccus from Marseille].
Cells are non-motile, regular, Gram-stain-positive cocci
with a diameter of about 850 nm. Methanogenic and
obligatory anaerobic member of the domain Archaea.
Mesophilic (25–45 uC) and slightly alkaliphilic, with
growth occurring with NaCl at 0.1–1.0 % (optimum
1.0 %). Produces methane from H2+methanol. The type
species is Methanomassiliicoccus luminyensis.
Description of Methanomassiliicoccus
luminyensis sp. nov.
Description of Methanomassiliicoccus gen. nov.
Methanomassiliicoccus luminyensis (lu.mi.ny.en9sis. N.L.
masc. adj. luminyensis belonging to Luminy, the place where
the type strain was isolated).
Methanomassiliicoccus [Me.tha.no.mas.si.li.i.coc9cus. N.L.
pref. methano- pertaining to methane; L. n. Massilia Latin
name for Marseille; N.L. masc. n. coccus (from Gr. masc. n.
In addition to the characters described for the genus, the
species is characterized by the following properties. Cells lyse
in 0.1 % (w/v) SDS or hypotonic distilled water. Produces
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B. Dridi and others
Table 1. Comparative characteristics of strain B10T, ‘Candidatus Aciduliprofundum boonei’ and Methanosphaera stadtmanae
Taxa: 1, strain B10T (data from this study); 2, ‘Candidatus Aciduliprofundum boonei’ DSM 19572 (Reysenbach et al., 2006); 3, Methanosphaera
stadtmanae DSM 3091T (Miller & Wolin, 1985). ND, No data available.
Characteristic
Source
Morphology
Diameter (mm)
Motility
Optima for growth:
Temperature (uC)
pH
NaCl (%, w/v)
DNA G+C content (mol%)
Cell-wall ultrastructure
1
2
3
Human faeces
Cocci, as single cells
0.7–1.0
Non-motile
Hydrothermal vent field
Pleiomorphic cocci
0.6–1.0
Motile
Human faeces
Cocci, as pairs or tetrads
1.0–1.2
Non-motile
70
4.5
37
6.5–6.9
37
7.6
1.0
59.93
Thin electron-dense layer and thick
transparent layer
Substrates for methane production
H2+methanol
ND
ND
39
Thick electron-dense layer
25.8
Thick electron-dense layer
None
H2+methanol
methane by reducing methanol with hydrogen as the electron
donor, although it cannot produce methane when hydrogen
or methanol is the sole energy source. Cannot produce
methane from formate, acetate, trimethylamine, 2-butanol,
2-propanol, cyclopentanol, 2-pentanol, ethanol, 1-propanol
or 2,3-butanediol, even with CO2 in the atmosphere.
Ferrari, A., Brusa, T., Rutili, A., Canzi, E. & Biavati, B. (1994). Isolation
The type strain, B10T (5DSM 24529T5CSUR P135T), was
isolated from human faeces collected in Marseille, France. The
genomic DNA G+C content of strain B10T is 59.93 mol%.
Hungate, R. E. & Macy, J. (1973). The roll-tube method for
and characterization of Methanobrevibacter oralis sp. nov. Curr Microbiol
29, 7–12.
Hallam, S. J., Girguis, P. R., Preston, C. M., Richardson, P. M. &
DeLong, E. F. (2003). Identification of methyl coenzyme M reductase
A (mcrA) genes associated with methane-oxidizing archaea. Appl
Environ Microbiol 69, 5483–5491.
cultivation of strict anaerobes. Bull Ecol Res Comm 17, 123–125.
Jones, J. B. & Stadtman, T. C. (1977). Methanococcus vannielii: culture
and effects of selenium and tungsten on growth. J Bacteriol 130, 1404–
1406.
Acknowledgements
Jones, J. B. & Stadtman, T. C. (1981). Selenium-dependent and
The authors thank Catherine Robert and Gislain Fournoux for
providing genomic data. The five co-authors would like to declare a
conflict of interest in that they hold a patent on a method for the
culture of Methanomassiliicoccus luminyensis gen. nov., sp. nov.
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