International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1672–1678
DOI 10.1099/ijs.0.000156
Sulfitobacter undariae sp. nov., isolated from a brown algae reservoir Sooyeon Park,1 Yong-Taek Jung,1,2 Sung-Min Won,1 Ji-Min Park1 and Jung-Hoon Yoon1 Correspondence Jung-Hoon Yoon [email protected]
1
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea
2
University of Science and Technology (UST), 113 Gwahak-ro, Yuseong-gu, Daejeon, Republic of Korea
A Gram-stain-negative, aerobic, non-spore-forming, non-flagellated and coccoid, ovoid or rodshaped bacterial strain, W-BA2T, was isolated from a brown algae reservoir in Wando of South Korea. Strain W-BA2T grew optimally at 25 6C, at pH 7.0–8.0 and in the presence of approximately 2.0–3.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain W-BA2T fell within the clade comprising the type strains of species of the genus Sulfitobacter, clustering coherently with the type strains of Sulfitobacter donghicola and Sulfitobacter guttiformis showing sequence similarity values of 98.0–98.1 %. Sequence similarities to the type strains of the other species of the genus Sulfitobacter were 96.0–97.4 %. Strain W-BA2T contained Q-10 as the predominant ubiquinone and C18 : 1v7c as the major fatty acid. The major polar lipids of strain W-BA2T were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain W-BA2T was 55.0 mol% and its DNA–DNA relatedness values with the type strains of Sulfitobacter donghicola, Sulfitobacter guttiformis and Sulfitobacter mediterraneus were 16–23 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain W-BA2T is separated from other species of the genus Sulfitobacter. On the basis of the data presented, strain W-BA2T is considered to represent a novel species of the genus Sulfitobacter, for which the name Sulfitobacter undariae sp. nov. is proposed. The type strain is W-BA2T (5KCTC 42200T5NBRC 110523T).
The genus Sulfitobacter, a member of the class Alphaproteobacteria, was proposed by Sorokin (1995) with the description of a single recognized species, Sulfitobacter pontiacus. At the time of writing, the genus Sulfitobacter comprises 14 species with validly published names (http://www.bacterio.net/ sulfitobacter.html) (Euze´by, 1997; Kwak et al., 2014; Hong et al., 2015). Members of the genus Sulfitobacter have been isolated from Antarctic lakes and marine environments and organisms (Sorokin, 1995; Pukall et al., 1999; Labrenz et al., 2000; Ivanova et al., 2004; Yoon et al., 2007a, b; Fukui et al., 2014; Kwak et al., 2014). In this study, we describe a bacterial strain, designated W-BA2T, which was isolated from a brown algae (Undaria pinnatifida) reservoir at Wando, an island of South Korea. Comparative 16S rRNA gene Abbreviations: DPG, diphosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain W-BA2T is KM275624. One supplementary figure is available with the online Supplementary Material.
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sequence analysis indicated that strain W-BA2T is phylogenetically most affiliated to the genus Sulfitobacter. The aim of the present work was to determine the exact taxonomic position of strain W-BA2T by using a polyphasic characterization that included the determination of chemotaxonomic and other phenotypic properties, detailed phylogenetic investigations based on 16S rRNA gene sequences and DNA–DNA hybridization. Leachate from a brown algae reservoir was collected from Wando, an island located on the South Sea in South Korea, and used as the source for the isolation of bacterial strains. Strain W-BA2T was isolated by the standard dilution plating technique at 25 uC on marine agar 2216 (MA; Becton Dickinson) and cultivated routinely under the same culture conditions. The type strains of four species of the genus Sulfitobacter were used as reference strains for phenotypic characterization, fatty acid and polar lipid analyses and DNA–DNA hybridization. Sulfitobacter donghicola DSW-25T was obtained from our previous study (Yoon et al., 2007b), and Sulfitobacter guttiformis DSM 11458T, Sulfitobacter mediterraneus DSM 12244T and 000156 G 2015 IUMS Printed in Great Britain
Sulfitobacter undariae sp. nov.
Table 1. Differential characteristics of strain W-BA2T and the type strains of three phylogenetically related species of the genus Sulfitobacter Strains: 1, W-BA2T; 2, Sulfitobacter donghicola DSW-25T (data taken from Yoon et al., 2007b) unless indicated otherwise; 3, Sulfitobacter guttiformis DSM 11458T (data obtained from this study unless indicated otherwise); 4, Sulfitobacter mediterraneus DSM 12244T (data obtained from this study unless indicated otherwise). +, Positive reaction; 2, negative reaction; W, weakly positive reaction. All strains are positive for activity of oxidase and catalase; growth on* yeast extract (0.2 %) and tryptone (weakly positive for Sulfitobacter guttiformis DSM 11458T); activity of alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and acid phosphatase; and susceptibility to ampicillin, carbenicillin, cefalotin, chloramphenicol, gentamicin, kanamycin, neomycin, novobiocin, penicillin G, polymyxin B and streptomycin. All strains are negative for hydrolysis of aesculin, casein, gelatin, starch, urea and xanthine; utilization of L-arabinose, cellobiose, D-fructose, D-galactose, D-glucose, maltose, Dmannose, sucrose, trehalose, D-xylose, acetate, benzoate, citrate, formate, succinate, L-glutamate and salicin; growth on* Casamino acids and yeast extract (0.005 %); activity of lipase (C 14), cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, aglucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase; and susceptibility to lincomycin. Characteristic
1
2
3
4
Motility Optimum temperature for growth (uC) Growth at 4 uC Maximum NaCl concentration (%) for growth Presence of Bacteriochlorophyll a Nitrate reduction Hydrolysis of: Hypoxanthine Tween 80 L-Tyrosine Utilization of: L-Malate Pyruvate Growth on: Peptone Phytone peptone Enzyme activity (API ZYM) Valine arylamidase Naphthol-AS-BI-phosphohydrolase Susceptibility to: Oleandomycin Tetracycline DNA G+C content (mol%)
2 25 + 10 2 2
2 25 2 5 2 2
+D 12–20D +D 4D +D +
+d 17–28d +d 8d 2d 2
+ + +
2 2
2 + +
+ 2 2
2 2
+ +
2 2
2 2
2 +
+* +*
W
2 2
W
2 2
W
W
W
W
2
2 + 55.0
+ + 56.9
2 2 55.0–56.3D
+ + 59d
W
W
*Data for Sulfitobacter donghicola DSW-25T obtained from this study. DData taken from Labrenz et al. (2000). dData taken from Pukall et al. (1999).
