International Journal of Systematic and Evolutionary Microbiology (2007), 57, 2874–2880
DOI 10.1099/ijs.0.65286-0
Pelagicoccus croceus sp. nov., a novel marine
member of the family Puniceicoccaceae within the
phylum ‘Verrucomicrobia’ isolated from seagrass
Jaewoo Yoon,1 Naoya Oku,2 Satoru Matsuda,2 Hiroaki Kasai2
and Akira Yokota1
Correspondence
1
Jaewoo Yoon
[email protected]
2
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku,
Tokyo 113-0032, Japan
Marine Biotechnology Institute Co. Ltd, 3-75-1, Heita, Kamaishi, Iwate 026-0001, Japan
An obligately aerobic, spherical, non-motile, pale-yellow pigmented bacterium was isolated from a
piece of leaf of seagrass, Enhalus acoroides (L.f.) Royle, grown in Okinawa, Japan and was
subjected to a polyphasic taxonomic study. Phylogenetic analyses based on 16S rRNA gene
sequences revealed that the novel isolate N5FB36-5T shared approximately 96–98 % sequence
similarity with the species of the genus Pelagicoccus of the family Puniceicoccaceae within the
phylum ‘Verrucomicrobia’. The DNA–DNA relatedness values of strain N5FB36-5T with
Pelagicoccus mobilis 02PA-Ca-133T and Pelagicoccus albus YM14-201T were below 70 %,
which is accepted as the phylogenetic definition of a novel species. b-Lactam antibiotic
susceptibility test and amino acid analysis of the cell wall hydrolysates indicated the absence of
muramic acid and diaminopimelic acid in the cell walls, which suggested that this strain lacks an
ordinary Gram-negative type of peptidoglycan in the cell wall. The DNA G+C content of
strain N5FB36-5T was 51.6 mol%; MK-7 was the major menaquinone; and the presence of
C16 : 0, C16 : 1v7c and anteiso-C15 : 0 as the major cellular fatty acids supported the identification
of the novel isolate as a member of the genus Pelagicoccus. On the basis of polyphasic taxonomic
data, it was concluded that this strain should be classified as a novel species of the genus
Pelagicoccus, for which the name Pelagicoccus croceus sp. nov. is proposed. The type strain is
N5FB36-5T (5MBIC08282T5KCTC 12903T).
The phylum ‘Verrucomicrobia’ (Hedlund et al., 1997;
Hugenholtz et al., 1998) represents a well defined, major
lineage within the domain Bacteria. A lot of cultureindependent studies based on the 16S rRNA gene
sequences revealed that this phylogenetic group is ubiquitous in nature (Hugenholtz et al., 1998; O’Farrell & Janssen,
1999; Joseph et al., 2003; Rappé & Giovannoni, 2003;
Kanokratana et al., 2004; Haukka et al., 2005; Dedysh et al.,
2006; Haukka et al., 2006). At present, it has been
informally classified into five subdivisions numbered 1 to
5 (Hugenholtz et al., 1998) and has also been classified into
six subdivisions numbered 1 to 6 (Vandekerckhove et al.,
2000). Among them, subdivision 1 is equivalent to the
family Verrucomicrobiaceae as described in the second
edition of Bergey’s Manual of Systematic Bacteriology
(Garrity & Holt, 2001). In addition, recently, the class
Opitutae, which is composed of two orders; the order
Puniceicoccales containing the family Puniceicoccaceae and
the order Opitutales containing the family Opitutaceae was
The GenBank/EMBL/DDBJ accession number for 16S rRNA gene
sequence of strain N5FB36-5T is AB297922.
2874
recently proposed for classification of species belonging to
subdivision 4 (Choo et al., 2007). Although verrucomicrobial 16S rRNA gene sequences have been identified from
marine animals and plants (Weidner et al., 2000; Alain
et al., 2002; Bowman & Nowak, 2004), only a few microorganisms isolated from marine organisms within subdivisions 1 and 4 have been cultivated and validly reported
(Scheuermayer et al., 2006; Choo et al., 2007; Kasai et al.,
2007). However, owing to the problem of uncultivability,
there are still only a few cultivated representatives so far
and their ecological niche also remains to be elucidated.
