Download Journal - International Journal of Systematic and Evolutionary

International Journal of Systematic and Evolutionary Microbiology (2011), 61, 2568–2572
DOI 10.1099/ijs.0.028274-0
Methylogaea oryzae gen. nov., sp. nov., a
mesophilic methanotroph isolated from a rice paddy
field
Estefanı́a Geymonat, Lucı́a Ferrando and Silvana E. Tarlera
Correspondence
Silvana E. Tarlera
[email protected]
Cátedra de Microbiologı́a, Facultad de Quı́mica y Facultad de Ciencias, Universidad de la
República-UDELAR, CC1157 Montevideo, Uruguay
A novel methanotroph, designated strain E10T, was isolated from a rice paddy field in Uruguay.
Strain E10T grew on methane and methanol as sole carbon and energy sources. Cells were
Gram-negative, non-motile, non-pigmented, slightly curved rods showing type I intracytoplasmic
membranes arranged in stacks. The strain was neutrophilic and mesophilic; optimum growth
occurred at 30–35 6C with no growth above 37 6C. The strain possessed only a particulate
methane monooxygenase (pmoA). Phylogenetic analysis based on 16S rRNA gene sequences
indicated that the strain was most closely related to the moderately thermophilic strains
Methylocaldum szegediense OR2T (91.6 % sequence similarity) and Methylococcus capsulatus
Bath (91.5 %). Comparative sequence analysis of pmoA genes also confirmed that strain E10T
formed a new lineage among the genera Methylocaldum and Methylococcus with 89 and 84 %
derived amino acid sequence identity to Methylococcus capsulatus Bath and Methylocaldum
gracile VKM-14LT, respectively. The DNA G+C content was 63.1 mol% and the major cellular
fatty acid was C16 : 0 (62.05 %). Thus, strain E10T (5JCM 16910T 5DSM 23452T) represents the
type strain of a novel species within a new genus, for which the name Methylogaea oryzae gen.
nov., sp. nov. is proposed.
Aerobic methane-oxidizing bacteria (MOB) are a highly
specialized and important group of ubiquitous bacteria that
are capable of utilizing methane as the sole source of carbon
and energy and, therefore, represent the largest global
methane sink. They inhabit environments where both
methane and oxygen are present, such as soils, wetlands,
freshwater and marine systems, lakes and sediments
(Bowman, 2006).
Methylocella and Methylocapsa, which are assigned to the
class Alphaproteobacteria. In addition, two filamentous
Gammaproteobacteria, Crenothrix polyspora and ‘Clonothrix
fusca’, are now considered to be type I methanotrophs
based on 16S rRNA analysis (Stoecker et al., 2006; Vigliotta
et al., 2007). Extreme methane oxidation by acidophilic
bacteria belonging to the phylum Verrucomicrobia has also
been reported (Op den Camp et al., 2009).
Based on their phylogeny, chemotaxonomy, internal
ultrastructure and metabolic pathways, MOB can be split
into two major subgroups (Hanson & Hanson, 1996). The
type I MOB include members of the genera Methylobacter,
Methylomicrobium, Methylomonas, Methylosphaera, Methylosarcina, Methylohalobius, Methylosoma, Methylovulum,
Methylothermus, Methylocaldum and Methylococcus, which
belong to the class Gammaproteobacteria. The type II MOB
include members of the genera Methylosinus, Methylocystis,
In wetland rice fields, MOB play a vital role functioning
as a bio-filter, oxidizing methane produced in anaerobic
environments by the methanogenic Archaea and thus
decreasing the release of methane into the atmosphere. In
a previous study, a novel bacterial strain, designated E10T,
was isolated from the soil–water interface of a flooded
Uruguayan rice field and was found to be moderately related
to the Methylococcus–Methylocaldum group (Ferrando &
Tarlera, 2009). To our knowledge, this is the first reported
case of the isolation of a type I MOB from a rice paddy.
Here, strain E10T is formally described and assigned an
accurate taxonomic rank.
Abbreviations: MOB, methane-oxidizing bacteria; MPN, most probable
number; pMMO, particulate methane monoxygenase; sMMO, soluble
methane monoxygenase.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA,
pmoA and nifH gene sequences of strain E10T are EU672873,
EU359002 and EU672874, respectively.
A supplementary figure is available with the online version of this
paper.
