INTERNATIONAL
JOURNALOF SYSTEMATICBACTERIOLOGY,
Apr. 1991, p. 301-305
0020-7713/91/020301-05$02.00/0
Copyright 0 1991, International Union of Microbiological Societies
Vol. 41, No. 2
Contribution of Genome Characteristics to Assessment of
Taxonomy of Obligate Methanotrophs
J. P. BOWMAN,” L. I. SLY, AND A. C. HAYWARD
Department of Microbiology, University of Queensland, Brisbane, Queensland, Australia 4072
The DNAs of a variety of obligate methanotrophic bacteria were analyzed for base composition and
nucleotide distribution. Genome molecular weights were determined for representative strains. Similarity
maps attained by plotting DNA base composition versus nucleotide distribution and genome molecular weight
showed that related species formed distinct clusters. Group I methanotrophs were found to form three clusters
in both nucleotide distribution and genome size analyses. The first cluster consisted of five Methylomonas
species, Methylomonas methanica, Methylomonas fodinarum, Methylomonas aurantiaca, “Methylomonas rubra,” and “Methylomonas agile.” The other clusters included both Methylomonas species and Methylococcus
species, indicating the heterogeneity within these genera. One cluster contained low-G+ C-content Methylococcus strains and included Methylococcus whittenburyi, Methylococcus bovis, Methylococcus vinelandii,
Methylococcus luteus, and ‘4Methylococcus ucrainicus. ” The type strains of Methylomonas pelagica and
“Methylomonas alba” and a marine methanotrophic strain also clustered with the low-G+C-content
Methylococcus group rather than with the genus Methylomonas. “Methylomonas gracilis” also appeared to be
genetically distinct from the true Methylomonas species and clustered with the high-G+ C-content Methylococcus strains. This cluster included Methylococcus capsulatus, Methylococcus thermophilus, and other moderately
thermophilic group I methanotrophic strains. The group I1 methanotrophs belonging to the genera “Methylosinus” and “Methylocystis” formed separate generic clusters according to genome molecular weight data but
not according to nucleotide distribution data.
Methanotrophic bacteria are a diverse group of gramnegative bacteria which obligately utilize C, compounds,
including methane, methanol, and methylamine, as sole
sources of carbon and energy (3, 5 , 11, 27). They are unable
to utilize as sole energy sources any compounds with carbon-carbon bonds. The taxonomy of methanotrophs is based
mainly on morphological characters, and the systematics of
this group remains confused and incomplete (26). Studies on
biochemical pathways, ultrastructure of intracytoplasmic
membranes (3, ll),16s rRNA sequences (25), and fatty acid
contents (1, 2, 20) have shown that methanotrophs form two
major groups (groups I and 11). The group I methanotrophs
include the genera Methylomonas and Methy Eococcus and
are characterized by the presence of stacked vesicular disks
of intracytoplasmic membrane, the ribulose monophosphate
pathway for C, compound incorporation, and mainly 16:l
and 16:O fatty acids; these organisms appear to belong to the
y subdivision of the proteobacteria. The group I1 methanotrophs (the genera “Methylosinus” and “Methylocystis”)
are characterized by intracytoplasmic membranes that are
arranged peripherally in the cell parallel to the cytoplasmic
membrane, the serine pathway for C, compounds, and
mainly 18:l fatty acid and belong to the 01 subdivision of the
proteobacteria. Currently only the group I genera Methylomonas and Methylococcus have valid taxonomic status
(17, 22). The other genera are not on the Approved Lists of
Bacterial Names (17, 22) and have no standing in bacterial
nomenclature. Chemotaxonomic and immunological analyses (4, 6, 8, 15, 16) have clearly shown that the group I
methanotrophs can be separated into three groups.
The physicochemical properties of DNA have proved to
be useful as a means of characterizing and identifying
budding and hyphal bacteria (9, 13). DNA compositional
nucleotide distribution studies (7) basically involve measur-
* Corresponding author.
,\‘
ing the shape of DNA melting profiles. Two measures can be
obtained. The first measure is the DNA melting transition
width, which is indicative of the nucleotide distribution in a
bacterial genome. The nucleotide distribution (al+ a,) is
determined from the time interval required for DNA to
denature one standard deviation to the left (a,)and right (a,)
of the melting temperature and is expressed as G + C content.
The second measure is the ratio (a,/a,), which is a measure
of the skewness or asymmetry of the melting curve. The
results of De Ley (7) showed that nucleotide distributions
within bacterial genera appear to be consistent and thus can
be used effectively in taxonomic studies. Likewise, the use
of DNA renaturation techniques has proved to be a useful
means to accurately determine genome molecular weight
(10). These properties of DNA were used in this study in an
attempt to observe the inter- and intrageneric relationships
of various representatives of methane-utilizing bacteria and
to assess the taxonomy of these organisms as a guide to
further study.
