Download Octadecabacter jejudonensis sp. nov., isolated from the junction

International Journal of Systematic and Evolutionary Microbiology (2014), 64, 719–724
DOI 10.1099/ijs.0.057513-0
Octadecabacter jejudonensis sp. nov., isolated
from the junction between the ocean and a
freshwater spring and emended description of the
genus Octadecabacter
Sooyeon Park and Jung-Hoon Yoon
Correspondence
Jung-Hoon Yoon
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon,
South Korea
[email protected]
A Gram-stain-negative, non-spore-forming, non-flagellated, rod-shaped bacterial strain, SSK21T, was isolated from the zone where the ocean and a freshwater spring meet at Jeju island, South
Korea, and subjected to a polyphasic taxonomic study. Strain SSK2-1T grew optimally at 30 6C,
at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Neighbour-joining, maximum-likelihood
and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that
strain SSK2-1T clustered with the type strains of two Octadecabacter species, showing 96.5–
96.8 % 16S rRNA gene sequence similarity. Strain SSK2-1T and Octadecabacter arcticus DSM
13978T contained Q-10 as the predominant ubiquinone and C18 : 1v7c as the common major
fatty acid. The polar lipid profile of strain SSK2-1T was similar to that of O. arcticus DSM 13978T
by having phosphatidylcholine, phosphatidylglycerol and one unidentified aminolipid as the major
components. The DNA G+C content of strain SSK2-1T was 60.1 mol%. Differential phenotypic
properties, particularly temperature range for growth, oxidase activity and nitrate reduction,
together with phylogenetic distinctiveness, revealed that strain SSK2-1T is separate from
recognized species of the genus Octadecabacter. On the basis of the data presented, strain
SSK2-1T is considered to represent a novel species of the genus Octadecabacter, for which the
name Octadecabacter jejudonensis sp. nov. is proposed. The type strain is SSK2-1T (5KCTC
32535T5CECT 8397T).
Soesokkak, located at Jeju island, South Korea, and
designated a Natural Environment Preservation Zone by
UNESCO, is a unique locality where the ocean and a
freshwater spring meet. During screening of bacteria from
this junction, many novel taxa have been isolated and
characterized taxonomically. One of these isolates, designated SSK2-1T, is described in this study, as it was found to
be phylogenetically related most closely to the genus
Octadecabacter, a member of the Alphaproteobacteria. The
genus Octadecabacter was proposed by Gosink et al. (1997,
1998) with the descriptions of two species, Octadecabacter
arcticus (the type species) and Octadecabacter antarcticus,
which were isolated from sea ice of the Arctic and
Antarctica, respectively. These are, at the time of writing,
the only recognized species of the genus. The aim of the
present work was to investigate if strain SSK2-1T represents
a third species of the genus Octadecabacter by using a
polyphasic characterization including chemotaxonomic
and other phenotypic analyses and a detailed phylogenetic
investigation based on 16S rRNA gene sequences.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain SSK2-1T is KF515220.
057513 G 2014 IUMS
Water was collected from the junction between the ocean
and a freshwater spring, called Soesokkak, and used as a
source for the isolation of bacterial strains. Strain SSK2-1T
was isolated by the standard dilution plating technique at
25 uC on marine agar 2216 (MA; Becton Dickinson) and
cultivated routinely on MA at 30 uC. O. arcticus DSM
13978T, which was used as a reference strain for fatty acid
and polar lipid analyses, was obtained from the Deutsche
Sammlung von Mikroorganismen und Zellkulturen
(DSMZ), Braunschweig, Germany. Cell morphology was
examined by light microscopy (BX51; Olympus) and
transmission electron microscopy (JEM1010; JEOL). The
latter technique was also used to assess the presence of
flagella on cells from an exponentially growing MA culture.
For this, the cells were negatively stained with 1 % (w/v)
phosphotungstic acid and the grids were examined after
being air-dried. The Gram reaction was determined by
using the bioMérieux Gram stain kit according to the
manufacturer’s instructions. Growth under anaerobic
conditions was determined after incubation for 10 days
in an anaerobic jar (MGC) with AnaeroPack (MGC) on
MA and on MA with potassium nitrate (0.1 %, w/v); the jar
was kept overnight at 4 uC to create anoxic conditions
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719
S. Park and J.-H. Yoon
before incubation at 30 uC. Growth at 4, 10, 20, 25, 30, 35,
37 and 40 uC was measured on MA to measure the optimal
temperature and temperature range for growth. The pH
range for growth was determined in marine broth 2216
(MB; Becton Dickinson) adjusted to pH 4.5–9.5 (using
increments of 0.5 pH units) by using sodium acetate/acetic
acid and Na2CO3 buffers. pH was verified after autoclaving.
