Thiovirga sulfuroxydans gen. nov., sp. nov., a
chemolithoautotrophic sulfur-oxidizing bacterium
isolated from a microaerobic waste-water biofilm
Tsukasa Ito,1Kenichi Sugita,1Isao Yumoto,2Yoshinobu Nodasaka3
and Satoshi Okabe1
1Department of Urban and Environmental Engineering, Graduate School of Engineering,
Hokkaido University, Kita-ku, Sapporo 060-8628, Japan
2Research Institute of Biological Resources and Function, Hokkaido Center, National Institute
of Advanced Industrial Science and Technology, Tsukisamu-Higashi, Toyohira-ku, Sapporo
3Laboratory of Electron Microscopy, Graduate School of Dental Medicine, Hokkaido University,
Kita-ku, Sapporo 060-8586, Japan
A novel mesophilic, chemolithoautotrophic, sulfur-oxidizing bacterium, designated strain SO07T,
was isolated from a microaerobic waste-water biofilm. Chemolithoautotrophic growth was
observed with elemental sulfur, sulfide and thiosulfate as sole electron donors and oxygen as
electron acceptor. Anaerobic and heterotrophic growth were not observed. Nitrate was not used
as a terminal electron acceptor. The optimum pH and temperature for growth were pH 7?5 and
306C, respectively. The major isoprenoid quinone was Q-8. The DNA G+C content of strain
SO07Twas 47?1 mol%. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that
strain SO07Tformed a monophyletic group in the c-Proteobacteria with only 89% similarity to
members of the genus Halothiobacillus, its nearest phylogenetic neighbours. In addition, the
isolate differed from members of the genus Halothiobacillus in its requirement for and
tolerance of NaCl; strain SO07Twas unable to grow in NaCl concentrations of more than
180 mM. On the basis of phylogenetic, chemotaxonomic and physiological data, it is proposed
that isolate SO07T(=JCM 12417T=ATCC BAA-1033T) represents the type strain of a novel
species in a new genus, Thiovirga sulfuroxydans gen. nov., sp. nov.
During investigations into the sulfur cycle in a microaero-
bic waste-water biofilm, a survey combining the molec-
ular techniques of 16S rRNA gene cloning followed by
fluorescence in situ hybridization revealed the presence
of a novel sulfur-oxidizing bacterium (approximately
at the oxic biofilm strata where high concentrations of ele-
mental sulfur (S0) accumulate. The use of S0as an electron
donor was an effective measure to establish an enrichment
culture of strain SO07Tand for further purification (Ito
et al., 2004). The 16S rRNA genes of large numbers of
uncultured bacterial clones that are closely related to strain
SO07Thave been found in GenBank (Benson et al., 2003),
suggesting the widespread distribution and ecological
importance of these types of bacteria in the environ-
ment. Analysis of phylogenetic and physiological character-
istics showed that strain SO07Trepresents the type strain
of the type species of a new genus; the name Thiovirga
sulfuroxydans gen. nov., sp. nov. is proposed.
The waste-water biofilm sample was collected from a sewer
line that transports primary settling tank effluent at the
Soseigawa municipal waste-water treatment plant, Sapporo,
Japan. Enrichment and isolation were performed using a
slightly modified version of medium used for neutrophilic
Thiobacillus species (Kuenen et al., 1991), designated SOB
medium in this study. The composition of SOB medium
(in g l21) was KH2PO4(0?5), K2HPO4(0?5), NH4Cl (0?5),
MgSO4.7H2O (0?1), CaCl2(0?05) and NaHCO3(1?0), plus
1 ml trace element solution l21(Kuenen et al., 1991). The
procedures for enrichment and isolation have been des-
cribed previously (Ito et al., 2004). Cells grown in liquid
SOB medium with thiosulfate (6?5 mM) as a sole electron
donor were used for studies on phenotypic properties and
chemotaxonomic traits unless otherwise specified. Aerobic
Abbreviation: EDS, energy-dispersive X-ray spectroscopy.
Published online ahead of print on 9 December 2004 as DOI 10.1099/
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of Thiovirga sulfuroxydans SO07Tis AB118236.
