International Journal of Systematic and Evolutionary Microbiology (2013), 63, 454–457
DOI 10.1099/ijs.0.042531-0
Porphyromonas crevioricanis is an earlier
heterotypic synonym of Porphyromonas cansulci
and has priority
Mitsuo Sakamoto and Moriya Ohkuma
Correspondence
Mitsuo Sakamoto
[email protected]
Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Tsukuba,
Ibaraki 305-0074, Japan
A DNA–DNA hybridization experiment was carried out to clarify the relationship between
Porphyromonas crevioricanis and Porphyromonas cansulci. The taxonomic standing of these two
species was unclear so far because of the high 16S rRNA gene sequence similarity value
(99.9 %). The DNA–DNA relatedness values between P. crevioricanis JCM 15906T and P.
cansulci JCM 13913T were above 91 % (91–99 %). In addition, P. crevioricanis JCM 15906T
exhibited high hsp60 gene sequence similarity with P. cansulci JCM 13913T (100 %). The hsp60
gene sequence analysis and the DNA–DNA relatedness values demonstrated that P.
crevioricanis JCM 15906T and P. cansulci JCM 13913T are a single species. Based on these
data, we propose Porphyromonas cansulci as a later heterotypic synonym of Porphyromonas
crevioricanis.
It has been reported that Porphyromonas cansulci JCM
13913T shows a very high hsp60 gene sequence similarity
value (100 %) to Porphyromonas crevioricanis JCM 15906T as
well as a very high 16S rRNA gene sequence similarity value
(99.9 %) (Sakamoto & Ohkuma, 2010). P. crevioricanis
(Hirasawa & Takada, 1994) and P. cansulci (Collins et al.,
1994) appeared in the same issue (vol. 44, no. 4) of
International Journal of Systematic Bacteriology. If the level of
DNA–DNA hybridization is sufficient to consider P.
crevioricanis and P. cansulci as members of the same species,
P. cansulci is a later synonym of P. crevioricanis because the
name P. crevioricanis Hirasawa and Takada 1994 has page
priority over the name P. cansulci Collins et al. 1994. To
resolve this issue, further studies were performed.
Porphyromonas crevioricanis JCM 15906T and Porphyromonas cansulci JCM 13913T were used in this study. The
strains were maintained on Eggerth Gagnon (EG) agar
(Merck) supplemented with 5 % (v/v) horse blood for 5–
7 days at 37 uC in an atmosphere containing 100 % CO2. P.
crevioricanis JCM 15906T and P. cansulci JCM 13913T were
obligately anaerobic, black-pigmented, asaccharolytic, nonspore-forming, non-motile, Gram-negative short rods or
rods. Cells on EG agar were 0.5 mm60.8–1.2 mm. Colonies
on EG agar plates after 5 days of incubation at 37 uC under
anaerobic conditions were 1–2 mm in diameter, circular,
entire, dome-shaped, smooth, and brown or black.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequences of P. crevioricanis JCM 15906T and P. cansulci JCM 13913T
are AB547658 and AB547655, respectively, and those for their partial
hsp60 gene sequences are AB547579 and AB547576, respectively.
454
Physiological and biochemical reactions were determined
in duplicate with the API 20A anaerobe test kit and the
Rapid ID 32A anaerobe identification kit, respectively
(bioMérieux), according to the manufacturer’s instructions. The phenotypic and biochemical characteristics of P.
crevioricanis JCM 15906T and P. cansulci JCM 13913T were
identical. Both strains failed to produce acid from Larabinose, cellobiose, glucose, glycerol, lactose, maltose, Dmannitol, D-mannose, melezitose, raffinose, L-rhamnose,
salicin, D-sorbitol, sucrose, trehalose and D-xylose. Both
strains were positive for indole production and gelatin
digestion. Both strains were negative for aesculin hydrolysis
and urease and catalase activities. In Rapid ID 32A tests,
both strains were positive for indole production, alkaline
phosphatase, alanine arylamidase, glutamyl glutamic acid
arylamidase and leucyl glycine arylamidase activities, but
negative for nitrate reduction, urease, b-N-acetylglucosaminidase, a-arabinosidase, arginine dihydrolase, a-fucosidase, a-galactosidase, b-galactosidase, b-galactosidase-6phosphate, a-glucosidase, b-glucosidase, b-glucuronidase,
mannose and raffinose fermentation and glutamic acid
decarboxylase, arginine arylamidase, glycine arylamidase,
histidine arylamidase, leucine arylamidase, phenylalanine
arylamidase, proline arylamidase, pyroglutamic acid arylamidase, serine arylamidase and tyrosine arylamidase
activities. All of these comparable data were in agreement
with the original data (Collins et al., 1994; Hirasawa &
Takada, 1994), with the exception of catalase activity which
differed from the original data of P. cansulci.