MATERIALS AND METHODS
Bacterial strains. The strains used in this study are listed in
Table 1.
Cultivation. Methane-utilizing strains were grown on solidified NMS medium ( 5 ) under a methane-air-CO, (5:4:1)
atmosphere. Marine methanotrophic strain A4 was cultivated on NMS medium supplemented with 0.5% NaCl and 5
ml of Staley vitamin solution (24) per liter. Methylomonas
pelagica was grown on NMS medium prepared with artificial
seawater (derived from Difco marine agar 2216 [catalog no.
0979]), supplemented with 5 ml of vitamin solution per liter,
and solidified with 1%agarose (type V) (21). Most strains
were incubated in the dark at 28°C; the exceptions were
Methylococcus capsulatus strains, “Methylomonas gracilis” strains, and Methylococcus sp. strains JB87, JB137,
JB146, and JB173, which were incubated at 45°C.
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BOWMAN ET AL.
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INT. J. SYST.BACTERIOL.
TABLE 1. Physicochemical properties of DNAs isolated from obligate methanotrophs
G+C content
(mol%)
Laboratory
no.
Strain (source)n
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Marine methanotrophic strain A4 (M. E. Lidstrom)b
Methylococcus bovis UQM 3504 (IMET 10593)
Methylococcus luteus UQM 3304T (NCIB 11914T)‘
Methylococcus luteus IMV 3098T (Methylococcus bovis VKM-6T)
“Methylococcus ucrainicus” UQM 3305* (NCIB 11915T)
Methylococcus vinelandii UQM 3586= (IMV 3030T)
Methylococcus whittenburyi UQM 3310 (NCIB 11128)
Methylococcus capsulatus UQM 3302 (NCIB 11132)
Methylococcus capsulatus JB175
Methylococcus sp. strain JB140
Methylococcus sp. strain JB87
Methylococcus sp. strain JB137
Methylococcus sp. strain JB146
Methylococcus sp. strain JB173
Methylococcus thermophilus UQM 3 5 S T (IMV 3037T)
“Methylocystis parvus” UQM 3309T (NCIB 11129T)
“Methylocystis minimus” UQM 3497 (IMET 10578)
“Methylocystis echinoides” UQM 3495 (IMET 10491)
“Methylocystis” sp. strain JB170
“Methylocystis” sp. strain JB190
“Methylocystis” sp. strain JB196
“Methylomonas agile” UQM 3308 (NCIB 11124)
“Methylomonas alba” UQM 3314T (NCIB 11123T)
Methylomonas aurantiaca UQM 3406T
Methylomonas aurantiaca JB104
Methylomonas aurantiaca JB177B
Methylomonas fodinarum UQM 3268T
Methylomonas fodinarum JB2
Methylomonas fodinarum JB3
Methylomonas fodinarum JB4
Methylomonas fodinarum JB6
Methylomonas fodinarum JB7
Methylomonas fodinarum JB70
“Methylomonas gracilis” UQM 3315T (NCIB 11912T)
“Methylomonas gracilis” JB185
“Methylomonas gracilis” JB187
Methylomonas methanica UQM 3307T (NCIB 11130T)
Methylomonas methanica JB5
Methylomonas methanica JB9
Methylomonas methanica JB199
Methylomonas methanica JB228
Methylomonas pelagica UQM 3505T (NCIMB 2265T)
“Methylomonas rubra” UQM 3303 (NCIB 11913)
“Methylosinus trichosporiurn” UQM 3311T (NCIB 11131T)
“Methylosinus sporiuin” UQM 3306T (NCIB 11126T)
“Methy losinus sporium” JB206
“Methylosinus” sp. strain JB120
“Methylosinus” sp. strain HB-D1C
55.4
51.9
50.8
49.8
48.7
52.8
53.7
62.5
62.3
63.4
65.1
65.2
64.9
65.6
60.7
66.0
63.4
62.0
64.4
63.6
66.9
59.6
54.4
56.2
56.0
56.9
57.8
59.0
58.7
58.6
58.1
58.2
59.1
60.1
59.5
59.3
53.4
51.4
51.3
53.0
51.4
48.5
52.8
62.9
67.1
66.4
64.5
67.0
Nucleotide
distribution:
Asymmetry:
+
U b r
Ul
ur
(mol% G+C)
14.4
14.2
13.5
13.6
13.9
13.3
13.1
10.9
10.9
11.4
10.3
10.7
10.2
11.5
10.2
9.4
8.6
9.7
9.4
8.6
9.7
12.1
14.5
11.8
11.9
11.8
11.2
12.0
11.6
12.0
11.3
11.8
11.5
11.1
10.5
10.8
10.6
10.5
10.7
10.5
11.0
13.8
10.6
8.8
9.2
9.0
9.2
7.5
1.5
1.4
1.3
1.5
1.3
1.3
1.4
1.1
1.1
1.1
1.0
1.1
1.1