Growth in the absence of NaCl and in the presence of 0.5,
1.0, 2.0 and 3.0 % (w/v) NaCl was investigated in trypticase
soy broth prepared according to the formula of the BD
medium except that NaCl was excluded and that 0.45 %
(w/v) MgCl2 . 6H2O was added. Growth in the presence of
2.0–10.0 % NaCl (at increments of 1.0 %) was investigated
in MB. Catalase and oxidase activities were determined as
described by Lányı́ (1987). Hydrolysis of casein, starch,
hypoxanthine, L-tyrosine and xanthine was tested on MA
using the substrate concentrations described by Barrow &
Feltham (1993). Hydrolysis of aesculin and Tweens 20, 40,
60 and 80 and nitrate reduction were investigated as
described by Lányı́ (1987) with the modification that
artificial seawater was used for the preparation of media.
Hydrolysis of gelatin and urea was investigated by using
nutrient gelatin and urea agar base media (Becton
Dickinson), respectively, with the modification that
artificial seawater was used for the preparation of media.
The artificial seawater contained (per litre distilled water)
23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g
MgSO4 . 7H2O and 1.3 g CaCl2 . 2H2O (Bruns et al.,
2001). Utilization of various substrates for growth was
tested according to Baumann & Baumann (1981), using
supplementation with 1 % (v/v) vitamin solution (Staley,
1968) and 2 % (v/v) Hutner’s mineral salts (Cohen-Bazire
et al., 1957). Acid production from carbohydrates was
tested as described by Leifson (1963). Susceptibility to
antibiotics was tested on MA plates using antibiotic discs
(Advantec) containing the following (mg per disc unless
otherwise stated): ampicillin (10), carbenicillin (100),
cephalothin (30), chloramphenicol (100), gentamicin
(30), kanamycin (30), lincomycin (15), neomycin (30),
novobiocin (5), oleandomycin (15), penicillin G (20 U),
polymyxin B (100 U), streptomycin (50) and tetracycline
(30). Enzyme activities were determined, after incubation
for 8 h at 30 uC, by using the API ZYM system
(bioMérieux).
Cell biomass of strain SSK2-1T for DNA extraction and for
the analyses of isoprenoid quinones and polar lipids was
obtained from cultures grown in MB for 5 days at 30 uC
and cell biomass of O. arcticus DSM 13978T for polar lipid
analysis was obtained from cultures grown in MB for
10 days at 10 uC. Chromosomal DNA was extracted and
purified according to the method described by Yoon et al.
(1996), with the modification that RNase T1 was used in
combination with RNase A to minimize contamination
of RNA. The 16S rRNA gene was amplified by PCR
as described previously (Yoon et al., 1998) using two
universal primers (59-GAGTTTGATCCTGGCTCAG-39
and 59-ACGGTTACCTTGTTACGACTT-39). Sequencing
720
of the amplified 16S rRNA gene was performed as
described by Yoon et al. (2003). Alignment of sequences
was carried out with CLUSTAL W software (Thompson et al.,
1994). Gaps at the 59 and 39 ends of the alignment were
omitted from further analysis. Phylogenetic analyses were
performed as described by Yoon et al. (2003).