2568
Strain E10T was isolated from the soil–water interface of a
flooded rice field in Treinta y Tres, South-east Uruguay
(32u 559 S 54u 509 W) from most probable number (MPN)
counts of methanotrophs followed by repeated dilution in
liquid nitrate mineral salts (NMS) medium (Whittenbury
et al., 1970), with added methane to achieve a 25 % (v/v)
Downloaded from www.microbiologyresearch.org by
028274 G 2011 IUMS
IP: 78.47.27.170
On: Thu, 13 Oct 2016 11:31:02
Printed in Great Britain
Methylogaea oryzae gen. nov., sp. nov.
methane atmospheric concentration, and on solid medium
as described by Ferrando & Tarlera (2009).
Cell morphology was observed using phase-contrast microscopy. The presence of Azotobacter-type cysts was determined using the staining procedure of Vela & Wyss (1964).
Cells were fixed and observed using electron microscopy,
which was performed at UMAT (Unidad de Microscopı́a
Electrónica de Transimisión), Facultad de Ciencias,
UDELAR, Uruguay. Exponentially growing cells were fixed
with 2.5 % glutaraldehyde and 4 % para-formaldehyde in
0.1 M phosphate buffer (pH 7.4) for 1 h at 4 uC. After
centrifugation, cells were washed twice with 0.05 M
cacodylate buffer (pH 6.4), fixed for 1 h with 1 % osmium
tetroxide in 0.025 M cacodylate buffer (pH 6.5), harvested,
washed three times with distilled water, dehydrated in
increasing concentrations of ethanol and embedded in SPIChem Araldite resin. Thin (300–500 nm) and ultrathin
(50–70 nm) sections were cut on an RMC MT-X ultramicrotome and stained with uranyl acetate and lead stain
solution (Sigma–Aldrich). The size of the cells was
determined by negative staining with 2 % (w/v) uranyl
acetate. Electron microscopy was performed in a JEOL
JEM-1010 transmission electron microscope at 80 kV with
calibrated magnifications. Images were obtained with a
digital Hamamatsu C-4742-95 camera.
Utilization of various carbon sources was studied in liquid
NMS medium supplemented with one of the following
filter-sterilized substrates (0.1 %, w/v): formate, formamide, methylamine, glucose, lactose, acetate or peptone.
Growth on methanol was tested at concentrations of 0.1,
0.5, 1.0 and 1.5 % (w/v). Nitrogen sources were tested in
liquid NMS medium by replacing KNO3 with one of the
following substrates (0.05 %, w/v): NH4Cl, urea, peptone,
yeast extract, lysine, glycine, threonine or tryptophan.
Temperature range for growth was determined at 15, 18,
20, 23, 28, 30, 35, 37 and 39 uC in liquid NMS medium. pH
range for growth was tested in liquid NMS medium
adjusted to pH 4–9 by using different buffer systems
(citrate/phosphate and phosphate buffers). Tolerance of
NaCl was determined by observing growth in mineral
medium with 0.1, 0.2, 0.5 and 1 % (w/v) NaCl.
Phospholipid and fatty acid analyses were performed by
the Identification Service of the DSMZ as described by
Kämpfer & Kroppenstedt (1996) using the fatty acid
database of the Microbial Identification System (MIDI) for
determining composition. The DNA G+C content was
measured at the DSMZ by using the HPLC method as
described by Mesbah et al. (1989).
DNA was extracted and PCR-mediated amplifications of
the 16S rRNA gene (1451 bp) and of a partial fragment of
the pmoA gene, encoding the 27 kDa peptide of particulate
methane monoxygenase (pMMO), was carried out using
primer sets 27F–1492R and A189f–mb661r, respectively,
as described previously (Ferrando & Tarlera, 2009).
Amplification of partial fragments of the mmoX gene,
encoding the a-subunit of soluble methane monoxygenase
http://ijs.sgmjournals.org
(sMMO), and the nifH gene, encoding dinitrogenase
reductase H, was performed using primer sets mmoXA–
mmoXB (Auman et al., 2000) and PolF–PolR (Poly et al.,
2001), respectively. All sequencing reactions were performed
at Macrogen Sequencing Service, Korea, using an ABI
PRISM 3730XL capillary sequencer (Applied Biosystems).