As part of our programme to construct a comprehensive
culture collection library of phylogenetically or functionally
novel bacteria and fungi (http://www.nedo.go.jp/activities/
portal/p02038.html), we have developed the in situ culture
technique for the collection of yet-uncultured microbes
from coastal waters (Yasumoto-Hirose et al., 2006). This
new approach enabled us to discover a clustered set of
novel marine verrucomicrobial species belonging to the
class Opitutae (subdivision 4), for which we proposed a
new genus Pelagicoccus (Yoon et al., 2007b). As another
Downloaded from www.microbiologyresearch.org by
65286 G 2007 IUMS
IP: 88.99.165.207
On: Thu, 15 Jun 2017 04:30:29
Printed in Great Britain
Pelagicoccus croceus sp. nov.
attempt towards the same mission, we designed a lectinsupplemented medium in the aim to collect bacteria in
symbiotic associations with eukaryotes, which is based on
the accumulating evidence that lectins are involved in
many host–symbiont associations (Hirsch, 1999; Koike
et al., 2004; McCowen et al., 1986; Venkataraman et al.,
1997). Applications of this medium to the marine plant
Enhalus acoroides (L.f.) Royle resulted in isolation of an
additional novel species of the genus Pelagicoccus.
Strain N5FB36-5T was isolated in October 2005 from a
piece of leaf of E. acoroides grown in the Kuira River
mangrove estuary, Iriomotejima, Okinawa, Japan (GPS
location; 24u 29.249 N, 123u 44.459 E). Isolation and
enrichment of strain N5FB36-5T was performed on 1/10
strength marine agar 2216 (Difco) supplemented with
10 mg jack bean lectin concanavalin A (Wako) ml21. To
prepare this medium, 3.74 g marine broth 2216, 15 g agar,
250 ml artificial seawater (Lyman & Fleming, 1940) and
750 ml distilled water were mixed and autoclaved, after
which a solution of 2.5 ml concanavalin A prepared at a
concentration of 4 mg ml21 in sterile distilled water was
added, before the agar solidified. A piece of the freshly
collected E. acoroides leaf (approx. 1 cm2) was vigorously
crushed in 5 ml sterile artificial seawater with a glass rod
and allowed to stand so that the solid particles could settle.
The supernatant was further diluted to 1021 dilution using
sterile artificial seawater, of which 50 ml aliquot was spread
on a 1/10 strength marine agar 2216 plate with concanavalin A. The enrichment agar medium was incubated
for 2 months at room temperature. The resulting translucent colony was transferred twice on the same medium to
purify the isolate and was used in this study. In the present
study, we attempted to elucidate the phylogenetic position
of strain N5FB36-5T using a polyphasic taxonomic
approach, including 16S rRNA gene sequence analysis. In
parallel, we performed physiological, biochemical and
chemotaxonomic analyses to characterize the novel isolate.
Based on these data, it is proposed that the isolate
represents a novel species within the phylum
‘Verrucomicrobia’.
The temperature and pH range for growth were determined by incubating the isolates on the 1/5 strength
marine agar 2216. The NaCl concentration for growth was
determined in a salt tolerance test medium containing 1 %
tryptone, 0.3 % yeast extract, 0.9 % MgCl2 . 6H2O, 0.9 %
MgSO4 . 7H2O, 0.2 % CaCl2 . 2H2O, 0.06 % KCl and 1.5 %
agar with 0–10 % (w/v) NaCl. Gram-staining was performed as described by Murray et al. (1994). Cell
morphology was observed using light microscopy (BX60;
Olympus). The cells of strain N5FB36-5T on 1/5 strength
marine agar 2216 were coccoid shaped ranging from 0.5 to
1.0 mm in diameter. Motility by flagellum and gliding
movement were not seen. Cell divisions by binary fission
were observed. Growth under anaerobic conditions was
determined after 2 weeks of incubation in an AnaeroPack
(Mitsubishi Gas Chemical) on 1/5 strength marine agar
2216. Catalase activity was determined by bubble formation
http://ijs.sgmjournals.org
in a 3 % H2O2 solution. Oxidase activity was determined by
cytochrome oxidase paper (Nissui Pharmaceutical). API
20E, API 50CH and API ZYM strips (bioMérieux) were used
to determine physiological and biochemical characteristics.