Fatty acid methyl esters (FAMEs) were obtained from
about 40 mg wet cells grown on EG agar at 37 uC for
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P. cansulci is a synonym of P. crevioricanis
5 days by saponification, methylation, and extraction using
minor modifications (Kuykendall et al., 1988) of the
method of Miller (1982). Cellular fatty acid profiles were
determined by the Sherlock Microbial Identification
System (MIDI, Inc.) version 2.99B with database BHIBLA
(version 3.80). Peaks were automatically integrated, fatty
acids were identified by equivalent chain-length (ECL), and
percentages of the total peak area were calculated. External
calibration was performed by using MIDI calibration
mixture 1 (FAMEs of straight-chain saturated fatty acids
from 9 to 20 carbons in length and five hydroxy acids). The
cellular fatty acid compositions of both strains were similar
(Table 1). The major fatty acid of P. crevioricanis JCM
15906T and P. cansulci JCM 13913T was iso-C15 : 0 though
the amount of this fatty acid in P. crevioricanis JCM 15906T
was lower than that of P. cansulci JCM 13913T. These
finding are in agreement with the original data (Collins
et al., 1994; Hirasawa & Takada, 1994).
Table 1. Cellular fatty acid content of P. crevioricanis and P.
cansulci
Strains: 1, P. crevioricanis JCM 15906T; 2, P. cansulci JCM 13913T.
Values are percentages of total fatty acids. tr, Trace amount (,0.5 %);
2, not detected.
Fatty acid
Saturated straight-chain
C14 : 0
C15 : 0
C16 : 0
C18 : 0
Unsaturated straight-chain
C16 : 1v7c
C18 : 1v9c
C18 : 2v6, 9c
Hydroxy
C16 : 0 3-OH
C17 : 0 3-OH
iso-C15 : 0 3-OH
iso-C17 : 0 3-OH
anteiso-C17 : 0 3-OH
Saturated branched-chain
iso-C13 : 0
iso-C15 : 0
anteiso-C15 : 0
iso-C16 : 0
iso-C17 : 0
Summed feature*
3
10
1
2
0.6
tr
7.7
2.3
0.9
tr
5.0
1.0
tr
19.1
5.4
2
11.3
2.2
1.4
2
1.9
22.7
0.6
1.2
tr
1.2
25.4
0.8
2
28.6
1.5
2
2.7
tr
39.6
2.3
tr
2.6
2.4
2.1
2.7
1.1
*Summed features represent groups of two or three fatty acids that
could not be separated using the MIDI system. Summed feature 3
contains one or more of an unknown fatty acid of ECL 13.570 and/or
iso-C15 : 0 ALDE. Summed feature 10 contains one or more of an
unknown fatty acid of ECL 17.834 and/or C18 : 1v11c/9t/6t fatty acid
methyl ester.
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Isoprenoid quinones were extracted as described by
Komagata & Suzuki (1987) and were analysed by HPLC
with a Cosmosil 5C18 column (4.66150 mm; Nacalai
Tesque). The elution solvent was a mixture of methanol
and 2-propanol (2 : 1, v/v). In this study, the menaquinone
compositions of both strains became clear for the first
time. The major menaquinones of P. crevioricanis JCM
15906T and P. cansulci JCM 13913T were MK-9 (33.9 and
30.7 %, respectively) and MK-10 (49.6 and 59.6 %,
respectively), and the minor menaquinones were MK-7
(3.1 and 3.0 %, respectively) and MK-8 (13.4 and 6.7 %,
respectively).