1.1
1.1
1.2
1.1
1.1
1.1
1.o
1.0
1.1
1.4
1.3
1.3
1.3
1.3
1.4
1.4
1.4
1.3
1.3
1.4
1.0
1.o
1.0
1.2
1.1
1.2
1.1
1.2
1.4
1.2
1.2
1.2
1.2
1.1
1.1
Genome size
(lo9 Da)
1.6
1.7
1.8
N D ~
1.8
1.5
1.6
2.8
2.4
2.7
ND
3.0
2.7
ND
2.5
2.5
2.3
2.2
2.0
ND
2.5
2.6
2.0
2.3
2.1
ND
1.8
ND
1.9
ND
ND
2.1
ND
2.8
ND
ND
2.0
ND
ND
ND
2.4
1.5
2.2
1.3
1.5
ND
1.5
1.4
Abbreviations: UQM, Culture Collection, Department of Microbiology, University of Queensland, Brisbane, Australia; ATCC, American Type Culture
Collection, Rockville, Md.; NCIB and NCIMB, National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland; DSM, German Collection of
Microorganisms, Braunschweig, Federal Republic of Germany; IMET, National Collection of Microorganisms, Institute for Microbiology, Jena, German
Democratic Republic; IMV, Institute of Microbiology and Virology, Academy of Sciences, Kiev, USSR; VKM, All-Union Collection of Microorganisms,
Academy of Science, Moscow, USSR; NCTC, National Collection of Type Cultures, Public Health Laboratory Service, London, United Kingdom; HB, Helen
Byers, Department of Microbiology, University of Queensland, Brisbane, Australia (environmental isolates); JB, J. Bowman, Department of Microbiology,
University of Queensland, Brisbane, Australia (environmental isolates).
See reference 14.
T = type strain.
ND, Not determined.
DNA extraction. Cultures were grown on agar plates for 3
to 5 days. Cell growth was harvested from plates with
distilled water and washed twice with saline-EDTA (0.1 M
disodium EDTA, 0.15 M NaC1; pH 8.0) by using centrifugation at 10,000 x g for 10 min. Cells of “Methylosinus” and
“Methylocystis” strains were pretreated with a solution
containing 1 mg of lysozyme per ml for 1 h at 37°C before
lysis. All strains were lysed by adding 2% (final concentration) sodium dodecyl sulfate at 60°C. DNAs were extracted
and purified from cell lysates by using a modified Marmur
technique (23).
DNA analyses. DNA samples were analyzed by using a
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METHANOTROPH GENOME CHARACTERISTICS
VOL. 41, 1991
303
70
60
-
55
-
50
I
Low GtC Methylococcus spp
45
7
1
1
I
I
I
I
I
8
9
10
11
12
13
14
15
DNA MELTING TRANSITION WIDTH (MOL% GtC)
FIG. 1. Similarity map of obligately methanotrophic bacteria based on DNA base compositions and nucleotide distributions. The numbers
are the laboratory numbers for strains given in Table 1.
microprocessor-controlled Gilford model 2600 spectrophotometer equipped with a thermoprogrammer. The Timethod
(23) was used to determine G + C contents; Escherichia coli
ATCC 11775 (G+C content, 51.7 mol%) was used as the
internal standard. The nucleotide distributions of DNA
samples were determined from the thermal denaturation
profiles generated by using the method of De Ley (7).
Genome molecular weights were determined by using a
DNA renaturation procedure in 5 x SSC (0.75 M NaCl, 0.075
M trisodium citrate; pH 7.0) containing 25% formamide
(Sigma Chemical Co.) (13). The molecular weight of E. coli
K-12 strain UQM 884 (= NCTC 10538) reference DNA was
taken to be 2.7 x lo9 (12). All procedures were repeated at
least four times.
RESULTS AND DISCUSSION
The DNA base composition, nucleotide distribution, and
genome molecular weight for each of the methanotrophs
which we studied are shown in Table 1. Similarity maps
constructed by plotting nucleotide distribution versus DNA
base composition (G+C content) (Fig. 1) and genome molecular weight versus DNA base composition (G+C content)
(Fig. 2) were created from the DNA analysis data.