Sulfitobacter pontiacus DSM 10014T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. The cell morphology, Gram reaction, pH range for growth and anaerobic growth were determined as described by Park et al. (2014). Growth at 4, 10, 20, 25, 28, 30, 35 and 37 uC was measured on MA to determine the optimal temperature and temperature range for growth. Growth at various concentrations of NaCl (0, 0.5 and 1.0–12.0 %, in increments of 1.0 %) was investigated by supplementing with appropriate concentrations of NaCl in marine broth 2216 (MB) prepared according to the formula of the Becton Dickinson medium except that NaCl was excluded. The requirement for Mg2+ ions was investigated by using MB, prepared according to the formula of the Becton http://ijs.sgmjournals.org
Dickinson medium, that comprised all of the constituents except MgCl2 and MgSO4. Catalase and oxidase activities were determined as described by La´nyı´ (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 Tween 80 and nitrate reduction were investigated as described by La´nyı´ (1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea were 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 (l21 distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g MgSO4 . 7H2O and 1.3 g 1673
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Sulfitobacter noctilucae NB-68T (KC428716)
97.7 96.9
Sulfitobacter noctilucicola NB-77T (KC428717) Sulfitobacter geojensis MM-124T (KC428714)
0.01
Sulfitobacter porphyrae SCM-1T (AB758574) Sulfitobacter mediterraneus CH-B427T (Y17387) Sulfitobacter donghicola DSW-25T (EF202614)
86.1
Sulfitobacter guttiformis EL-38T (Y16427)
89.7
Sulfitobacter undariae W-BA2T (KM275624) Sulfitobacter marinus SW-265T (DQ683726) Sulfitobacter litoralis Iso 3T (DQ097527)
60.7 53.6
Sulfitobacter pontiacus ChLG 10T (Y13155) Sulfitobacter brevis EL-162T (Y16425) Sulfitobacter delicatus KMM 3584T (AY180103)
51.2 100
Oceanibulbus indolifex HEL-45T (AJ550939) Sulfitobacter dubius KMM 3554T (AY180102) Roseobacter litoralis Och 149T (X78312)
99.3
Roseobacter denitrificans Och 114T (M59063) Pelagimonas varians SH4-1T (FJ882053) 100
Octadecabacter arcticus 238T (U73725) Octadecabacter antarcticus 307T (U14583)
93.1
Litoreibacter meonggei MA1-1T (JN021667) 100
Litoreibacter albidus KMM 3851T (AB518881) Tateyamaria omphalii MKT107T (AB193438)
51.8
Aestuariihabitans beolgyonensis BB-MW15T (KC577450) Primorskyibacter sedentarius KMM 9018T (AB550558) Seohaeicola saemankumensis SD-15T (EU221274)
97.9 82.6
Leisingera methylohalidivorans MB2T (AY005463) Phaeobacter gallaeciensis BS107T (Y13244) Pelagicola litoralis CL-ES2T (EF192392) Ruegeria conchae TW15T (HQ171439)
99.8
Ruegeria atlantica IAM 14463T (D88526) Stappia stellulata IAM 12621T (D88525)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain W-BA2T, the type strains of species of the genus Sulfitobacter and representatives of some other related taxa. Bootstrap values (expressed as percentages of 1000 replications) of .50 % are shown at branching 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 (GenBank accession number, D88525) was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
CaCl2 . 2H2O (Bruns et al., 2001). Utilization of various substrates for growth was tested according to the method of 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). Growth on various substrates (Casamino acids, peptone, phytone peptone, tryptone and yeast extract) was tested on basal agar medium with the following constituents (l21): 0.1 g C6H5FeO7, 19.45 g NaCl, 5.9 g MgCl2 . 6H2O, 3.24 g MgSO4 . 7H2O, 1.8 g CaCl2 . 2H2O, 0.55 g KCl, 0.16 g NaHCO3, 0.08 g KBr, 0.034 g SrCl2 . 6H2O, 0.022 g H3BO3, 0.008 g Na2H2PO4, 0.004 g Na2SiO3, 0.0024 g NaF, 0.0016 g NH4NO3 and 15 g Noble agar
(Becton Dickinson). The substrates were added at a concentration of 0.2 % (w/v); yeast extract was added at concentrations of both 0.005 and 0.2 % (w/v). The ability to oxidize sulfite was investigated as described by Pukall et al. (1999) with the modification that 20 mM glucose was used. 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), cefalotin (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 25 uC, by using the API ZYM system (bioMe´rieux);
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Table 2. Cellular fatty acid compositions of strain W-BA2T and the type strains of three phylogenetically related species of the genus Sulfitobacter and Sulfitobacter pontiacus Strains: 1, W-BA2T; 2, Sulfitobacter donghicola DSW-25T; 3, Sulfitobacter guttiformis DSM 11458T; 4, Sulfitobacter mediterraneus DSM 12244T; 5, Sulfitobacter pontiacus DSM 10014T. All data obtained from this study (percentages of total fatty acids). Fatty acids that represented ,0.5 % in all strains were omitted. TR, Traces (,0.5 %); 2, Not detected. Fatty acid Straight-chain C16 : 0 C18 : 0 Unsaturated C18 : 1v7c Hydroxy C10 : 0 3-OH C12 : 0 3-OH C14 : 0 2-OH 11-Methyl C18 : 1v7c Summed features* 3 7
1
2
3
4
5
7.9 TR
10.3 2.7
5.2 0.8
5.0 0.8
6.9 2
87.1
79.7
86.4
66.8
83.2
3.1 2 2 2
4.4 2 2 2.5
2.6 2 2 2.4
3.2 2 16.5 6.4
2.7 0.8 2 4.1
1.5 2
2 2
TR
TR
1.9
2
1.1 1.3
*Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c; summed feature 7 contained unknown fatty acid (equivalent chain length, 18.846), C19 : 1v6c and/or C19 : 0 cyclo v10c.