63467 G 2005 IUMS Printed in Great Britain1059
International Journal of Systematic and Evolutionary Microbiology (2005), 55, 1059–1064
growth was examined in SOB medium supplemented
with sulfide (13 mM), thiosulfate (6?5 mM), elemental
sulfur (416 mg l21), methanol (1 mM), formate (1 mM),
acetate (1 mM) or propionate (1 mM) as electron donor.
Anaerobic growth was examined in SOB medium contain-
ing the same electron donors as in the aerobic growth
test and with nitrate (0?74 mM) as electron acceptor; the
headspace gas contained only N2 gas (99?99%, v/v).
Fermentative growth was tested in SOB medium containing
the same electron donors as in the aerobic growth test under
anoxic conditions without nitrate. Mixotrophic growth was
tested in SOB medium containing thiosulfate (6?5 mM)
together with one of formate (13 mM), acetate (13 mM)
or propionate (13 mM). Substrate utilization was evaluated
by measuring each substrate concentration and by DAPI
direct counting as described by Hobbie et al. (1977). The
concentrations of sulfate, thiosulfate, nitrite, nitrate, for-
mate, acetate and propionate in the culture solutions were
determined with an ion chromatograph equipped with
either an IonPac AS9-HCC column for sulfate, thiosulfate,
nitrite and nitrate or an ICE-AS1 column for formate,
acetate and propionate (model DX-100; Nippon Dionex).
The concentration of total dissolved sulfide (H2S, HS2and
S22) was determined by the methylene blue method (Cline,
1969). The temperature range for growth was examined in
SOB medium containing thiosulfate (6?5 mM) at 5, 10, 15,
20, 25, 30, 34, 37, 42, 47 and 55uC. The pH range for growth
(4?0–10?0) was tested in SOB medium containing thio-
sulfate at 30uC. The NaCl requirement and tolerance of the
isolate were examined in SOB medium containing various
NaCl concentrations (0?03, 0?08, 0?13, 0?18, 0?23, 1?0, 2?0,
3?0 and 4?0 M). Growth of strain SO07Twas monitored
by measuring the optical density at 540 nm. All the growth
tests described above were conducted at 30uC and pH 7?0
unless otherwise specified.
For morphological observations, cells grown on solid SOB
medium containing 1?5% agar at 25uC for 1 day were
negatively stained with 1% (w/v) phosphotungstic acid
and then observed under a Hitachi model H-800 transmis-
sion electron microscope at an acceleration voltage of
75 kV. For ultrastructure analysis, cells grown on SOB agar
medium were immersed in 2% (v/v) glutaraldehyde in
0?1 M phosphate buffer for 2 h; cells were then washed
twice for 10 min and then for 1 h in 0?1 M phosphate
buffer. Cells were fixed in 1% OsO4in 0?1 M phosphate
buffer for 2 h and then washed again in the same way. The
fixed cells were dehydrated in a graduated acetone series
(70–100%) and subsequently embedded into the resin.
Thin sections were cut with a Reichert model ultracut S
ultramicrotome, placed on copper grids and stained with
10% (w/v) uranyl acetate and 1% (w/v) lead citrate. The
stained thin sections were examined by transmission
electron microscopy (TEM) and energy-dispersive X-ray
spectroscopy (TEM-EDS) with a JEOL model JEM-2000ES
at an acceleration voltage of 200 kV. For scanning electron
microscope observations, cells incubated in liquid SOB
medium containing thiosulfate at 25uC for 1 day were fixed
with 2% (v/v) glutaraldehyde in 0?1 M phosphate buffer
for 2 h. Further preparation steps were conducted accord-
ing to Yumoto et al. (2001). The cultures were mounted
on aluminium stubs and observed under a Hitachi model
S-4000 scanning electron microscope at an acceleration
voltage of 3?5 kV.