The 16S rRNA gene sequences (1451 nt) and the deduced
amino acid sequences of the pmoA gene (149 amino acids)
were compared with closely related sequences of reference
organisms initially carried out by using the BLAST (Altschul
et al., 1997) and megaBLAST (Zhang et al., 2000) programs.
Sequences with the highest scores were then selected for
the calculation of pairwise sequence similarities using a
global alignment algorithm, which was implemented at
the EzTaxon server (http://www.eztaxon.org/; Chun et al.,
2007). Sequences were then aligned with CLUSTAL W version
1.8 (Thompson et al., 1994) and corrected manually where
necessary. Phylogenetic analyses were conducted using the
neighbour-joining method, with the Kimura two-parameter
model, and the maximum-parsimony algorithm, and
clustering was determined by using bootstrap values based
on 1000 replications as implemented in the MEGA version
4 software package (Tamura et al., 2007). Phylogenetic
analysis of the pmoA gene was performed as described
previously (Ferrando & Tarlera, 2009).
Strain E10T was isolated from the highest positive dilutions
(1025) from the MPN counts of the soil–water interface
samples after further transfers on NMS liquid and solid
media (Ferrando & Tarlera, 2009). The strain was purified
after repeated subculturing in liquid NMS medium until it
was isolated from a mixed culture, in which it was
associated with Hyphomicrobium-like cells. The purity of
the culture was verified by phase-contrast microscopy and
failure to grow on diluted TSB medium, in liquid NMS
medium without methane and on nutrient agar plates.
Strain E10T did not survive in glycerol stock cultures at
280 uC or as freeze-dried cultures.
Colonies of strain E10T grown on plates for 1 week were 1–
2 mm in diameter, round, convex, white, semi-transparent
and smooth with entire edges. Cells were non-motile,
slightly curved rods that were 2.0–2.260.5–0.7 mm (Fig.
1a). No cysts or cyst-like cells were detected in cell
preparations, even after 1 month of incubation, and
resistance to desiccation was not observed. The strain was
not heat resistant (when treated at 50–80 uC for 10 min)
and did not form exospores. Electron microscopic analysis
of ultrathin sections of cells showed a typical Gramnegative structure of the cell wall and the presence of type I
MOB intracytoplasmic membranes arranged as stacks of
vesicular disks (Fig. 1b, c). Cells contained large inclusions
of low electron density, probably comprising poly-bhydroxybutyrate granules, and also glycogen inclusions
which stained dark (Fig. 1b, c).
Strain E10T grew only on methane and methanol (0.1–
1.0 %, w/v) as sole carbon and energy sources. None of the
other carbon substrates tested supported growth. Nitrate,
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 11:31:02
2569
E. Geymonat, L. Ferrando and S. E. Tarlera
Table 1. Cellular fatty acid profiles of strain E10T and members
of genera comprising type I methanotrophs
Taxa: 1, strain E10T (data from this study); 2, Methylothermus
(Tsubota et al., 2005); 3, Methylocaldum (Eshinimaev et al., 2004); 4,
Methylococcus (Bowman et al., 1995); 5, Methylovulum (Iguchi et al.,
2011). Values are percentages of the total fatty acids. 2, Not detected;
NR, not reported.
Fatty acid
T
Fig. 1. Morphology of strain E10 depicted by a transmission
electron micrograph of negatively stained cells (a) and ultrathin
sections of cells showing cell-wall (CW) structure, glycogen
inclusions (G), the arrangement of intracytoplasmic membranes
(ICM) and the presence of poly-b-hydroxybutyrate (PHB) (b, c).
Bars, 500 nm (a, c) and 100 nm (b).
ammonia, urea, lysine, peptone and yeast extract were
utilized as sole nitrogen sources. No growth was observed
in nitrogen-free medium under a reduced oxygen level and
the acetylene reduction test was negative, although the nifH
gene was amplified by PCR. A positive control assay under
the same atmospheric conditions, where acetylene reduction was verified, was carried out using Methylococcus
capsulatus strain ‘Bath’. NaCl concentrations above 0.5 %
(w/v) were inhibitory. Growth occurred at 20–37 uC but
not at 18 uC or at 39 uC. Optimal growth occurred at 30–
35 uC with a specific growth rate of 0.14 h21 (equivalent to
a doubling time of 5 h). The pH range for growth was
pH 5–8 with optimum growth at pH 6.5–6.8.