Isoprenoid quinones were extracted and analysed as
described by Komagata & Suzuki (1987), using a
reversed-phase HPLC machine equipped with a YMC
ODS-A (25064.6 mm) column. The isoprenoid quinones
were eluted by a mixture of methanol/2-propanol (2 : 1, v/
v) using a flow rate of 1 ml min21 at room temperature
and detected by UV absorbance at 275 nm. For cellular
fatty acid analysis, cell mass of strain SSK2-1T and O.
arcticus DSM 13978T was harvested from MA plates after
cultivation for 5 days at 30 uC and for 10 days at 10 uC,
respectively. The physiological age of the cell masses was
standardized by observing the growth development of
colonies on the agar plates followed by harvesting them
from the same quadrant on the agar plates according to the
standard MIDI protocol (Sherlock Microbial Identification
System, version 6.1). Fatty acids were saponified, methylated and extracted using the standard protocol of the
MIDI (Sherlock Microbial Identification System, version
6.1). The fatty acids were analysed by GC (Hewlett Packard
6890) and identified by using the TSBA6 database of the
Microbial Identification System (Sasser, 1990). Polar lipids
were extracted according to the procedures described by
Minnikin et al. (1984) and separated by two-dimensional
TLC using chloroform/methanol/water (65 : 25 : 3.8, by
vol.) for the first dimension and chloroform/methanol/
acetic acid/water (40 : 7.5 : 6 : 1.8, by vol.) for the second
dimension as described by Minnikin et al. (1977).
Individual polar lipids were identified by spraying with
molybdophosphoric acid, molybdenum blue, ninhydrin
and a-naphthol reagents (Minnikin et al., 1984; Komagata
& Suzuki, 1987) and with Dragendorff’s reagent (Sigma).
The DNA G+C content was determined by the method of
Tamaoka & Komagata (1984) with the modification that
DNA was hydrolysed and the resultant nucleotides were
analysed with a reversed-phase HPLC machine equipped
with a YMC ODS-A (25064.6 mm) column. The nucleotides were eluted by a mixture of 0.55 M NH4H2PO4
(pH 4.0) and acetonitrile (40 : 1, v/v), using flow rate of
1 ml min21 at room temperature and detected by UV
absorbance at 270 nm.
Morphological, cultural, physiological and biochemical
characteristics of strain SSK2-1T are given in the species
description and in Table 1. The almost-complete 16S rRNA
gene sequence of strain SSK2-1T determined in this study
comprised 1384 nt, representing approximately 95 % of the
Escherichia coli 16S rRNA gene sequence. In the neighbourjoining phylogenetic tree based on 16S rRNA gene
sequences, strain SSK2-1T joined the cluster comprising
the type strains of O. arcticus and O. antarcticus by a
bootstrap resampling value of 99.3 % (Fig. 1). The relationships among strain SSK2-1T and the type strains of O.
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Octadecabacter jejudonensis sp. nov.
Table 1. Differential phenotypic characteristics between strain
SSK2-1T and the type strains of the two recognized species of
the genus Octadecabacter
Strains: 1, SSK2-1T; 2, O. arcticus 238T; 3, O. antarcticus 307T. Data of
reference strains are taken from Gosink et al. (1997). +, Positive
reaction; 2, negative reaction; W, weakly positive reaction. All strains
are positive for catalase activity, and utilization of D-glucose and
succinate (weakly positive for the two reference strains). All strains
are negative for anaerobic growth, Gram-staining, motility and
hydrolysis of gelatin.
Characteristic
Growth at:
4 and 10 uC
20 and 30 uC
Oxidase
Nitrate reduction
Hydrolysis of starch
Utilization of:
D-Fructose
Acetate
Citrate
Pyruvate
L-Glutamate
DNA G+C content
(mol%)
1
2
3
2
+
+
+
+
+
2
2
2
2
+
2
2
2
2
+
+
+
2
2
60.1
2
2
2
2
2
2
+
56
W
W
W
57
arcticus and O. antarcticus were also maintained in the trees
constructed using the maximum-likelihood and maximumparsimony algorithms (Fig. 1). Strain SSK2-1T exhibited 16S
rRNA gene sequence similarity values of 96.5 and 96.8 % to
O. arcticus 238T and O. antarcticus 307T, respectively, and of
less than 96.3 % to the type strains of the other recognized
species used in the analyses.
The predominant isoprenoid quinone detected in strain
SSK2-1T and O. arcticus DSM 13978T was ubiquinone-10
(Q-10), which is typical of the vast majority of the class
Alphaproteobacteria; minor amounts of Q-8 (approx. 1 %)
and Q-9 (approx. 2 %) were also present strain SSK2-1T.