API 20E, API 50CH and API ZYM were read after 72 h
incubation at 30 uC and 4 h incubation at 37 uC, respectively. Determination of the respiratory quinone system and
cellular fatty acid composition was carried out as described
previously (Katsuta et al., 2005). DNA was prepared
according to the method of Marmur (1961) from cells
grown on 1/5 strength marine agar 2216 and the DNA base
composition was determined by using the HPLC method of
Mesbah et al. (1989). DNA–DNA hybridizations were
carried out with photobiotin-labelled probes in microplate
wells as described by Ezaki et al. (1989). The hybridization
temperature was set at 48 uC. Hybridization was performed
using five replications for each. Of the values obtained, the
highest and lowest for each sample were excluded and the
mean of the remaining three values is quoted as the DNA–
DNA relatedness value. b-Lactam antibiotic susceptibility
test against the novel isolate was checked on 1/5 strength
marine agar 2216, using 8 mm paper disc (Advantec) at the
following antibiotic concentrations: 1, 10, 100, 500 and
1000 mg ml21 ampicillin and 1, 10, 100, 500 and 1000 mg
ml21 penicillin G. Cell walls were prepared by the methods
described by Schleifer & Kandler (1972), and amino acids in
an acid hydrolysate of the cell walls were identified by TLC
(Harper & Davis, 1979) and HPLC, as their phenylthiocarbamoyl derivatives, with a model LC-10AD HPLC
apparatus (Shimazu) equipped with a Wakopak WS-PTC
column (Wako Pure Chemical Industries) (Yokota et al.,
1993). An approximately 1500 bp fragment of the 16S rRNA
gene was amplified from the extracted DNA by using
bacterial universal primers specific to the 16S rRNA gene:
27F and 1492R (Escherichia coli numbering system;
Weisburg et al., 1991). To ascertain the phylogenetic
position of the novel isolate, the 16S rRNA gene sequence
of strain N5FB36-5T was compared with the sequences
obtained from GenBank (National Center for Biotechnology
Information, http://www.ncbi.nlm.nih.gov). Multiple alignments of the sequences were performed using CLUSTAL_X
(version 1.83) (Thompson et al., 1997). Alignment gaps and
ambiguous bases were not taken into consideration when
the 1187 bases of the 16S rRNA gene nucleotides were
compared. The phylogenetic relationships were determined
by using the maximum-likelihood method (Felsenstein,
1985) and the Ratchet model (Sikes & Lewis, 2001) of
evolution in PAUP* 4.0b10 (Swofford, 2002). Bootstrap
analysis was performed by using 1000 trial replications to
provide confidence estimates for tree topologies. The
similarity values were calculated using the MEGA 3.1 software
(Kumar et al., 2004).
An evolutionary tree based on the maximum-likelihood
method generated a comparison of the 16S rRNA gene
sequences and revealed that strain N5FB36-5T was
phylogenetically affiliated with the Pelagicoccus species of
the family Puniceicoccaceae belonging to the order
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 04:30:29
2875
J. Yoon and others
Puniceicoccales within the phylum ‘Verrucomicrobia’ with a
bootstrap confidence value of 100 % (Fig. 1). Comparative
analysis of the 16S rRNA gene sequences revealed that the
sequence of strain N5FB36-5T had a similarity of 98.7 % to
that of Pelagicoccus mobilis 02PA-Ca-133T, 97.9 % to
Pelagicoccus albus YM14-201T and 96.8 % to the three
strains (H-MN57T, H-MN48 and MN1-156) of
Pelagicoccus litoralis. All other cultivated species of the
class Opitutae (subdivision 4) with currently published
names were more distantly related, showing a 16S rRNA
gene sequence similarity of less than 90 %.
DNA–DNA hybridization values between strain N5FB365T and Pelagicoccus mobilis 02PA-Ca-133T and Pelagicoccus
albus YM14-201T were 1.4 and 2.2 %, respectively. These
results strongly suggest that strain N5FB36-5T should be
classified as a separate species, if it can be distinguished by
phenotypic traits (Wayne et al., 1987).
As shown in Table 1, the predominant cellular fatty acids of
five novel strains were C15 : 0 (21.2 %), C16 : 0 (20.7 %),
C16 : 1v7c (12.7 %) and anteiso-C15 : 0 (25.4 %), which are
similar to other members of the genus Pelagicoccus. On the
other hand, strain N5FB36-5T is distinguished from the
other species of the genus Pelagicoccus by a different
proportion of C15 : 0. Furthermore, on the basis of their
fatty acid composition, this strain is differentiated
from Coraliomargarita akajimensis 04OKA010-24T, Cerasicoccus arenae YM26-026T and Puniceicoccus vermicola
IMCC1545T, their phylogenetically neighbouring taxa,
indicating that strain N5FB36-5T probably represents an
independent species of the genus Pelagicoccus of the family
Puniceicoccaceae within the phylum ‘Verrucomicrobia’.