the strip was inoculated with cells suspended in artificial seawater from which CaCl2 was excluded. For spectral analysis of in vivo pigment absorption, strain W-BA2T was cultivated aerobically in the dark at 25 uC in MB. The culture was washed twice by centrifugation with a MOPS buffer (0.01 M MOPS/NaOH; 0.1 M KCl; 0.001 M MgCl2; pH 7.5) and disrupted by means of sonication (VC505; Sonics & Materials). After removal of cell debris by centrifugation, the absorption spectrum of the supernatant was examined on an Eon Microplate spectrophotometer (Biotek). Cell biomass of strain W-BA2T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 2 days in MB at 25 uC, and cell biomass of Sulfitobacter donghicola DSW25T, Sulfitobacter guttiformis DSM 11458T, Sulfitobacter mediterraneus DSM 12244T and Sulfitobacter pontiacus DSM 10014T for DNA extraction and for polar lipid analysis was obtained from cultures grown under the same culture conditions. Chromosomal DNA was extracted and purified according to the protocol of Hunter (1985), with the modification that RNase T1 was used in combination with RNase A to minimize contamination by RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al., 1998) using two universal primers, 9F (59-GAGTTTGATCCTGGCTCAG-39) and 1512R (59ACGGTTACCTTGTTACGACTT-39). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were http://ijs.sgmjournals.org
performed as described by Yoon et al. (2003). DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes in microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values are quoted as DNA–DNA relatedness values. Isoprenoid quinones were extracted and analysed as described by Komagata & Suzuki (1987), using reversedphase HPLC and 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 W-BA2T, Sulfitobacter donghicola DSW-25T, Sulfitobacter guttiformis DSM 11458T, Sulfitobacter mediterraneus DSM 12244T and Sulfitobacter pontiacus DSM 10014T was harvested from MA plates after cultivation for 3 days at 25 uC. The physiological age of the cell masses was standardized by observing the 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 6.2B). Fatty acids were saponified, methylated and extracted using the standard MIDI protocol (Sherlock Microbial Identification System, version 6.2B). The fatty acids were analysed by GC (model 6890; Hewlett Packard) and identified 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 Embley & Wait (1994). Individual polar lipids were identified by spraying the plates with 10 % ethanolic 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 by reversed-phase HPLC 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 a 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 W-BA2T are given in the species description and in Table 1 or Fig. S1 (available in the online Supplementary Material). The almost complete 16S rRNA gene sequence of strain W-BA2T determined in this study comprised 1385 nt, approximately 95 % of the Escherichia coli 16S rRNA gene sequence. In the neighbour-joining phylogenetic tree based on 16S rRNA gene 1675
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(c)
(b)
(a) L2
L1
DPG PG
PL1
PL1
L1
L5
L2
L3
PL1
L3 L4
L4
L1
DPG AL1
PE
PG
PC AL2 AL3
PG AL1
PE
PC
PC
(d)
AL1
PL2 PL3 PL4
(e) L2
L1
PG
PC
L2
PL1
PE AL1
AL4
L5
PL1 L1
DPG
PG
PE AL1
PC L6 AL3
Fig. 2. Thin layer chromatograms of the total polar lipids of strain W-BA2T (a), Sulfitobacter donghicola DSW-25T (b), Sulfitobacter guttiformis DSM 11458T (c), Sulfitobacter mediterraneus DSM 12244T (d) and Sulfitobacter pontiacus DSM 10014T (e). Spots were revealed by spraying the plates with 10 % ethanolic molybdophosphoric acid. AL1–4, unidentified aminolipids; L1–6, unidentified lipids; PL1–4, unidentified phospholipids.
sequences, strain W-BA2T fell within the clade comprising the type strains of species of the genus Sulfitobacter, joining the cluster comprising the type strains of Sulfitobacter donghicola and Sulfitobacter guttiformis by a bootstrap resampling value of 89.7 % (Fig. 1). The relationships among strain W-BA2T, Sulfitobacter donghicola DSW-25T and Sulfitobacter guttiformis EL-38T were also maintained in the trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain WBA2T exhibited 16S rRNA gene sequence similarity values of 98.1, 98.0 and 97.3 % to Sulfitobacter donghicola DSW25T, Sulfitobacter guttiformis EL-38T and Sulfitobacter mediterraneus CH-B427T, respectively, and of 96.0–97.4 % to the type strains of the other species of the genus Sulfitobacter. The predominant isoprenoid quinone detected in strain W-BA2T was ubiquinone-10 (Q-10), which is typical of the vast majority of the class Alphaproteobacteria including the genus Sulfitobacter (Yoon et al., 2007b; Fukui et al., 2014). In Table 2, the fatty acid profile of strain W-BA2T is compared with those of the type strains of Sulfitobacter donghicola, Sulfitobacter guttiformis, Sulfitobacter mediterraneus and Sulfitobacter pontiacus, which were grown and analysed under
identical conditions in this study. The major fatty acid (.10 % of the total fatty acids) detected in strain W-BA2T was C18 : 1v7c (87.1 %) (Table 2). The fatty acid profile of strain W-BA2T was similar to those of the type strains of the three species of the genus Sulfitobacter, although there were differences in the proportions of some fatty acids (Table 2). The major polar lipids found in strain W-BA2T were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified aminolipid (AL1) and one unidentified lipid (L1); minor amounts of diphosphatidylglycerol (DPG), one additional unidentified lipid, one unidentified phospholipid and two unidentified aminolipids were also present (Fig. 2). The polar lipid profile of strain W-BA2T was similar to those of the type strains of Sulfitobacter donghicola, Sulfitobacter guttiformis, Sulfitobacter mediterraneus and Sulfitobacter pontiacus in that PC, PG, AL1 and L1 were major polar lipids (Fig. 2). However, the polar lipid profile of strain W-BA2T was distinguishable from that of Sulfitobacter donghicola DSW-25T by the presence of PE and from those of Sulfitobacter guttiformis DSM 11458T and Sulfitobacter mediterraneus DSM 12244T by the presence of DPG (Fig. 2). The DNA G+C content of strain W-BA2T was 55.0 mol%, a value in the range reported for members of the