Nucleotide distributions. The group I methanotrophs
formed three clusters on the nucleotide distribution-DNA
base composition map. Strains of Methylococcus spp. produced two separate clusters. The first cluster contained
Methylococcus capsulatus, Methylococcus thermophilus,
and other moderately thermophilic strains with DNAs having high G + C contents (59 to 66 mol%) and transition widths
of 10.2 to 11.5 mol% G+C. Strains of “Methylomonas
gracilis” also clustered with these strains. The second
cluster consisted of low-G+C-content (48 to 55 mol%)
mesophilic strains which had DNAs with broader melting
transition widths (13.1 to 14.5 mol% G+C). This cluster
included strains of Methylococcus whittenburyi, Methylococcus bovis , Methy lococcus vinelandii, Methylococcus luteus, and “Methylococcus ucrainicus.” The low-G+C-content Methylococcus cluster also included “Methylomonas
alba ,” Methylomonas pelagica (21), and marine methan-
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INT. J. SYST.BACTERIOL.
BOWMAN ET AL.
70
Me thy losinus
SPP.
Methylomonas
SPP.
1.o
1.5
2.0
2.5
3.0
3.5
GENOME MOLECULAR WEIGHT ( 10 ’daltons)
FIG. 2. Similarity map of obligately methanotrophic bacteria based on DNA base compositions and genome molecular weights. The
numbers are the laboratory numbers for strains given in Table 1.
otrophic strain A4 of Lidstrom (14). The species Methylomonas methanica, Methylomonas fodinarum, Methylomonas
aurantiaca, “Methylomonas rubra,” and “Methylomonas
agile” exhibited DNA melting transition widths of 10.5 to
12.1 mol% G + C and formed a single cluster. The DNA base
compositions of the members of this cluster ranged from 51
to 60 mol% G+C.
The group I1 methanotrophs (members of the genera
“Methylosinus” and “Methylocystis”) exhibited similar nucleotide distributions and DNA base compositions and
formed two overlapping clusters (Fig. 1). For these strains
the G + C contents ranged from 62 to 67 mol% and the
transition widths ranged from 7.5 to 9.7 mol% G+C.
Genome molecular weights. The genome molecular weights
of the methanotrophs which we studied were useful for
demonstrating relationships between taxa; these relationships were consistent with the relationships determined by
using nucleotide distribution data.
The Methylomonas species again fell into separate clusters. The genome sizes of Methylomonas methanica strains
were similar to those of Methylomonas fodinarum, Methylomonas aurantiaca, “Methylomonas rubra ,’’ and “Methylomonas agile” strains, varying from 1.8 X lo9 to 2.6 x lo9
daltons. On the other hand, “Methylomonas gracilis” appeared to be genotypically similar to Methylococcus thermo-
philus and Methylococcus capsulatus (Table l ) , with which
it shares phenotypic characteristics that are common to
other moderately thermophilic group I methanotrophs (19,
20) and are not present in most mesophilic group I methanotrophs. These characteristics include the ability to fix
nitrogen and the ability to fix CO, via Calvin-Benson cycle
enzymes in the presence of methane (18, 19, 26). Methylomonas pelagica also appeared to be unrelated to Methylomonas methanica and clustered with the low-G+C-content Methylococcus strains because of its significantly
smaller genome size and the broad DNA melting transition
widths of these organisms. The genome size of “Methylomonas alba” overlapped the genome sizes determined for
members of the Methylomonas cluster, but this organism
had a significantly higher transition width, which was similar
to the transition widths determined for the low-G +C-content
Methylococcus strains. These observations indicate that the
description given previously for the genus Methylomonas
(19) may be imprecise; this description has led to the
assignment of morphologically similar but genotypically
unrelated species to the genus.
Methylococcus capsulatus , Methylococcus thermophilus,
and moderately thermophilic strains JB137, JB140, and
JB146 have genome sizes (2.4 x lo9 to 3.0 x lo9 daltons) that
are considerably larger than the genome sizes of the other
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VOL.41, 1991
METHANOTROPH GENOME CHARACTERISTICS
species of the genus Methylococcus (19), which also have
lower G + C contents (55 mol% or less). The latter species
have genome sizes that range from 1.6 x lo9 to 2.0 x lo9
daltons. The genus Methylococcus was defined on the basis
of purely phenotypic characteristics (19) and is clearly
genotypically heterogeneous. The results of several limited
studies of both phenotypic and chemotaxonomic properties
(1, 2, 6, 8, 20) support this view.
The group I1 methanotrophs (the genera “Methylosinus”
and “Methylocystis”) can be distinguished by their different
genome molecular weights. “Methylocystis” genome sizes
range from 2.0 x lo9 to 2.5 x lo9 daltons and are on the
average 50 to 100% larger than the genome sizes of “Methylosinus” strains (range, 1.3 x lo9 to 1.5 x lo9 daltons).
This study of the physicochemical properties of genomic
DNAs provided information that is helpful for assessing the
taxonomic relationships among the genera and species of the
obligately methanotrophic bacteria; in particular, it demonstrated the genetic heterogeneity in the genera Methylomonus and Methylococcus. This approach was undertaken as
the first stage of an extensive taxonomic study of these
organisms, and the results highlight the areas that need
nomenclatural revision.
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