Approximately 1500 bases of the 16S rRNA gene sequence
have been determined for both strains (Sakamoto &
Ohkuma, 2010). For phylogenetic analysis of 16S rRNA
gene sequences, 1429 bp sequences of each species were
used. 558 bp nucleotide sequences of the hsp60 gene
determined by Sakamoto & Ohkuma (2010) were also
used. Phylogenetic analysis was performed as described
previously (Sakamoto & Ohkuma, 2010). P. crevioricanis
JCM 15906T and P. cansulci JCM 13913T formed a single
cluster and a distinct line of descent (Fig. 1) as reported
previously (Sakamoto & Ohkuma, 2010). Both strains
exhibited 99.9 % 16S rRNA gene sequence similarity with
each other as mentioned above. There were no discrepancies between the 16S rRNA gene sequence of P.
crevioricanis JCM 15906T and the original 16S rRNA gene
sequence deposited in the public databases. On the other
hand, the 16S rRNA gene sequence of P. cansulci JCM
13913T differs in 3 positions (plus 2 unknown bases) from
the originally reported sequence (GenBank accession no.
X76260). Since the original 16S rRNA gene sequence of P.
cansulci has 2 unknown bases, the quality of this original
sequence is doubtful. In addition, hsp60 gene sequence
analysis suggested that both strains are the same species
(Fig. 1). The hsp60 gene has been found to be a useful
alternative phylogenetic marker (Sakamoto & Ohkuma,
2010, 2011; Sakamoto et al., 2010). More recently,
Sakamoto & Ohkuma (2012) used the hsp60 gene sequence
to reclassify Bacteroides chinchillae (Kitahara et al., 2011).
Chromosomal DNA was extracted using a Genomic-tip
100/G kit (Qiagen). The DNA base composition was
determined by the HPLC method of Tamaoka & Komagata
(1984). The elution solvent was a mixture of 0.02 M
NH4H2PO4 and acetonitrile (20 : 1, v/v). The DNA–DNA
hybridization experiment was carried out in microplate
wells, as described by Ezaki et al. (1989). Hybridization was
performed at 43 uC for 16 h. The DNA G+C contents of
P. crevioricanis JCM 15906T and P. cansulci JCM 13913T
were 45.9 and 45.6 mol%, respectively. The value for P.
crevioricanis JCM 15906T was slightly higher than the
original data (44 to 45 mol% by HPLC method) (Hirasawa
& Takada, 1994). On the other hand, the value for P.
cansulci JCM 13913T was slightly lower than the original
data (49 to 51 mol% by the thermal denaturation method)
(Collins et al., 1994). The DNA–DNA relatedness values
between P. crevioricanis JCM 15906T and P. cansulci JCM
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455
M. Sakamoto and M. Ohkuma
(b)
(a)
P. endodontalis JCM 8526T (AB547659)
100
0.01
0.02
P. gingivicanis JCM 15907T (AB547662)
100
100
P. levii JCM 13866T (AB547585)
81
P. circumdentaria JCM 13864T (AB547657)
P. uenonis JCM
13868T
P. uenonis JCM 13868T (AB547589)
100
(AB547668)
P. asaccharolytica JCM 6326T
(AB547573)
P. canoris JCM 16132T (AB547575)
100
P. asaccharolytica JCM 6326T
(AB547653)
P. salivosa JCM 15984T (AB547666)
75
100
P. macacae JCM
13914T
71
71
P. cangingivalis JCM 15983T (AB547574)
(AB547665)
P. catoniae JCM 13863T (AB547577)
P. canoris JCM 16132T (AB525416)
P. endodontalis JCM 8526T (AB547580)
P. cangingivalis JCM 15983T (AB547654)
95
96
P. bennonis JCM 16335T (AB588020)
97
P. gingivicanis JCM 15907T (AB547583)
100
P. circumdentaria JCM 13864T (AB547578)
P. somerae JCM 13867T (AB547667)
100
77
P. crevioricanis JCM 15906T (AB547579)
79
P. levii JCM 13866T (AB547664)
100 P. cansulci JCM 13913T (AB547576)
P. crevioricanis JCM 15906T (AB547658)
100
P. gulae JCM 13865T (AB547584)
P. cansulci JCM 13913T (AB547655)
100
P. gulae JCM 13865T (AB547663)
100
P. somerae JCM 13867T (AB547588)
100
P. gingivalis JCM 12257T (AB547581)
P. salivosa JCM 15984T (AB547587)
P. gingivalis JCM 12257T (AB547660)
100
P. catoniae JCM 13863T (AB547656)
P. macacae JCM 13914T (AB547586)
Tannerella forsythia JCM 10827T (AB547635)
Tannerella forsythia JCM 10827T (AB547708)
Fig. 1. Phylogenetic trees based on the 16S rRNA gene (a) and hsp60 (b) sequences showing the relationships between P.
crevioricanis and P. cansulci. The tree was constructed by the neighbour-joining method. Numbers at nodes indicate the
percentage bootstrap values of 1000 replicates. Bars, 0.01 (a) or 0.02 (b) substitutions per nucleotide position.