When the isolate was grown in the presence of increasing
concentrations (1–1000 mg ml21) of b-lactam antibiotics,
it showed a remarkable resistance to ampicillin and
Fig. 1. Maximum-likelihood phylogenetic tree showing the position of strain N5FB36-5T. Numbers at branch nodes are
bootstrap values based on 1000 replications. The sequence determined in this study is shown in bold. The sequence of
Escherichia coli ATCC 11775T was used as the outgroup reference. Subdivisions of the phylum ‘Verrucomicrobia’ are shown
as SD 1–6. Bar, 0.1 substitutions per site.
2876
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 57
IP: 88.99.165.207
On: Thu, 15 Jun 2017 04:30:29
Pelagicoccus croceus sp. nov.
Table 1. Cellular fatty acid content (%) of Pelagicoccus
croceus sp. nov. and the related taxa of the family
Puniceicoccaceae
Taxa: 1, strain N5FB36-5T; 2, Pelagicoccus mobilis 02PA-Ca-133T (data
from Yoon et al., 2007b); 3, Pelagicoccus albus YM14-201T (Yoon et
al., 2007b); 4, Pelagicoccus litoralis H-MN57T (Yoon et al., 2007b); 5,
Coraliomargarita akajimensis 04OKA010-24T (Yoon et al., 2007a); 6,
Cerasicoccus arenae YM26-026T (Yoon et al., 2007c); 7, Puniceicoccus
vermicola IMCC1545T (Choo et al., 2007). Data are expressed as
percentages of total fatty acids. Fatty acids representing less than 1 %
are not shown. –, Not detected; ND, not described; tr, trace.
Fatty acid
1
2
3
4
5
6
7
1.3
21.2
20.7
6.8
tr
5.8
5.9
23.3
2.1
tr
4
1.7
23.8
tr
1.2
4.4
4.5
14.3
1.3
1.4
24.2
1.7
3.3
1.5
15.6
38.7 4.9
tr
ND
2.3 7.9
–
7
1.5 24.7
2.6
12.7
1.5
1
15.1
–
tr
14.5
–
1.2
20.7
–
–
tr
23.5
–
–
43.3
tr
1.8
25.4
1.5
2.1
2.3
29.8
tr
tr
2.3
37.5
3.9
1.1
1.7
38.1
1.4
8.2
1.8
3
tr
3.5 5.3
–
ND
tr 30.9
–
3.6
–
tr
–
–
tr
tr
5.5
tr
1.4
5.7
tr
tr
–
1.3
tr
–
–
1.3
Based on the results of the phylogenetic analysis and its
biochemical and physiological properties, strain N5FB365T isolated from a piece of leaf of seagrass E. acoroides
should be classified as a novel species of the genus
Pelagicoccus of the family Puniceicoccaceae within the
phylum ‘Verrucomicrobia’, for which the name
Pelagicoccus croceus sp. nov. is proposed.
Description of Pelagicoccus croceus sp. nov.
Pelagicoccus croceus (cro.ce9us. L. masc. adj. croceus,
saffron-coloured, yellow, golden, referring to the paleyellow colour of colonies).
penicillin G. The cell wall was prepared by disrupting cells,
followed by heating with 3 % SDS, washing and centrifugation. Amino acid analysis of the cell wall hydrolysate
indicated the absence of muramic acid and diaminopimelic
acid in the cell wall, suggesting that the strain does not
contain an ordinary Gram-negative type of peptidoglycan
in the cell walls. This taxonomic trait, consistently observed
in all verrucomicrobial species within the class Opitutae
(subdivision 4), is relevant to parasitic bacteria such as
mycoplasma and chlamydia. Although the natural habitat
of Pelagicoccus has not been specified yet, it is quite possible
that they are symbionts of certain marine eukaryotic hosts
in at least some parts of their life history. This speculation
is supported by the recent reports that both maternally
inherited cytoplasmic endosymbionts in nematodes of the
genus Xiphinema and defensive extrusive ectosymbionts on
hypotrich ciliates were identified as novel groups of the
phylum ‘Verrucomicrobia’ (Petroni et al., 2000;
Vandekerckhove et al., 2000).
Cells are obligately aerobic, non-motile, cocci 0.5–1.0 mm
in diameter. Neither cellular gliding movement nor
swarming growth is observed. Colonies grown on 1/5
strength marine agar 2216 are circular, convex and paleyellow. The temperature range for growth is 20–30 uC,
optimally at 25–30 uC, but no growth occurs at 4 or 45 uC.