Analyses of whole-cell fatty acids and isoprenoid quinones
were performed as described previously (Yumoto et al.,
2001). Genomic DNA for the analysis of G+C content was
prepared as described by Marmur (1961). The G+C
content (mol%) of the genomic DNA was determined by
HPLC according to the method of Tamaoka & Komagata
(1984). The levels of DNA relatedness were determined
fluorometrically by the method of Ezaki et al. (1989) using
photobiotin-labelled DNA probes and microplates. The
DNA–DNA hybridization study was conducted only against
Halothiobacillus neapolitanus (strain JCM 3861), the type
species of the genus Halothiobacillus and most closely
related species to strain SO07T, because 16S rRNA gene
similarities between strain SO07Tand all members of the
genus Halothiobacillus are low (less than 89%).
PCR amplification, purification of PCR products and 16S
rRNA gene sequencing were carried out as described
previously (Ito et al., 2004). Phylogenetic inferences were
made with the 16S rRNA gene sequence database associated
with the ARB software package (Ludwig et al., 2004).
Phylogenetic trees were then constructed by using the ARB
neighbour-joining and maximum-parsimony algorithms.
Bootstrap analysis was performed to establish a confidence
level for nodes. 16S rRNA gene sequence similarity values
were calculated by using the program Similarity_Matrix in
the Ribosomal Database Project II (Cole et al., 2003).
Chemolithoautotrophic growth of strain SO07T
observed on sulfide, thiosulfate and elemental sulfur.
Growth on thiosulfate reduced the pH to a minimum of
6?0. The pH range for growth of strain SO07Twas 6?0–9?0,
with optimal growth at pH 7?5. The temperature range for
growth was 15–42uC, with an optimum growth tempera-
ture of 30–34uC. This strain grew at NaCl concentrations
of 30–180 mM. No growth was observed in 230 mM
NaCl. Heterotrophic growth was not observed when tested
with acetate, formate, propionate or methanol. Anaerobic
growth did not occur in the presence of any organic or
inorganic substrates when nitrate was used as an electron
acceptor. No fermentative growth was observed on
methanol, formate, acetate or propionate. Mixotrophic
tests on thiosulfate with formate, acetate or propionate
revealed growth inhibition.
Colonies of strain SO07Tgrown on solid SOB medium
containing thiosulfate were 0?5–1?0 mm in diameter,
morphology of the colonies did not change during
incubation for 5 days. The isolate was Gram-negative,
catalase-positive and oxidase-positive. Cells were rod-
shaped (0?5–0?861?0–2?0 mm), non-spore-forming and
1060 International Journal of Systematic and Evolutionary Microbiology 55
T. Ito and others
motile by means of a single polar flagellum (Fig. 1).
Structure of the cell wall was a typical Gram-negative-
stained type (Fig. 1b). Scanning electron microscope
observations revealed that the cells had large surface areas
with cobble-like structures on their surfaces (Fig. 2).
Carboxysome-like inclusions were observed in the cells
(Fig. 1b). The number of carboxysome-like inclusions in
exponential-growth-phase cells incubated with thiosulfate
averaged about 6±3 (n=20) per cell, whereas that with
sulfide was 2±1 (n=20) per cell. It has been reported
that the number of carboxysomes per cell in Thiomonas
intermedia (formerly Thiobacillus intermedius) is propor-
tional to the specific activity of ribulose-1,5-bisphosphate
carboxylase (Purohit et al., 1976). The higher number of
carboxysomes in cells incubated with thiosulfate may
explain the higher maximum specific growth rate on
thiosulfate of 0?41 h21, compared with 0?30 h21on sulfide.
Storage of polyphosphate-like inclusions was observed
when strain SO07Twas grown with sulfide as electron
donor (Fig. 1b). The polyphosphate-like inclusions con-
tained phosphorous, which was determined by TEM-EDS
Analysis of quinone compounds revealed Q-8 to be the
major isoprenoid quinone. Predominant cellular fatty
acids of strain SO07Twere C12:0 (2%), C16:0 (19%),
C18:0(16%), C16:1(30%) and C18:1(31%). The DNA
G+C content of strain SO07Twas 47?1 mol%. The DNA–
DNA relatedness between strain SO07Tand H. neapolitanus
JCM 3861 was less than 2%.