13913T are shown in Table 2 and demonstrate that these
both strains represent a single species. Consequently, P.
cansulci Collins et al. 1994 is a later heterotypic synonym of
P. crevioricanis Hirasawa and Takada 1994.
(6.7–13.4 %) are present. The DNA G+C content is in the
range 44–45.9 mol% (by HPLC method).
Emended description of Porphyromonas
crevioricanis Hirasawa & Takada 1994
We thank Ms. Natsuko Suzuki and Ms. Misako Matsuda for their
technical assistance. This work was supported by a research grant
(2009–2011) of IFO (Institute for Fermentation, Osaka, Japan), and
also by a Grant-in-Aid for Scientific Research from the Japan Society
for the Promotion of Science (Grant No. 23580126) to M. S.
The description is as given by Hirasawa & Takada (1994)
and Collins et al. (1994) with the following modification:
Catalase activity is variable. The predominant respiratory
quinones are MK-9 (30.7–33.9 %) and MK-10 (49.6–
59.6 %); minor amounts of MK-7 (3.0–3.1 %) and MK-8
Table 2. DNA G+C content and DNA–DNA relatedness
values
Strain
1. P. crevioricanis
JCM 15906T
2. P. cansulci
JCM 13913T
456
DNA G+C
content (mol%)
DNA–DNA
relatedness
(%) with:
Acknowledgements
References
Collins, M. D., Love, D. N., Karjalainen, J., Kanervo, A., Forsblom, B.,
Willems, A., Stubbs, S., Sarkiala, E., Bailey, G. D. & other authors
(1994). Phylogenetic analysis of members of the genus Porphyromonas
and description of Porphyromonas cangingivalis sp. nov. and
Porphyromonas cansulci sp. nov. Int J Syst Bacteriol 44, 674–679.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
1
2
deoxyribonucleic acid–deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in
which radioisotopes are used to determine genetic relatedness among
bacterial strains. Int J Syst Bacteriol 39, 224–229.
45.9
100
91
Hirasawa, M. & Takada, K. (1994). Porphyromonas gingivicanis sp.
45.6
99
100
nov. and Porphyromonas crevioricanis sp. nov., isolated from beagles.
Int J Syst Bacteriol 44, 637–640.
Kitahara, M., Tsuchida, S., Kawasumi, K., Amao, H., Sakamoto, M.,
Benno, Y. & Ohkuma, M. (2011). Bacteroides chinchillae sp. nov. and
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 63
IP: 88.99.165.207
On: Fri, 16 Jun 2017 15:28:07
P. cansulci is a synonym of P. crevioricanis
Bacteroides rodentium sp. nov., isolated from chinchilla (Chinchilla
lanigera) faeces. Int J Syst Evol Microbiol 61, 877–881.
Sakamoto, M. & Ohkuma, M. (2011). Identification and classification
Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in
of the genus Bacteroides by multilocus sequence analysis. Microbiology
157, 3388–3397.
bacterial systematics. Methods Microbiol 19, 161–207.
Sakamoto, M. & Ohkuma, M. (2012). Bacteroides sartorii is an earlier
Kuykendall, L. D., Roy, M. A., O’neill, J. J. & Devine, T. E. (1988). Fatty
heterotypic synonym of Bacteroides chinchillae and has priority. Int J
Syst Evol Microbiol 62, 1241–1244.
acids, antibiotic resistance, and deoxyribonucleic acid homology
groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 38, 358–
361.
Sakamoto, M., Suzuki, N. & Benno, Y. (2010). hsp60 and 16S rRNA
of bacterial whole-cell fatty acid methyl esters, including hydroxy
acids. J Clin Microbiol 16, 584–586.
gene sequence relationships among species of the genus Bacteroides
with the finding that Bacteroides suis and Bacteroides tectus are
heterotypic synonyms of Bacteroides pyogenes. Int J Syst Evol Microbiol
60, 2984–2990.
Sakamoto, M. & Ohkuma, M. (2010). Usefulness of the hsp60 gene for
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
the identification and classification of Gram-negative anaerobic rods.
J Med Microbiol 59, 1293–1302.
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Miller, L. T. (1982). Single derivatization method for routine analysis
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
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