The pH range for growth is 6.5–9.0. NaCl is required for
growth and cells can tolerate up to 5 % (w/v). Growth
occurs in the presence of ampicillin (1–1000 mg ml21) and
penicillin G (1–1000 mg ml21). Catalase- and oxidasepositive. Nitrate is not reduced. Aesculin is hydrolysed but
agar, DNA, starch, gelatin and urea are not. The reaction
for ONPG is positive, but acetoin, tryptophan deaminase,
citrate utilization, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, hydrogen sulfide and indole
production are negative. Acid is produced from methyl bD-xylopyranoside, methyl a-D-mannnopyranoside, aesculin
ferric citrate, lactose, melibiose, D-turanose, D-lyxose, Dtagatose and 5-ketogluconate, but not from sucrose,
glycerol, galactose, fructose, mannose, mannitol, sorbitol,
trehalose, D-fucose, L-fucose, D-arabitol, L-arabitol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose,
adonitol, glucose, sorbose, rhamnose, dulcitol, inositol,
methyl a-D-glucopyranoside, N-acetyl-D-glucosamine,
amygdalin, arbutin, salicin, cellobiose, maltose, inulin,
melezitose, raffinose, starch, glycogen, xylitol, gentiobiose,
gluconate or 2-ketogluconate. Alkaline phosphatase, leucine arylamidase and acid phosphatase are positive, but
b-galactosidase, naphthol-AS-BI-phosphohydrolase, agalactosidase, a-glucosidase, valine arylamidase, trypsin,
esterase (C4), esterase lipase (C8), lipase (C4), cystine
arylamidase, chymotrypsin, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase are negative. The usual components of bacterial
cell walls such as muramic acid and diaminopimelic acid
could not be detected. Major fatty acid components
(.1.0 %) include C14 : 0 (1.3 %), C15 : 0 (21.2 %), C16 : 0
(20.7 %), C17 : 0 (6.8 %), C15 : 1v6c (2.6 %), C16 : 1v7c
(12.7 %), C18 : 1v9c (1.5 %), iso-C16 : 0 (1.8 %), anteisoC15 : 0 (25.4 %) and anteiso-C17 : 0 (1.5 %). The G+C
content of DNA is 51.6 mol%.
Strain N5FB36-5T also showed distinct phenotypic features
that discriminated it from the cultivated members of the
class Opitutae given in Table 2.
The type strain, N5FB36-5T (5MBIC08282T5KCTC
12903T), was isolated from the leaf surface of seagrass E.
acoroides (L.f.) Royle.
Saturated
C14 : 0
C15 : 0
C16 : 0
C17 : 0
C18 : 0
Unsaturated
C15 : 1v6c
C16 : 1v7c
C18 : 1v9c
Branched
iso-C14 : 0
iso-C16 : 0
anteiso-C15 : 0
anteiso-C17 : 0
Hydroxy
C13 : 0 2-OH
C12 : 0 3-OH
C16 : 0 3-OH
http://ijs.sgmjournals.org
ND
ND
ND
ND
2.1
ND
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 04:30:29
2877
J. Yoon and others
Table 2. Differential phenotypic characteristics of Pelagicoccus croceus sp. nov. and the related genera of the class Opitutae within
the phylum ‘Verrucomicrobia’
Taxa: 1, strain N5FB36-5T; 2, Pelagicoccus mobilis 02PA-Ca-133T (data from Yoon et al., 2007b); 3, Pelagicoccus albus YM14-201T (Yoon et al.,
2007b); 4, Pelagicoccus litoralis H-MN57T (Yoon et al., 2007b); 5, Coraliomargarita akajimensis 04OKA010-24T (Yoon et al., 2007a); 6, Cerasicoccus
arenae YM26-026T (Yoon et al., 2007c); 7, Puniceicoccus vermicola IMCC1545T (Choo et al., 2007); 8, Alterococcus agarolyticus BCRC 19135T (Shieh
& Jean, 1998); 9, Opitutus terrae DSM 11246T (Chin et al., 2001). +, Positive; W, weakly positive; 2, negative; ND, no data.