The major fatty acids (.10 % of the total) found in strain
SSK2-1T were C18 : 1v7c (66.6 %) and 11-methyl C18 : 1v7c
(14.3 %). In Table 2, the fatty acid profile of strain SSK21T is compared with that of O. arcticus DSM 13978T
analysed under identical conditions. The fatty acid profile
of strain SSK2-1T was similar to that of O. arcticus DSM
13978T in that C18 : 1v7c is the predominant fatty acid and
a significant amount of 11-methyl C18 : 1v7c is present,
although there were differences in the proportions of
some fatty acids (Table 2). The major polar lipids found
in strain SSK2-1T were phosphatidylcholine, phosphatidylglycerol and one unidentified aminolipid (Fig. 2). In
Fig. 2, the polar lipid profile of strain SSK2-1T is compared
with that of O. arcticus DSM 13978T. The polar lipid
profile of strain SSK2-1T was similar to that of O. arcticus
DSM 13978T in that phosphatidylcholine, phosphatidylglycerol and one unidentified aminolipid are major
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polar lipids, but was distinguishable from that of O.
arcticus DSM 13978T in that one additional major polar
lipid (L4) is absent. The DNA G+C content of strain
SSK2-1T was 60.1 mol%, a value slightly higher than
those reported for species of the genus Octadecabacter
(Table 1).
From the results obtained from the chemotaxonomic
analysis and the phylogenetic analysis based on 16S rRNA
gene sequences, it is reasonable to classify strain SSK2-1T as
a member of the genus Octadecabacter (Figs 1 and 2; Table
1). Strain SSK2-1T was distinguishable from the type
strains of O. arcticus and O. antarcticus by differences in
some phenotypic characteristics, including temperature
range for growth, oxidase activity, nitrate reduction, starch
hydrolysis and utilization of several substrates (Table 1).
These differences, in combination with the phylogenetic
distinctiveness of strain SSK2-1T, suggest that the novel
strain is separate from the two recognized species of the
genus Octadecabacter (Stackebrandt & Goebel, 1994). On
the basis of the data presented, therefore, strain SSK2-1T is
considered to represent a novel species of the genus
Octadecabacter, for which the name Octadecabacter jejudonensis sp. nov. is proposed.
Emended description of the genus
Octadecabacter Gosink et al. 1997
The description of the genus Octadecabacter is as given by
Gosink et al. (1997) with the following amendments.
Oxidase activity is variable. Nitrate reduction is variable.
The predominant ubiquinone is Q-10. The major polar
lipids are phosphatidylcholine, phosphatidylglycerol and
one unidentified aminolipid. The major fatty acid is
C18 : 1v7c. The G+C content of the chromosomal DNA
is 56.0–60.1 mol%.
Description of Octadecabacter jejudonensis sp.
nov.
Octadecabacter jejudonensis (je.ju.do.nen9sis. N.L. masc.
adj. jejudonensis pertaining to Jeju island of South Korea,
from where the type strain was isolated).
Cells are Gram-stain-negative, non-spore-forming, nonflagellated and rod-shaped, approximately 0.2–0.5 mm in
diameter and 0.8–5.0 mm in length. Gas vesicles are
produced. Colonies on MA are circular, slightly convex,
smooth, glistening, reddish orange and 0.5–1.0 mm in
diameter after incubation for 5 days at 30 uC. Optimal
growth occurs at 30 uC; growth occurs at 15 and 35 uC, but
not at 10 or 37 uC. Optimal pH for growth is between 7.0
and 8.0; growth occurs at pH 6.0, but not at pH 5.5.
Growth occurs in the presence of 1.0–6.0 % (w/v) NaCl
with an optimum of approximately 2.0 % (w/v) NaCl.
Mg2+ ions are required for growth. Anaerobic growth does
not occur on MA or on MA supplemented with nitrate.
Catalase- and oxidase-positive. Nitrate is reduced to nitrite.