Strain E10T revealed a unique fatty acid profile compared
with other type I MOB, although the profiles of members
of the genera Methylocaldum and Methylococcus were the
most similar (Table 1). The high level and predominance of
C16 : 0 was a feature shared with members of the genera
Methylococcus, Methylocaldum and Methylovulum; however, strain E10T also contained unsaturated C16 : 1v9c,
which, to our knowledge, has not previously been reported in significant amounts in other type I or type II
methanotrophs. Summed feature 3, which most probably
corresponds to C16 : 1v7c since this is a common fatty acid
among many methanotrophs while iso-C15 : 0 2-OH has
never been detected in MOB, was the other major
component detected. Strain E10T also showed minor
amounts of iso-C16 : 0 3-OH and C20 : 0 fatty acids, which
have not yet been found in other type I MOB.
2570
C12 : 0
C14 : 0
C15 : 0
C16 : 0
iso-C16 : 0 3-OH
C16 : 0 3-OH
C16 : 1
C16 : 1v7c
C16 : 1v9c
C17:0 cyclo
C18 : 1v9c
C20 : 0
Summed feature 3*
1
2.11
5.84
1.03
62.05
3.69
2.93
2
2
7.36
2
2
2.66
10.33
2
3
4
5
NR
NR
NR
1.24
2.07
37.24
1.97
3.51
63.67
1–6
0–13
34–56
NR
NR
NR
NR
NR
NR
NR
11.90
8.00
2
NR
2
11–46
NR
NR
NR
NR
0–15
0–3
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
2
3.46
NR
4.71
35.16
8.99
NR
34.20
2.97
46.90
NR
*Summed feature 3 comprises C16 : 1v7c and/or iso-C15 : 0 2-OH,
which could not be separated by the MIDI System. However, it most
likely represents C16 : 1v7c, a frequent fatty acid in MOB.
The nearly complete 16S rRNA gene sequence and partial
pmoA gene sequence of strain E10T were determined in a
previous study (Ferrando & Tarlera, 2009). The phylogenetic
analysis based on the 16S rRNA gene clearly showed that
strain E10T represents a new distinct line of descent within
the type I methanotrophs, clustering between the lineages
defined by the genera Methylocaldum and Methylococcus (Fig.
2). According to pairwise nucleotide sequence similarity
values calculated using the EzTaxon web-based tool that
employs the Myers algorithm, the closest taxonomically
described methanotrophs were Methylocaldum szegediense
OR2T (91.6 % 16S rRNA gene sequence similarity) and
Methylococcus capsulatus Bath (91.5 %). The pmoA nucleotide sequence of strain E10T differed by ~18 % from the pmoA
sequences of members of the genera Methylococcus and
Methylocaldum, which is similar to genus-level differences
among other methanotrophic bacteria. The deduced amino
acid sequences showed 89 % similarity to Methylococcus
capsulatus and 81–84 % similarity to the other described
species of the genus Methylocaldum. The pmoA-based phylogeny agrees with the 16S rRNA-based phylogeny as to the
unique position of the strain E10T in a novel branch among
the genera Methylocaldum and Methylococcus (Fig. S1). The
DNA G+C content of strain E10T was 63.1 mol% as
determined by HPLC.
Morphological and physiological characteristics of strain
E10T and those of other phylogenetically related members
of methanotroph-containing genera are shown in Table 2.
The absence of resting cellular stages in strain E10T also
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 61
IP: 78.47.27.170
On: Thu, 13 Oct 2016 11:31:02
Methylogaea oryzae gen. nov., sp. nov.
Fig. 2. Neighbour-joining tree based on 16S
rRNA gene sequences (1388 nt) showing the
phylogenetic position of strain E10T relative to
other type I methanotrophs. Bootstrap values
,70 % are not shown. Filled circles indicate that
the corresponding nodes were also recovered in
the tree generated with the maximum-parsimony
algorithm. GenBank accession numbers are
given in parentheses. Bar, 0.01 substitutions
per nucleotide position.
differentiated it from members of the genera Methylococcus
and Methylocaldum. Another typical feature of the
members of the genus Methylocaldum is cell polymorphism, which was not observed in strain E10T. Furthermore,
unlike all the species with validly published names
belonging to the abovementioned genera, which are
moderately thermophilic or thermotolerant, strain E10T
is clearly a mesophile. Beyond this, differences in the 16S
rRNA gene sequences between strain E10T and closely
related members of the genera Methylocaldum and
Methylococcus were .8 % and are thus too large to allow
inclusion of this strain in these genera. Due to its
distinctive phenotypic traits, characteristic fatty acid profile
and phylogenetic position, strain E10T represents a novel
species of a new genus within the Gammaproteobacteria
(type I methanotrophs), for which the name Methylogaea
oryzae gen. nov., sp. nov. is proposed.