Characteristic
Isolation source
Cell diameter (mm)
Colony colour
Flagella
Appendage
Motility
Anaerobic growth
Production of:
Catalase
Oxidase
ONPG
Nitrate reduction
Temperature range (uC)
Growth at 4 uC
Growth at 37 uC
pH range
NaCl range (%)
Hydrolysis of:
Agar
DNA
Starch
Urea
Aesculin
Acid production from:
D-Arabinose
Cellobiose
Galactose
Glucose
Lactose
Mannitol
Mannose
Melibiose
Enzyme activity of:
Acid phosphatase
a-Galactosidase
a-Glucosidase
N-Acetyl-bglucosaminidase
Antibiotic resistance
(mg ml21) to:
Ampicillin (1–1000)
Penicillin G (1–1000)
Cell wall component of:
Muramic acid
meso-Diaminopimelic
acid
DNA G+C content
(mol%)
Major quinone
2878
1
2
3
4
5
6
7
8
9
Seagrass
Seawater
Seawater
Seawater
Seawater
0.5–1.0
Pale-yellow
2
2
2
2
0.5–0.7
White
+
+
+
2
0.8–1.2
White
2
2
2
+
1.0–1.2
White
2
2
2
2
0.5–1.2
White
2
2
2
2
Marine
sand
0.8–1.0
Pale-pink
2
2
2
2
Sea
polychaete
0.6–1.0
Pale-red
2
2
2
+
Hot
springs
0.8–0.9
White
+
2
+
+
Rice paddy
soil
0.4–0.6
Unpigmented
+
2
+
2
+
+
+
2
20–30
2
2
6.5–9.0
1.0–5.0
2
+
2
2
20–37
2
+
7.0–9.0
1.0–4.0
+
+
+
2
15–37
2
+
6.5–9.0
3.0–7.0
+
+
+
2
4–30
+
2
6.5–9.0
1.0–4.0
2
+
+
2
20–30
2
2
7.0–9.0
1.0–5.0
+
+
+
2
10–30
2
2
6.0–9.0
0.0–8.0
2
2
2
2
8–37
2
+
5.0–12.0
1.0–7.5
+
+
2
2
ND
ND
2
2
2
2
+
+
+
+
+
+
2
+
2
2
+
2
2
2
2
+
2
2
2
+
2
+
2
2
ND
2
2
ND
+
2
ND
ND
2
2
2
2
w
2
2
w
2
+
2
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
2
+
+
2
2
2
+
+
+
2
+
2
2
+
+
+
+
2
2
2
2
+
+
+
+
2
w
2
+
+
+
+
+
+
+
+
+
2
2
2
+
2
2
+
+
+
+
2
2
2
2
2
+
2
2
2
2
2
2
2
+
2
2
2
ND
ND
ND
ND
ND
ND
ND
ND
+
+
+
+
+
+
+
+
+
+
+
+
+
+
2
2
ND
2
2
2
2
2
2
2
2
2
2
2
2
2
2
ND
ND
ND
ND
51.6
57.4
57.2
56.4
53.9
54
52.1±0.5
65.5–67.0
74
MK-7
MK-7
MK-7
MK-7
MK-7
MK-7
MK-7
ND
ND
W
2
+
2
2
2
W
ND
38–58
ND
ND
7.0–8.5
2.0–2.5
+
2
w
+
10–37
2
+
5.5–9.0
0.0–3.0
ND
ND
ND
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 57
IP: 88.99.165.207
On: Thu, 15 Jun 2017 04:30:29
Pelagicoccus croceus sp. nov.
Acknowledgements
We thank Sachiko Kawasaki, Atsuko Katsuta, Ayako Matsuzaki,
Tomomi Haga and Yukiko Itazawa for their technical assistance. This
work was supported by the New Energy and Industrial Technology
Development Organization (NEDO).
previously uncultured soil bacteria. Appl Environ Microbiol 69,
7210–7215.
Kanokratana, P., Chanapan, S., Pootanakit, K. & Eurwilaichitr, L.
(2004). Diversity and abundance of Bacteria and Archaea in the Bor
Khlueng hot spring in Thailand. J Basic Microbiol 44, 430–444.
Kasai, H., Katsuta, A., Sekiguchi, H., Matsuda, S., Adachi, K.,
Kazutoshi, S., Yoon, J., Yokota, A. & Shizuri, Y. (2007). Rubritalea
References
Alain, K., Olagnon, M., Desbruyères, D., Pagé, A., Barbier, G.,
Juniper, S. K., Quérellou, J. & Cambon-Bonavita, M.-A. (2002).
Phylogenetic characterization of the bacterial assemblage associated
with mucous secretions of the hydrothermal vent polychaete
Paralvinella palmiformis. FEMS Microbiol Ecol 42, 463–476.
Bowman, J. P. & Nowak, B. (2004). Salmonid gill bacteria and their
relationship to amoebic gill disease. J Fish Dis 27, 483–492.
Chin, K.-J., Liesack, W. & Janssen, P. H. (2001). Opitutus terrae gen.
nov., sp. nov., to accommodate novel strain of the division
‘Verrucomicrobia’ isolated from rice paddy soil. Int J Syst Evol
Microbiol 51, 1965–1968.