Comparison of 16S rRNA gene sequences revealed that
strain SO07Tformed a monophyletic group within the c-
Proteobacteria, as supported by high bootstrap values
(Fig. 3), and can clearly be distinguished from members
of the genus Halothiobacillus (less than 89% sequence
similarity between them). A slightly higher level of 16S
rRNA gene sequence similarity was observed between strain
SO07Tand ‘Thiobacillus baregensis’ (90%) than between
strain SO07Tand members of the genus Halothiobacillus
(87–89%). High sequence similarities were obtained
between strain SO07Tand partial sequences of the as-
yet-uncultured bacterial clones SRang2.5 (98%) and
bacteriap48 (97%), which were retrieved from sulfurous
environments, i.e. Sulphur River in Parker Cave and a
muddy hot pool in Kuirau Park, respectively (Angert et al.,
1998; Sunna & Bergquist, 2003). Thus, the ability of these
clones to oxidize sulfur could be inferred. Characterization
of strain SO07Tstrongly indicated that the cluster including
these environmental clones comprised chemolithoauto-
trophic sulfur-oxidizing bacteria. As in waste-water bio-
films, the source of strain SO07T, oxygen concentrations in
these habitats would be low due to the relatively high
concentrations of organic matter and sulfide.
Strain SO07Tshares the same phenotypic properties as
members of the genus Halothiobacillus: mesophilic, neu-
trophilic and obligately chemolithoautotrophic, obtaining
energy from reduced inorganic sulfur compounds. Both
strain SO07Tand members of the genus Halothiobacillus
contain ubiquinone Q-8 as the major isoprenoid quinone.
Fig. 1. Transmission electron micrographs of cells of strain
SO07T. (a) Negatively stained cell showing the single polar fla-
gellum (bar, 0?4 mm). (b) An ultrathin section of a cell stained
with uranyl acetate and lead citrate (bar, 0?2 mm). Cells in (a)
and (b) were grown on thiosulfate and sulfide, respectively. CA,
carboxysome; PI, polyphosphate-like inclusion. The carboxysome
was enveloped by a thin membrane-like structure.
Fig. 2. Scanning electron micrograph of cells of strain SO07T
grown on SOB medium containing thiosulfate. Bar, 0?2 mm.
Thiovirga sulfuroxydans gen. nov., sp. nov.
The presence of carboxysome-like inclusions in the cells of
strain SO07Thas also been described for H. neapolitanus by
Shively et al. (1973). In contrast to members of the genus
Halothiobacillus, strain SO07Tdid not require NaCl for
growth and the growth of this strain was completely
inhibited at NaCl concentrations greater than 180 mM
(Table 1). In fact, all Halothiobacillus species have high
NaCl tolerances (more than 860 mM NaCl and up to
4000 mM) and their optimal NaCl concentrations for
growth are relatively high (more than 400 mM NaCl)
(Kelly & Wood, 2000; Sievert et al., 2000). These distinctive
characteristics of strain SO07Tallow it to be differentiated
Fig. 3. Phylogenetic tree based on analysis of 16S rRNA gene sequences of strain SO07Tand representative species of
some distantly related sulfur-oxidizing genera of the c-Proteobacteria, constructed by using the neighbour-joining method.
Nearly complete 16S rRNA gene sequences were used. Numbers at the nodes represent bootstrap values. Bar, 0?02 inferred
nucleotide substitutions per nucleotide position. Further isolation and characterization of bacteria represented by environ-
mental clones, including BPC028, SRE59 or SRang1.28, might expand the coverage of cluster SO07, as shown by the
Table 1. Main phenotypic characteristics that differentiate strain SO07Tfrom mesophilic, neutrophilic and chemolithoauto-
trophic sulfur-oxidizing bacteria belonging to the c-Proteobacteria
Species: 1, Thiovirga sulfuroxydans SO07T; 2, Halothiobacillus neapolitanus DSM 15147T; 3, Halothiobacillus hydrothermalis DSM 7121T; 4,
Halothiobacillus halophilus DSM 6132T; 5, Halothiobacillus kellyi DSM 13162T; 6, Thiobacillus sp. W5; 7, Thiomicrospira frisia DSM 12351T;
8, Thiomicrospira chilensis DSM 12352T. All species were obligately chemolithoautotrophic, mesophilic, neutrophilic motile rods and oxidized
thiosulfate, sulfur and sulfide. Data from Kelly & Wood (2000), Sievert et al. (2000) and Brinkhoff et al. (1999a, b).