Description of Methylogaea gen. nov.
Methylogaea (Me.thy.lo.ga9e.a. N.L. neut. n. methyl the
methyl group; N.L. fem. n. Gaea the mother goddess of the
earth in Greek mythology; N.L. fem. n. Methylogaea referring
to the terrestrial origin of a methyl-using bacterium).
Cells are Gram-negative, aerobic, non-motile, curved rods.
Resting stages are not present. Cells possess a typical type
I intracytoplasmic membrane system forming stacks of
membrane vesicles. pMMO is present but sMMO is not.
Mesophilic and non-thermotolerant. Obligately methanotrophic, utilizing methane or methanol. The NifH gene is
present. Major cellular fatty acids are C16 : 0, followed by
summed feature 3 (C16 : 1v7c and/or iso-C15 : 0 2-OH) and
C16 : 1v9c. Most closely related to the genera Methylococcus
and Methylocaldum (type I methanotrophs) in the class
Gammaproteobacteria. The type species is Methylogaea oryzae.
Description of Methylogaea oryzae sp. nov.
Methylogaea oryzae (o.ry9za.e. L. fem. n. Oryza genus name
of rice; L. gen. n. oryzae of rice, referring to the isolation of
the type strain from a flooded rice field).
Displays the following properties in addition to those given
in the genus description. Cells are 2.0–2.260.5–0.7 mm.
Grows on nitrate, ammonia, urea, lysine, peptone and yeast
extract as sole nitrogen sources. Optimum growth occurs at
30–35 uC and at pH 7. Sensitive to NaCl concentrations
above 0.5 % (w/v).
Table 2. Characteristics of strain E10T and other phylogenetically related type I methanotrophs
Taxa: 1, strain E10T (data from this study); 2, Methylothermus (Tsubota et al., 2005); 3, Methylocaldum (Eshinimaev et al., 2004; Bodrossy et al.,
1997); 4, Methylococcus (Bowman et al., 1993). +, Positive; 2, negative; NR, not reported.
Characteristic
Cell morphology
Motility
Cyst formation
Pigmentation
Growth temperature range (uC)*
Presence of mmoX gene
Presence of nifH gene
DNA G+C content (mol%)
1
2
3
4
Curved rods
2
2
White
20–37 (30–35)
2
+
63.1
Cocci
2
2
White
37–67 (57–59)
2
2
62.5
Rods–pleomorphic
+
+
Brown
20–61 (42–55)
2
Cocci–rods
Variable
+
White to creamy
30–55 (37–50)
+
+
62–65
NR
56–60
*Optimum values are given in parentheses.
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 11:31:02
2571
E. Geymonat, L. Ferrando and S. E. Tarlera
The type strain, E10T (5JCM 16910T 5DSM 23452T), was
isolated from a rice paddy field located in Treinta y Tres,
Uruguay. The DNA G+C content of the type strain is
63.1 mol%.
Iguchi, H., Yurimoto, H. & Sakai, Y. (2011). Methylovulum
References
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z.,
Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res 25,
3389–3402.
Auman, A. J., Stolyar, S., Costello, A. M. & Lidstrom, M. E. (2000).
Molecular characterization of methanotrophic isolates from freshwater lake sediment. Appl Environ Microbiol 66, 5259–5266.
Bodrossy, L., Holmes, E. M., Holmes, A. J., Kovács, K. L. & Murrell,
J. C. (1997). Analysis of 16S rRNA and methane monooxygenase gene
sequences reveals a novel group of thermotolerant and thermophilic
methanotrophs, Methylocaldum gen. nov. Arch Microbiol 168, 493–503.