Choo, Y.-J., Lee, K., Song, J. & Cho, J.-C. (2007). Puniceicoccus
vermicola gen. nov., sp. nov., a new marine bacterium and description
of Puniceicoccaceae fam. nov., Puniceicoccales ord. nov., Opitutaceae
fam. nov., Opitutales ord. nov., and Opitutae classis nov. in the
phylum ‘Verrucomicrobia’. Int J Syst Evol Microbiol 57, 532–537.
squalenifaciens sp. nov., a squalene-producing marine bacterium
belonging to subdivision 1 of the phylum ‘Verrucomicrobia’. Int J Syst
Evol Microbiol 57, 1630–1634.
Katsuta, A., Adachi, K., Matsuda, S., Shizuri, Y. & Kasai, H. (2005).
Ferrimonas marina sp. nov. Int J Syst Evol Microbiol 55, 1851–1855.
Koike, K., Jimbo, M., Sakai, R., Kaeriyama, M., Muramoto, K., Ogata, T.,
Maruyama, T. & Kamiya, H. (2004). Octocoral chemical signaling
selects and controls dinoflagellate symbionts. Biol Bull 207, 80–86.
MEGA3: integrated software
for molecular evolutionary genetics analysis and sequence alignment.
Brief Bioinform 5, 150–163.
Kumar, S., Tamura, K. & Nei, M. (2004).
Lyman, J. & Fleming, R. H. (1940). Composition of sea water. J Mar
Res 3, 134–146.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic
acid from micro-organisms. J Mol Biol 3, 208–218.
McCowen, S. M., MacArthur, L. & Gates, J. E. (1986). Azolla fern
lectins that specifically recognize endosymbiotic cyanobacteria. Curr
Microbiol 14, 329–333.
Dedysh, S. N., Pankratov, T. A., Belova, S. E., Kulichevskaya, I. S. &
Liesack, W. (2006). Phylogenetic analysis and in situ identification of
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
bacteria community composition in an acidic Sphagnum peat bog.
Appl Environ Microbiol 72, 2110–2117.
measurement of the G+C content of deoxyribonucleic acid by
high-performance liquid chromatography. Int J Syst Bacteriol 39,
159–167.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
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.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994).
Determinative and cytological light microscopy. In Methods for
General and Molecular Bacteriology, pp. 21–41. Edited by P. Gerhardt,
R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC:
American Society for Microbiology.
using the bootstrap. Evolution 39, 783–791.
O’Farrell, K. A. & Janssen, P. H. (1999). Detection of Verrucomicrobia
Garrity, G. M. & Holt, J. G. (2001). The road map to the Manual. In
in a pasture soil by PCR-mediated amplification of 16S rRNA genes.
Appl Environ Microbiol 65, 4280–4284.
Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 119–
166. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity.
New York: Springer.
Harper, J. J. & Davis, G. H. G. (1979). Two-dimensional thin-layer
chromatography for amino acid analysis of bacterial cell walls. Int J
Syst Bacteriol 29, 56–58.
Haukka, K., Heikkinen, E., Kairesalo, T., Karjalainen, H. & Sivonen, K.
(2005). Effect of humic material on the bacterioplankton community
composition in boreal lakes and mesocosms. Environ Microbiol 7,
620–630.
Petroni, G., Spring, S., Schleifer, K.-H., Verni, F. & Rosati, G. (2000).
Defensive extrusive ectosymbionts of Euplotidium (Ciliophora) that
contain microtubule-like structures are bacteria related to
Verrucomicrobia. Proc Natl Acad Sci U S A 97, 1813–1817.
Rappé, M. S. & Giovannoni, S. J. (2003). The uncultured microbial
majority. Annu Rev Microbiol 57, 369–394.
Scheuermayer, M., Gulder, T. A., Bringmann, G. & Hentschel, U.
(2006). Rubritalea marina gen. nov., sp. nov., a marine representative
of the phylum ‘Verrucomicrobia’, isolated from a sponge (Porifera).
Int J Syst Evol Microbiol 56, 2119–2124.
Haukka, K., Kolmonen, E., Hyder, R., Hietala, J., Vakkilainen, K.,
Kairesalo, T., Haario, H. & Sivonen, K. (2006). Effect of nutrient
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of
loading on bacterioplankton community composition in lake
mesocosms. Microb Ecol 51, 137–146.
bacterial cell walls and their taxonomic implications. Bacteriol Rev
36, 407–477.