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genus Sulfitobacter (Table 1; Yoon et al., 2007b). The results obtained from the chemotaxonomic analyses are sufficient to support the result of the phylogenetic analysis, i.e. showing that strain W-BA2T is a member of the genus Sulfitobacter (Table 2; Fig. 2). Strain W-BA2T exhibited DNA–DNA relatedness values of 23, 16 and 19 % to Sulfitobacter donghicola DSW-25T, Sulfitobacter guttiformis DSM 11458T and Sulfitobacter mediterraneus DSM 12244T, respectively. Strain W-BA2T could be distinguished from the type strains of Sulfitobacter donghicola, Sulfitobacter guttiformis and Sulfitobacter mediterraneus by differences in several phenotypic characteristics, including motility, growth at 4 uC, maximum NaCl concentration for growth, nitrate reduction, hydrolysis and utilization of some substrates and susceptibility to two antibiotics (Table 1). These differences, in combination with the phylogenetic and genetic distinctiveness of strain WBA2T, suggest that the novel strain is separated from other species of the genus Sulfitobacter (Wayne et al., 1987; Stackebrandt & Goebel, 1994). On the basis of the data presented, therefore, strain W-BA2T is considered to represent a novel species of the genus Sulfitobacter, for which the name Sulfitobacter undariae sp. nov. is proposed. Description of Sulfitobacter undariae sp. nov. Sulfitobacter undariae (un.da9ri.ae. N.L. gen. n. undariae of Undaria, named after the generic name of the brown algae Undaria pinnatifida, from whose reservoir the type strain was isolated). Cells are Gram-stain-negative, non-spore-forming, nonflagellated and coccoid, ovoid or rod-shaped, approximately 0.2–0.5 mm in diameter and 0.5–3.0 mm in length. Colonies on MA are circular, slightly convex, smooth, glistening, yellowish-white in colour and 0.5–1.0 mm in diameter after incubation for 3 days at 25 uC. Optimal growth occurs at 25 uC; growth occurs at 4 and 30 uC, but not at 35 uC. Optimal pH for growth is between pH 7.0 and 8.0; growth occurs at pH 5.5, but not at pH 5.0. Growth occurs in the presence of 0–10.0 % (w/v) NaCl with an optimum of approximately 2.0–3.0 % (w/v) NaCl. Mg2+ ions are not required for growth. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Catalase- and oxidase-positive. Nitrate is not reduced to nitrite. Bacteriochlorophyll a is not produced. Hypoxanthine, Tween 80 and L-tyrosine are hydrolysed, but aesculin, casein, gelatin, starch, urea and xanthine are not. L-Arabinose, cellobiose, D-fructose, D-galactose, Dglucose, maltose, D-mannose, sucrose, trehalose, D-xylose, acetate, benzoate, citrate, formate, L-malate, pyruvate, succinate, salicin and L-glutamate are not utilized as carbon and energy sources. Acid is not produced from Larabinose, cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, melezitose, melibiose, raffinose, L-rhamnose, D-ribose, sucrose, trehalose, D-xylose, myo-inositol, D-mannitol or D-sorbitol. In assays with the API ZYM system, activity of alkaline phosphatase, esterase http://ijs.sgmjournals.org
(C 4), esterase lipase (C 8), leucine arylamidase and acid phosphatase is present and activity of valine arylamidase and naphthol-AS-BI-phosphohydrolase is weakly present, but activity of lipase (C 14), cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase is absent. Oxidation of sulfite is not observed. Susceptible to ampicillin, carbenicillin, cefalotin, chloramphenicol, gentamicin, kanamycin, neomycin, novobiocin, penicillin G, polymyxin B, streptomycin and tetracycline, but not to lincomycin or oleandomycin. The predominant ubiquinone is Q-10. The major fatty acid (.10 % of the total fatty acids) is C18 : 1v7c. The major polar lipids are PC, PG, PE, one unidentified aminolipid and one unidentified lipid. The type strain, W-BA2T (5KCTC 42200T5NBRC 110523T), was isolated from leachate from a brown algae reservoir in Wando, an island located on the South Sea in South Korea. The DNA G+C content of the type strain is 55.0 mol%.
Acknowledgements This work was supported by the project on survey of indigenous species of Korea of the National Institute of Biological Resources (NIBR) under the Ministry of Environment (MOE) of the Republic of Korea and the Program for Collection, Management and Utilization of Biological Resources from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea (grant NRF2013M3A9A5075953).
References Barrow, G. I. & Feltham, R. K. A. (editors) (1993). Cowan and Steel’s
Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press. Baumann, P. & Baumann, L. (1981). The marine Gram-negative
eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes. In The Prokaryotes, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Tru¨per, A. Balows & H. G. Schlegel. Berlin: Springer. Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda
ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006. Cohen-Bazire, G., Sistrom, W. R. & Stanier, R. Y. (1957). Kinetic
studies of pigment synthesis by nonsulfur purple bacteria. J Cell Comp Physiol 49, 25–68. Embley, T. M. & Wait, R. (1994). Structural lipids of eubacteria. In
Chemical Methods in Prokaryotic Systematics, pp. 121–161. Edited by M. Goodfellow & A. G. O’Donnell. Chichester: Wiley. Euze´by, J. P. (1997). List of Bacterial Names with Standing in
Nomenclature: a folder available on the Internet. Int J Syst Bacteriol 47, 590–592. 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. 1677
S. Park and others
Fukui, Y., Abe, M., Kobayashi, M., Shimada, Y., Saito, H., Oikawa, H., Yano, Y. & Satomi, M. (2014). Sulfitobacter porphyrae sp. nov.,
isolated from the red alga Porphyra yezoensis. Int J Syst Evol Microbiol 64, 438–443.
Pukall, R., Buntefuss, D., Fru¨hling, A., Rohde, M., Kroppenstedt, R. M., Burghardt, J., Lebaron, P., Bernard, L. & Stackebrandt, E. (1999). Sulfitobacter mediterraneus sp. nov., a new sulfite-oxidizing member of the a-Proteobacteria. Int J Syst Bacteriol 49, 513–519.
Hong, Z., Lai, Q., Luo, Q., Jiang, S., Zhu, R., Liang, J. & Gao, Y. (2015).
Sasser, M. (1990). Identification of bacteria by gas chromatography
Sulfitobacter pseudonitzschiae sp. nov., isolated from the toxic marine diatom Pseudo-nitzschia multiseries. Int J Syst Evol Microbiol 65, 95– 100.
of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Sorokin, D. Y. (1995). [Sulfitobacter pontiacus gen. nov., sp. nov. – a new heterotrophic bacterium from the Black Sea, specialized on sulfite oxidation]. Mikrobiologiya 64, 354–365 (in Russian).
Hunter, I. S. (1985). Gene cloning in Streptomyces. In DNA Cloning: a Practical Approach, vol. 2, p. 19–44. Edited by D. M. Glover. Oxford: IRL Press.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place
Ivanova, E. P., Gorshkova, N. M., Sawabe, T., Zhukova, N. V., Hayashi, K., Kurilenko, V. V., Alexeeva, Y., Buljan, V., Nicolau, D. V. & other authors (2004). Sulfitobacter delicatus sp. nov. and Sulfitobacter
for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846– 849.
dubius sp. nov., respectively from a starfish (Stellaster equestris) and sea grass (Zostera marina). Int J Syst Evol Microbiol 54, 475–480.