Conditions for optimum growth:
Upper NaCl concn for growth (mM)
Major fatty acids
C12:0, C16:0, C18:0,
C16:0, C16:1, C18:1,
NDND ND ND NDND
DNA G+C content (mol%) 67?4 64?2 62?0 56?0 39?6 49?9
ND, Not determined.
1062 International Journal of Systematic and Evolutionary Microbiology 55
T. Ito and others
as a freshwater species. Analysis of nearly complete 16S
rRNA gene sequences revealed low levels of similarity
(87–89%) between strain SO07Tand Halothiobacillus
species. The level of DNA–DNA hybridization between
strain SO07Tand H. neapolitanus was less than 2%.
In addition, the DNA G+C content of strain SO07T
(47?1 mol%) is significantly lower than those of Halothio-
bacillus species, i.e. 56?0–67?4 mol% (Kelly & Wood, 2000;
Sievert et al., 2000). Because of the distinct differences in
phylogeny, chemotaxonomy and the requirement for and
tolerance of NaCl between strain SO07Tand members of
the genus Halothiobacillus, it is proposed that strain SO07T
represents the type strain of the type species of a new genus,
Thiovirga sulfuroxydans gen. nov., sp. nov.
Description of Thiovirga gen. nov.
Thiovirga (Thi.o.vir9ga. Gr. n. thion sulfur; L. fem. n. virga
rod; N.L. fem n. Thiovirga sulfur rod).
Obligately chemolithoautotrophic, Gram-negative rod.
Motile, obtaining energy from reduced inorganic sulfur
compounds. Oxidase- and catalase-positive. No spore
formation. No anaerobic or heterotrophic growth observed.
Cells contain carboxysome inclusions. Cells store poly-
phosphate inclusions when grown on sulfide. Contains
ubiquinone Q-8. Major fatty acids are C16:0, C18:0, C16:1
and C18:1. Member of the c-Proteobacteria, which is
distantly related to halotolerant sulfur-oxidizing bacteria,
the members of the genus Halothiobacillus.
The type species is Thiovirga sulfuroxydans.
Description of Thiovirga sulfuroxydans sp. nov.
Thiovirga sulfuroxydans (sul.fur.ox9y.dans. L. n. sulfur
sulfur; N.L. part. adj. oxydans oxidizing; N.L. part. adj.
Cells are rod-shaped, 0?5–0?861?0–2?0 mm. Colonies
medium) are white in colour and lens-shaped (diameter
of 0?5–1?0 mm). Gram-negative. Cells occur singly or in
pairs and are motile by single polar flagella. Strictly aerobic.
Grows chemolithoautotrophically on thiosulfate, sulfur
and sulfide. Nitrate is not used as terminal electron accep-
tor. No heterotrophic growth occurs. Optimum growth
temperature is 30–34uC; optimum pH is 7?5; and optimum
NaCl concentration is 30 mM. No growth is observed in
NaCl concentrations of more than 180 mM or above 42uC.
Cells contain carboxysome inclusions. Stores polyphos-
phate inclusions in cells when grown on sulfide. Major
isoprenoid quinone isubiquinone Q-8.Majorfattyacidsare
C16:0(19%), C18:0(16%), C16:1(30%) and C18:1(31%);
C12:0(2%) is present as a minor fatty acid.
The type strain is SO07T(=JCM 12417T=ATCC BAA-
1033T). The DNA G+C content of the type strain is
47?1 mol%. Isolated from a microaerobic waste-water
biofilm at a waste-water treatment plant at Sapporo, Japan.
This work was partially supported by a grant-in-aid (no. 13650593) for
Developmental Scientific Research from the Ministry of Education,
Science and Culture of Japan. This study was also carried out as a
part of ‘The Project for Development of Technologies for Analysing
and Controlling the Mechanism of Biodegrading and Processing’,
which was entrusted by the New Energy and Industrial Technology
Development Organization (NEDO). T.I. is supported by a research
fellowship of the Japan Society for the Promotion of Science.
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