Hedlund, B. P., Gosink, J. J. & Staley, J. T. (1997). Verrucomicrobia
Shieh, W. Y. & Jean, W. D. (1998). Alterococcus agarolyticus, gen. nov.,
div. nov., a new division of the bacteria containing three new species
of Prothecobacter. Antonie Van Leeuwenhoek 72, 29–38.
sp. nov., a halophilic thermophilic bacterium capable of agar
degradation. Can J Microbiol 44, 637–645.
Hirsch, A. M. (1999). Role of lectins (and rhizobial exopolysacchar-
Sikes, D. S. & Lewis, P. O. (2001). Beta software, version 1. PAUPRat:
ides) in legume nodulation. Curr Opin Plant Biol 2, 320–326.
PAUP
Hugenholtz, P., Goebel, B. M. & Pace, N. R. (1998). Impact of
implementation of the parsimony ratchet. Department of
Ecology and Evolutionary Biology, University of Connecticut, Storrs.
PAUP*: Phylogenetic analysis using parsimony
(*and other methods). Sunderland, MA: Sinauer Associates.
culture-independent studies on the emerging phylogenetic view of
bacterial diversity. J Bacteriol 180, 4765–4774.
Swofford, D. L. (2002).
Joseph, S. J., Hugenholtz, P., Sangwan, P., Osborne, C. A. &
Janssen, P. H. (2003). Laboratory cultivation of widespread and
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &
Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 04:30:29
2879
J. Yoon and others
strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res 25, 4876–4882.
Yasumoto-Hirose, M., Nishijima, M., Ngirchechol, M. K., Kanoh, K.,
Shizuri, Y. & Miki, W. (2006). Isolation of marine bacteria by in situ
Vandekerckhove, T. T. M., Willems, A., Gillis, M. & Coomans, A.
(2000). Occurrence of novel verrucomicrobial species, endosymbiotic
culture on media-supplemented polyurethane foam. Mar Biotechnol
(NY) 8, 227–237.
and associated with parthenogenesis in Xiphinema americanum-group
species (Nematoda, Longidoridae). Int J Syst Evol Microbiol 50,
2197–2205.
Yokota, A., Tamura, T., Nishii, T. & Hasegawa, T. (1993). Kineococcus
Venkataraman, C., Haack, B. J., Bondada, S. & Kwaik, Y. A. (1997).
Identification of a Gal/GalNAc lectin in the protozoan Hartmannella
vermiformis as a potential receptor for attachment and invasion by the
legionnaires’ disease bacterium. J Exp Med 186, 537–547.
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O.,
Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other
authors (1987). International Committee on Systematic Bacteriology.
Report of the ad hoc committee on reconciliation of approaches to
bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Weidner, S., Arnold, W., Stackebrandt, E. & Pühler, A. (2000).
Phylogenetic analysis of bacterial communities associated with leaves
of the seagrass Halophila stipulacea by a culture-independent small
subunit rRNA gene approach. Microb Ecol 39, 22–31.
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S
ribosomal DNA amplification for phylogenetic study. J Bacteriol 173,
697–703.
2880
aurantiacus gen. nov., sp. nov., a new aerobic, gram-positive, motile
coccus with meso-diaminopimelic acid and arabinogalactan in the cell
wall. Int J Syst Bacteriol 43, 52–57.
Yoon, J., Yasumoto-Hirose, M., Katsuta, A., Sekiguchi, H., Matsuda, S.,
Kasai, H. & Yokota, A. (2007a). Coraliomargarita akajimensis gen. nov.,
sp. nov, a novel member of the phylum ‘Verrucomicrobia’ isolated from
seawater in Japan. Int J Syst Evol Microbiol 57, 959–963.
Yoon, J., Yasumoto-Hirose, M., Matsuo, Y., Nozawa, M., Matsuda, S.,
Kasai, H. & Yokota, A. (2007b). Pelagicoccus mobilis gen. nov., sp.
nov., Pelagicoccus albus sp. nov. and Pelagicoccus litoralis sp. nov.,
three novel members of subdivision 4 within the phylum
‘Verrucomicrobia’ isolated from seawater by in situ cultivation. Int J
Syst Evol Microbiol 57, 1377–1385.
Yoon, J., Matsuo, Y., Matsuda, S., Adachi, K., Kasai, H. & Yokota, A.
(2007c). Cerasicoccus arenae gen. nov., sp. nov., a carotenoid-
producing marine representative of the family Puniceicoccaceae within
the phylum ‘Verrucomicrobia’ isolated from marine sand. Int J Syst
Evol Microbiol 57, 2067–2072.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 57
IP: 88.99.165.207
On: Thu, 15 Jun 2017 04:30:29