Staley, J. T. (1968). Prosthecomicrobium and Ancalomicrobium: new prosthecate freshwater bacteria. J Bacteriol 95, 1921–1942.
Komagata, K. & Suzuki, K.-I. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
Kwak, M.-J., Lee, J.-S., Lee, K. C., Kim, K. K., Eom, M. K., Kim, B. K. & Kim, J. F. (2014). Sulfitobacter geojensis sp. nov., Sulfitobacter
composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
noctilucae sp. nov., and Sulfitobacter noctilucicola sp. nov., isolated from coastal seawater. Int J Syst Evol Microbiol 64, 3760–3767.
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
Labrenz, M., Tindall, B. J., Lawson, P. A., Collins, M. D., Schumann, P. & Hirsch, P. (2000). Staleya guttiformis gen. nov., sp. nov. and Sulfitobacter brevis sp. nov., a-3-Proteobacteria from hypersaline,
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific
Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
heliothermal and meromictic antarctic Ekho Lake. Int J Syst Evol Microbiol 50, 303–313.
phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194.
La´nyı´, B. (1987). Classical and rapid identification methods for
Yoon, J.-H., Kim, I.-G., Shin, D.-Y., Kang, K. H. & Park, Y.-H. (2003).
medically important bacteria. Methods Microbiol 19, 1–67. Leifson, E. (1963). Determination of carbohydrate metabolism of
marine bacteria. J Bacteriol 85, 1183–1184. Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
Microbulbifer salipaludis sp. nov., a moderate halophile isolated from a Korean salt marsh. Int J Syst Evol Microbiol 53, 53–57. Yoon, J.-H., Kang, S.-J. & Oh, T.-K. (2007a). Sulfitobacter marinus sp.
nov., isolated from seawater of the East Sea in Korea. Int J Syst Evol Microbiol 57, 302–305. Yoon, J.-H., Kang, S.-J., Lee, M.-H. & Oh, T.-K. (2007b). Description
aquaemixtae sp. nov., isolated from the junction between the ocean and a freshwater spring. Int J Syst Evol Microbiol 64, 1378–1383.
of Sulfitobacter donghicola sp. nov., isolated from seawater of the East Sea in Korea, transfer of Staleya guttiformis Labrenz et al. 2000 to the genus Sulfitobacter as Sulfitobacter guttiformis comb. nov. and emended description of the genus Sulfitobacter. Int J Syst Evol Microbiol 57, 1788–1792.
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Park, S., Park, D.-S., Bae, K. S. & Yoon, J.-H. (2014). Phaeobacter
DOI 10.1099/ijs.0.000156
Sulfitobacter undariae sp. nov., isolated from a brown algae reservoir Sooyeon Park,1 Yong-Taek Jung,1,2 Sung-Min Won,1 Ji-Min Park1 and Jung-Hoon Yoon1 Correspondence Jung-Hoon Yoon [email protected]
1
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea
2
University of Science and Technology (UST), 113 Gwahak-ro, Yuseong-gu, Daejeon, Republic of Korea
A Gram-stain-negative, aerobic, non-spore-forming, non-flagellated and coccoid, ovoid or rodshaped bacterial strain, W-BA2T, was isolated from a brown algae reservoir in Wando of South Korea. Strain W-BA2T grew optimally at 25 6C, at pH 7.0–8.0 and in the presence of approximately 2.0–3.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain W-BA2T fell within the clade comprising the type strains of species of the genus Sulfitobacter, clustering coherently with the type strains of Sulfitobacter donghicola and Sulfitobacter guttiformis showing sequence similarity values of 98.0–98.1 %. Sequence similarities to the type strains of the other species of the genus Sulfitobacter were 96.0–97.4 %. Strain W-BA2T contained Q-10 as the predominant ubiquinone and C18 : 1v7c as the major fatty acid. The major polar lipids of strain W-BA2T were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain W-BA2T was 55.0 mol% and its DNA–DNA relatedness values with the type strains of Sulfitobacter donghicola, Sulfitobacter guttiformis and Sulfitobacter mediterraneus were 16–23 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain W-BA2T is separated from other species of the genus Sulfitobacter. On the basis of the data presented, strain W-BA2T is considered to represent a novel species of the genus Sulfitobacter, for which the name Sulfitobacter undariae sp. nov. is proposed. The type strain is W-BA2T (5KCTC 42200T5NBRC 110523T).
The genus Sulfitobacter, a member of the class Alphaproteobacteria, was proposed by Sorokin (1995) with the description of a single recognized species, Sulfitobacter pontiacus. At the time of writing, the genus Sulfitobacter comprises 14 species with validly published names (http://www.bacterio.net/ sulfitobacter.html) (Euze´by, 1997; Kwak et al., 2014; Hong et al., 2015). Members of the genus Sulfitobacter have been isolated from Antarctic lakes and marine environments and organisms (Sorokin, 1995; Pukall et al., 1999; Labrenz et al., 2000; Ivanova et al., 2004; Yoon et al., 2007a, b; Fukui et al., 2014; Kwak et al., 2014). In this study, we describe a bacterial strain, designated W-BA2T, which was isolated from a brown algae (Undaria pinnatifida) reservoir at Wando, an island of South Korea. Comparative 16S rRNA gene Abbreviations: DPG, diphosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain W-BA2T is KM275624. One supplementary figure is available with the online Supplementary Material.
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sequence analysis indicated that strain W-BA2T is phylogenetically most affiliated to the genus Sulfitobacter. The aim of the present work was to determine the exact taxonomic position of strain W-BA2T by using a polyphasic characterization that included the determination of chemotaxonomic and other phenotypic properties, detailed phylogenetic investigations based on 16S rRNA gene sequences and DNA–DNA hybridization. Leachate from a brown algae reservoir was collected from Wando, an island located on the South Sea in South Korea, and used as the source for the isolation of bacterial strains. Strain W-BA2T was isolated by the standard dilution plating technique at 25 uC on marine agar 2216 (MA; Becton Dickinson) and cultivated routinely under the same culture conditions. The type strains of four species of the genus Sulfitobacter were used as reference strains for phenotypic characterization, fatty acid and polar lipid analyses and DNA–DNA hybridization. Sulfitobacter donghicola DSW-25T was obtained from our previous study (Yoon et al., 2007b), and Sulfitobacter guttiformis DSM 11458T, Sulfitobacter mediterraneus DSM 12244T and 000156 G 2015 IUMS Printed in Great Britain
Sulfitobacter undariae sp. nov.