Bowman, J. (2006). The Methanotrophs – the families Methylococcaceae
and Methylocystaceae. In The Prokaryotes: a Handbook on the Biology of
Bacteria, 3rd edn, vol. 5, pp. 266–289. Edited by M. Dworkin, S. Falkow,
E. Rosenberg, K. H. Schleifer & E. Stackebrandt. New York: Springer.
Bowman, J. P., Sly, L. I., Nichols, P. D. & Hayward, A. C. (1993). Revised
taxonomy of the Methanotrophs: description of Methylobacter gen.
nov., emendation of Methylococcus, validation of Methylosinus and
Methylocystis species, and a proposal that the family Methylococcaceae
includes only the group I Methanotrophs. Int J Syst Bacteriol 43, 735–
753.
Bowman, J. P., Sly, L. I. & Stackebrandt, E. (1995). The phylogenetic
position of the family Methylococcaceae. Int J Syst Bacteriol 45, 182–
185.
Chun, J., Lee, J.-H., Jung, Y., Kim, M., Kim, S., Kim, B. K. & Lim, Y. W.
(2007). EzTaxon: a web-based tool for the identification of
prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst
Evol Microbiol 57, 2259–2261.
Eshinimaev, B. Ts., Medvedkova, K. A., Khmelenina, V. N., Suzina,
N. E., Osipov, G. A., Lysenko, A. M. & Trotsenko, IuA. (2004).
miyakonense gen. nov., sp. nov., a type I methanotroph isolated from
forest soil. Int J Syst Evol Microbiol 61, 810–815.
Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of
fatty acid patterns of the coryneform bacteria and related taxa. Can J
Microbiol 42, 989–1005.
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Op den Camp, H. J. M., Islam, T., Stott, M. B., Harhangi, H. R.,
Hynes, A., Schouten, S., Jetten, M. S. M., Birkeland, N.-K., Pol, A. &
Dunfield, P. F. (2009). Environmental, genomic and taxonomic
perspectives on methanotrophic Verrucomicrobia. Environ Microbiol
Reports 1, 293–306.
Poly, F., Monrozier, L. J. & Bally, R. (2001). Improvement in the RFLP
procedure for studying the diversity of nifH genes in communities of
nitrogen fixers in soil. Res Microbiol 152, 95–103.
Stoecker, K., Bendinger, B., Schöning, B., Nielsen, P. H., Nielsen,
J. L., Baranyi, C., Toenshoff, E. R., Daims, H. & Wagner, M. (2006).
Cohn’s Crenothrix is a filamentous methane oxidizer with an unusual
methane monooxygenase. Proc Natl Acad Sci U S A 103, 2363–2367.
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular
evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol
Evol 24, 1596–1599.
CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
Tsubota, J., Eshinimaev, B. Ts., Khmelenina, V. N. & Trotsenko, Y. A.
(2005). Methylothermus thermalis gen. nov., sp. nov., a novel
moderately thermophilic obligate methanotroph from a hot spring
in Japan. Int J Syst Evol Microbiol 55, 1877–1884.
Vela, G. R. & Wyss, O. (1964). Improved stain for visualization of
Azotobacter encystment. J Bacteriol 87, 476–477.
Vigliotta, G., Nutricati, E., Carata, E., Tredici, S. M., De Stefano, M.,
Pontieri, P., Massardo, D. R., Prati, M. V., De Bellis, L. & Alifano, P.
(2007). Clonothrix fusca Roze 1896, a filamentous, sheathed,
methanotrophic c-Proteobacterium. Appl Environ Microbiol 73,
[New thermophilic methanotrophs of the genus Methylocaldum].
Mikrobiologiia 73, 530–539 (in Russian).
3556–3565.
Ferrando, L. & Tarlera, S. (2009). Activity and diversity of
Whittenbury, R., Phillips, K. C. & Wilkinson, J. F. (1970). Enrichment,
methanotrophs in the soil-water interface and rhizospheric soil from
a flooded temperate rice field. J Appl Microbiol 106, 306–316.
isolation and some properties of methane-utilizing bacteria. J Gen
Microbiol 61, 205–218.
Hanson, R. S. & Hanson, T. E. (1996). Methanotrophic bacteria.
Zhang, Z., Schwartz, S., Wagner, L. & Miller, W. (2000). A greedy
Microbiol Rev 60, 439–471.
algorithm for aligning DNA sequences. J Comput Biol 7, 203–214.
2572
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 61
IP: 78.47.27.170
On: Thu, 13 Oct 2016 11:31:02