Aesculin, casein, hypoxanthine, starch, Tweens 20, 40, 60
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721
S. Park and J.-H. Yoon
Loktanella litorea DPG-5T (JN885197)
77.9
75.8
0.01
Loktanella koreensis GA2-M3T (DQ344498)
Loktanella salsilacus LMG 21507T (AJ440997)
Jannaschia helgolandensis Hel 10T (AJ438157)
65.6
Jannaschia pohangensis H1-M8T (DQ643999)
100
87.6
94.7
Jannaschia seosinensis CL-SP26T (AY906862)
99.8
Litoreibacter janthinus KMM 3842T (AB518880)
Litoreibacter meonggei MA1-1T (JN021667)
99.9
Litoreibacter arenae GA2-M15T (EU342372)
100
84.3
Octadecabacter arcticus 238T (U73725)
Octadecabacter antarcticus 307T (U14583)
99.3
Octadecabacter jejudonensis SSK2-1T (KF515220)
99.6
97.2
Litoreibacter albidus KMM 3851T (AB518881)
Sulfitobacter pontiacus CHLG 10T (Y13155)
Sulfitobacter marinus SW-265T (DQ683726)
Roseobacter litoralis ATCC 49566T (X78312)
63.3
83.9
60.3
100
Nereida ignava 2SM4T (AJ748748)
Marivita cryptomonadis CL-SK44T (EU512919)
Marivita litorea CL-JM1T (EU512918)
Marivita geojedonensis DPG-138T (JN885198)
63.0
Marivita hallyeonensis DPG-28T (JF260872)
Marivita byunsanensis SMK-114T (FJ467624)
53.2
Thalassobius maritimus GSW-M6T (HM748766)
99.6
Donghicola eburneus SW-277T (DQ667965)
Donghicola xiamenensis Y-2T (DQ120728)
Primorskyibacter sedentarius KMM 9018T (AB550558)
Thalassobius gelatinovorus IAM 12617T (D88523)
Thalassobius mediterraneus CECT 5383T (AJ878874)
Thalassobius aestuarii JC2049T (AY442178)
Shimia marina CL-TA03T (AY962292)
99.7
Shimia isoporae SW6T (FJ976449)
Stappia stellulata IAM 12621T (D88525)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain SSK2-1T, the
type strains of the two recognized species of the genus Octadecabacter and representatives of some other members of the
Alphaproteobacteria. Bootstrap values (expressed as percentages of 1000 replications) of .50 % are shown at branch points.
Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and
maximum-parsimony algorithms. Stappia stellulata IAM 12621T was used as an outgroup. Bar, 0.01 substitutions per nucleotide
position.
and 80, urea and L-tyrosine are hydrolysed, but gelatin and
xanthine are not. Cellobiose, D-fructose, D-galactose, Dglucose, D-xylose, acetate, citrate, L-malate and succinate
are utilized, but L-arabinose, maltose, D-mannose, sucrose,
trehalose, benzoate, formate, pyruvate, L-glutamate and
salicin are not. Acid is produced from L-arabinose,
D-galactose, D-glucose, maltose, D-ribose and D-xylose,
but not from cellobiose, D-fructose, lactose, D-mannose,
melezitose, melibiose, raffinose, L-rhamnose, sucrose,
722
trehalose, myo-inositol, D-mannitol or D-sorbitol. In assays
with the API ZYM system, alkaline phosphatase, esterase
(C4), esterase lipase (C8), leucine arylamidase and acid
phosphatase activities are present, but lipase (C14), valine
arylamidase, cystine arylamidase, trypsin, a-chymotrypsin,
naphthol-AS-BI-phosphohydrolase, a-galactosidase, bgalactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase activities are absent. Susceptible to ampicillin,
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Octadecabacter jejudonensis sp. nov.
Table 2. Cellular fatty acid compositions (%) of strain SSK21T and the type strain of Octadecabacter arcticus
All data were obtained from this study. Fatty acids that represented
,0.5 % in both strains were omitted. TR, Trace (,0.5 %); 2, not
detected.
SSK2-1T
Fatty acid
Acknowledgements
This work was supported by a grant from the National Institute
of Biological Resources (NIBR) funded by the Ministry of
Environment (MOE) and the Program for Collection, Management
and Utilization of Biological Resources and BK 21 program from the
Ministry of Science, ICT & Future Planning (MSIP) of the Republic of
Korea.
O. arcticus DSM
13978T
References
Straight-chain
C16 : 0
C18 : 0
Unsaturated
C18 : 1v7c
Hydroxy
C10 : 0 3-OH
C12 : 1 3-OH
11-methyl
C18 : 1v7c
Summed
feature 3*
6.5
4.4
4.4
2
66.6
74.9
2
4.9
14.3
2.9
9.1
1.1
7.6
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T
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