Table 1. Differential characteristics of strain W-BA2T and the type strains of three phylogenetically related species of the genus Sulfitobacter Strains: 1, W-BA2T; 2, Sulfitobacter donghicola DSW-25T (data taken from Yoon et al., 2007b) unless indicated otherwise; 3, Sulfitobacter guttiformis DSM 11458T (data obtained from this study unless indicated otherwise); 4, Sulfitobacter mediterraneus DSM 12244T (data obtained from this study unless indicated otherwise). +, Positive reaction; 2, negative reaction; W, weakly positive reaction. All strains are positive for activity of oxidase and catalase; growth on* yeast extract (0.2 %) and tryptone (weakly positive for Sulfitobacter guttiformis DSM 11458T); activity of alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and acid phosphatase; and susceptibility to ampicillin, carbenicillin, cefalotin, chloramphenicol, gentamicin, kanamycin, neomycin, novobiocin, penicillin G, polymyxin B and streptomycin. All strains are negative for hydrolysis of aesculin, casein, gelatin, starch, urea and xanthine; utilization of L-arabinose, cellobiose, D-fructose, D-galactose, D-glucose, maltose, Dmannose, sucrose, trehalose, D-xylose, acetate, benzoate, citrate, formate, succinate, L-glutamate and salicin; growth on* Casamino acids and yeast extract (0.005 %); activity of lipase (C 14), cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, aglucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase; and susceptibility to lincomycin. Characteristic
1
2
3
4
Motility Optimum temperature for growth (uC) Growth at 4 uC Maximum NaCl concentration (%) for growth Presence of Bacteriochlorophyll a Nitrate reduction Hydrolysis of: Hypoxanthine Tween 80 L-Tyrosine Utilization of: L-Malate Pyruvate Growth on: Peptone Phytone peptone Enzyme activity (API ZYM) Valine arylamidase Naphthol-AS-BI-phosphohydrolase Susceptibility to: Oleandomycin Tetracycline DNA G+C content (mol%)
2 25 + 10 2 2
2 25 2 5 2 2
+D 12–20D +D 4D +D +
+d 17–28d +d 8d 2d 2
+ + +
2 2
2 + +
+ 2 2
2 2
+ +
2 2
2 2
2 +
+* +*
W
2 2
W
2 2
W
W
W
W
2
2 + 55.0
+ + 56.9
2 2 55.0–56.3D
+ + 59d
W
W
*Data for Sulfitobacter donghicola DSW-25T obtained from this study. DData taken from Labrenz et al. (2000). dData taken from Pukall et al. (1999).
Sulfitobacter pontiacus DSM 10014T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. The cell morphology, Gram reaction, pH range for growth and anaerobic growth were determined as described by Park et al. (2014). Growth at 4, 10, 20, 25, 28, 30, 35 and 37 uC was measured on MA to determine the optimal temperature and temperature range for growth. Growth at various concentrations of NaCl (0, 0.5 and 1.0–12.0 %, in increments of 1.0 %) was investigated by supplementing with appropriate concentrations of NaCl in marine broth 2216 (MB) prepared according to the formula of the Becton Dickinson medium except that NaCl was excluded. The requirement for Mg2+ ions was investigated by using MB, prepared according to the formula of the Becton http://ijs.sgmjournals.org
Dickinson medium, that comprised all of the constituents except MgCl2 and MgSO4. Catalase and oxidase activities were determined as described by La´nyı´ (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 Tween 80 and nitrate reduction were investigated as described by La´nyı´ (1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea were 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 (l21 distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g MgSO4 . 7H2O and 1.3 g 1673
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Sulfitobacter noctilucae NB-68T (KC428716)
97.7 96.9
Sulfitobacter noctilucicola NB-77T (KC428717) Sulfitobacter geojensis MM-124T (KC428714)
0.01
Sulfitobacter porphyrae SCM-1T (AB758574) Sulfitobacter mediterraneus CH-B427T (Y17387) Sulfitobacter donghicola DSW-25T (EF202614)
86.1
Sulfitobacter guttiformis EL-38T (Y16427)
89.7
Sulfitobacter undariae W-BA2T (KM275624) Sulfitobacter marinus SW-265T (DQ683726) Sulfitobacter litoralis Iso 3T (DQ097527)
60.7 53.6
Sulfitobacter pontiacus ChLG 10T (Y13155) Sulfitobacter brevis EL-162T (Y16425) Sulfitobacter delicatus KMM 3584T (AY180103)
51.2 100
Oceanibulbus indolifex HEL-45T (AJ550939) Sulfitobacter dubius KMM 3554T (AY180102) Roseobacter litoralis Och 149T (X78312)
99.3
Roseobacter denitrificans Och 114T (M59063) Pelagimonas varians SH4-1T (FJ882053) 100
Octadecabacter arcticus 238T (U73725) Octadecabacter antarcticus 307T (U14583)
93.1
Litoreibacter meonggei MA1-1T (JN021667) 100
Litoreibacter albidus KMM 3851T (AB518881) Tateyamaria omphalii MKT107T (AB193438)
51.8
Aestuariihabitans beolgyonensis BB-MW15T (KC577450) Primorskyibacter sedentarius KMM 9018T (AB550558) Seohaeicola saemankumensis SD-15T (EU221274)
97.9 82.6
Leisingera methylohalidivorans MB2T (AY005463) Phaeobacter gallaeciensis BS107T (Y13244) Pelagicola litoralis CL-ES2T (EF192392) Ruegeria conchae TW15T (HQ171439)
99.8
Ruegeria atlantica IAM 14463T (D88526) Stappia stellulata IAM 12621T (D88525)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain W-BA2T, the type strains of species of the genus Sulfitobacter and representatives of some other related taxa. Bootstrap values (expressed as percentages of 1000 replications) of .50 % are shown at branching 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 (GenBank accession number, D88525) was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
CaCl2 . 2H2O (Bruns et al., 2001). Utilization of various substrates for growth was tested according to the method of 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). Growth on various substrates (Casamino acids, peptone, phytone peptone, tryptone and yeast extract) was tested on basal agar medium with the following constituents (l21): 0.1 g C6H5FeO7, 19.45 g NaCl, 5.9 g MgCl2 . 6H2O, 3.24 g MgSO4 . 7H2O, 1.8 g CaCl2 . 2H2O, 0.55 g KCl, 0.16 g NaHCO3, 0.08 g KBr, 0.034 g SrCl2 . 6H2O, 0.022 g H3BO3, 0.008 g Na2H2PO4, 0.004 g Na2SiO3, 0.0024 g NaF, 0.0016 g NH4NO3 and 15 g Noble agar
(Becton Dickinson). The substrates were added at a concentration of 0.2 % (w/v); yeast extract was added at concentrations of both 0.005 and 0.2 % (w/v). The ability to oxidize sulfite was investigated as described by Pukall et al. (1999) with the modification that 20 mM glucose was used. 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), cefalotin (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 25 uC, by using the API ZYM system (bioMe´rieux);
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Table 2. Cellular fatty acid compositions of strain W-BA2T and the type strains of three phylogenetically related species of the genus Sulfitobacter and Sulfitobacter pontiacus Strains: 1, W-BA2T; 2, Sulfitobacter donghicola DSW-25T; 3, Sulfitobacter guttiformis DSM 11458T; 4, Sulfitobacter mediterraneus DSM 12244T; 5, Sulfitobacter pontiacus DSM 10014T. All data obtained from this study (percentages of total fatty acids). Fatty acids that represented ,0.5 % in all strains were omitted. TR, Traces (,0.5 %); 2, Not detected. Fatty acid Straight-chain C16 : 0 C18 : 0 Unsaturated C18 : 1v7c Hydroxy C10 : 0 3-OH C12 : 0 3-OH C14 : 0 2-OH 11-Methyl C18 : 1v7c Summed features* 3 7
1
2
3
4
5
7.9 TR
10.3 2.7
5.2 0.8
5.0 0.8
6.9 2
87.1
79.7
86.4
66.8
83.2
3.1 2 2 2
4.4 2 2 2.5
2.6 2 2 2.4
3.2 2 16.5 6.4
2.7 0.8 2 4.1
1.5 2
2 2
TR
TR
1.9
2
1.1 1.3
*Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c; summed feature 7 contained unknown fatty acid (equivalent chain length, 18.846), C19 : 1v6c and/or C19 : 0 cyclo v10c.
the strip was inoculated with cells suspended in artificial seawater from which CaCl2 was excluded. For spectral analysis of in vivo pigment absorption, strain W-BA2T was cultivated aerobically in the dark at 25 uC in MB. The culture was washed twice by centrifugation with a MOPS buffer (0.01 M MOPS/NaOH; 0.1 M KCl; 0.001 M MgCl2; pH 7.5) and disrupted by means of sonication (VC505; Sonics & Materials). After removal of cell debris by centrifugation, the absorption spectrum of the supernatant was examined on an Eon Microplate spectrophotometer (Biotek). Cell biomass of strain W-BA2T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 2 days in MB at 25 uC, and cell biomass of Sulfitobacter donghicola DSW25T, Sulfitobacter guttiformis DSM 11458T, Sulfitobacter mediterraneus DSM 12244T and Sulfitobacter pontiacus DSM 10014T for DNA extraction and for polar lipid analysis was obtained from cultures grown under the same culture conditions. Chromosomal DNA was extracted and purified according to the protocol of Hunter (1985), with the modification that RNase T1 was used in combination with RNase A to minimize contamination by RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al., 1998) using two universal primers, 9F (59-GAGTTTGATCCTGGCTCAG-39) and 1512R (59ACGGTTACCTTGTTACGACTT-39). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were http://ijs.sgmjournals.org
performed as described by Yoon et al. (2003). DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes in microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values are quoted as DNA–DNA relatedness values. Isoprenoid quinones were extracted and analysed as described by Komagata & Suzuki (1987), using reversedphase HPLC and 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 W-BA2T, Sulfitobacter donghicola DSW-25T, Sulfitobacter guttiformis DSM 11458T, Sulfitobacter mediterraneus DSM 12244T and Sulfitobacter pontiacus DSM 10014T was harvested from MA plates after cultivation for 3 days at 25 uC. The physiological age of the cell masses was standardized by observing the 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 6.2B). Fatty acids were saponified, methylated and extracted using the standard MIDI protocol (Sherlock Microbial Identification System, version 6.2B). The fatty acids were analysed by GC (model 6890; Hewlett Packard) and identified 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 Embley & Wait (1994). Individual polar lipids were identified by spraying the plates with 10 % ethanolic 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 by reversed-phase HPLC 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 a 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 W-BA2T are given in the species description and in Table 1 or Fig. S1 (available in the online Supplementary Material). The almost complete 16S rRNA gene sequence of strain W-BA2T determined in this study comprised 1385 nt, approximately 95 % of the Escherichia coli 16S rRNA gene sequence. In the neighbour-joining phylogenetic tree based on 16S rRNA gene 1675
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(c)
(b)
(a) L2
L1
DPG PG
PL1
PL1
L1
L5
L2
L3
PL1
L3 L4
L4
L1
DPG AL1
PE
PG
PC AL2 AL3
PG AL1
PE
PC
PC
(d)
AL1
PL2 PL3 PL4
(e) L2
L1
PG
PC
L2
PL1
PE AL1
AL4
L5
PL1 L1
DPG
PG
PE AL1
PC L6 AL3
Fig. 2. Thin layer chromatograms of the total polar lipids of strain W-BA2T (a), Sulfitobacter donghicola DSW-25T (b), Sulfitobacter guttiformis DSM 11458T (c), Sulfitobacter mediterraneus DSM 12244T (d) and Sulfitobacter pontiacus DSM 10014T (e). Spots were revealed by spraying the plates with 10 % ethanolic molybdophosphoric acid. AL1–4, unidentified aminolipids; L1–6, unidentified lipids; PL1–4, unidentified phospholipids.
sequences, strain W-BA2T fell within the clade comprising the type strains of species of the genus Sulfitobacter, joining the cluster comprising the type strains of Sulfitobacter donghicola and Sulfitobacter guttiformis by a bootstrap resampling value of 89.7 % (Fig. 1). The relationships among strain W-BA2T, Sulfitobacter donghicola DSW-25T and Sulfitobacter guttiformis EL-38T were also maintained in the trees reconstructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain WBA2T exhibited 16S rRNA gene sequence similarity values of 98.1, 98.0 and 97.3 % to Sulfitobacter donghicola DSW25T, Sulfitobacter guttiformis EL-38T and Sulfitobacter mediterraneus CH-B427T, respectively, and of 96.0–97.4 % to the type strains of the other species of the genus Sulfitobacter. The predominant isoprenoid quinone detected in strain W-BA2T was ubiquinone-10 (Q-10), which is typical of the vast majority of the class Alphaproteobacteria including the genus Sulfitobacter (Yoon et al., 2007b; Fukui et al., 2014). In Table 2, the fatty acid profile of strain W-BA2T is compared with those of the type strains of Sulfitobacter donghicola, Sulfitobacter guttiformis, Sulfitobacter mediterraneus and Sulfitobacter pontiacus, which were grown and analysed under
identical conditions in this study. The major fatty acid (.10 % of the total fatty acids) detected in strain W-BA2T was C18 : 1v7c (87.1 %) (Table 2). The fatty acid profile of strain W-BA2T was similar to those of the type strains of the three species of the genus Sulfitobacter, although there were differences in the proportions of some fatty acids (Table 2). The major polar lipids found in strain W-BA2T were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified aminolipid (AL1) and one unidentified lipid (L1); minor amounts of diphosphatidylglycerol (DPG), one additional unidentified lipid, one unidentified phospholipid and two unidentified aminolipids were also present (Fig. 2). The polar lipid profile of strain W-BA2T was similar to those of the type strains of Sulfitobacter donghicola, Sulfitobacter guttiformis, Sulfitobacter mediterraneus and Sulfitobacter pontiacus in that PC, PG, AL1 and L1 were major polar lipids (Fig. 2). However, the polar lipid profile of strain W-BA2T was distinguishable from that of Sulfitobacter donghicola DSW-25T by the presence of PE and from those of Sulfitobacter guttiformis DSM 11458T and Sulfitobacter mediterraneus DSM 12244T by the presence of DPG (Fig. 2). The DNA G+C content of strain W-BA2T was 55.0 mol%, a value in the range reported for members of the
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Sulfitobacter undariae sp. nov.
genus Sulfitobacter (Table 1; Yoon et al., 2007b). The results obtained from the chemotaxonomic analyses are sufficient to support the result of the phylogenetic analysis, i.e. showing that strain W-BA2T is a member of the genus Sulfitobacter (Table 2; Fig. 2). Strain W-BA2T exhibited DNA–DNA relatedness values of 23, 16 and 19 % to Sulfitobacter donghicola DSW-25T, Sulfitobacter guttiformis DSM 11458T and Sulfitobacter mediterraneus DSM 12244T, respectively. Strain W-BA2T could be distinguished from the type strains of Sulfitobacter donghicola, Sulfitobacter guttiformis and Sulfitobacter mediterraneus by differences in several phenotypic characteristics, including motility, growth at 4 uC, maximum NaCl concentration for growth, nitrate reduction, hydrolysis and utilization of some substrates and susceptibility to two antibiotics (Table 1). These differences, in combination with the phylogenetic and genetic distinctiveness of strain WBA2T, suggest that the novel strain is separated from other species of the genus Sulfitobacter (Wayne et al., 1987; Stackebrandt & Goebel, 1994). On the basis of the data presented, therefore, strain W-BA2T is considered to represent a novel species of the genus Sulfitobacter, for which the name Sulfitobacter undariae sp. nov. is proposed. Description of Sulfitobacter undariae sp. nov. Sulfitobacter undariae (un.da9ri.ae. N.L. gen. n. undariae of Undaria, named after the generic name of the brown algae Undaria pinnatifida, from whose reservoir the type strain was isolated). Cells are Gram-stain-negative, non-spore-forming, nonflagellated and coccoid, ovoid or rod-shaped, approximately 0.2–0.5 mm in diameter and 0.5–3.0 mm in length. Colonies on MA are circular, slightly convex, smooth, glistening, yellowish-white in colour and 0.5–1.0 mm in diameter after incubation for 3 days at 25 uC. Optimal growth occurs at 25 uC; growth occurs at 4 and 30 uC, but not at 35 uC. Optimal pH for growth is between pH 7.0 and 8.0; growth occurs at pH 5.5, but not at pH 5.0. Growth occurs in the presence of 0–10.0 % (w/v) NaCl with an optimum of approximately 2.0–3.0 % (w/v) NaCl. Mg2+ ions are not required for growth. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Catalase- and oxidase-positive. Nitrate is not reduced to nitrite. Bacteriochlorophyll a is not produced. Hypoxanthine, Tween 80 and L-tyrosine are hydrolysed, but aesculin, casein, gelatin, starch, urea and xanthine are not. L-Arabinose, cellobiose, D-fructose, D-galactose, Dglucose, maltose, D-mannose, sucrose, trehalose, D-xylose, acetate, benzoate, citrate, formate, L-malate, pyruvate, succinate, salicin and L-glutamate are not utilized as carbon and energy sources. Acid is not produced from Larabinose, cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, melezitose, melibiose, raffinose, L-rhamnose, D-ribose, sucrose, trehalose, D-xylose, myo-inositol, D-mannitol or D-sorbitol. In assays with the API ZYM system, activity of alkaline phosphatase, esterase http://ijs.sgmjournals.org
(C 4), esterase lipase (C 8), leucine arylamidase and acid phosphatase is present and activity of valine arylamidase and naphthol-AS-BI-phosphohydrolase is weakly present, but activity of lipase (C 14), cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase is absent. Oxidation of sulfite is not observed. Susceptible to ampicillin, carbenicillin, cefalotin, chloramphenicol, gentamicin, kanamycin, neomycin, novobiocin, penicillin G, polymyxin B, streptomycin and tetracycline, but not to lincomycin or oleandomycin. The predominant ubiquinone is Q-10. The major fatty acid (.10 % of the total fatty acids) is C18 : 1v7c. The major polar lipids are PC, PG, PE, one unidentified aminolipid and one unidentified lipid. The type strain, W-BA2T (5KCTC 42200T5NBRC 110523T), was isolated from leachate from a brown algae reservoir in Wando, an island located on the South Sea in South Korea. The DNA G+C content of the type strain is 55.0 mol%.
Acknowledgements This work was supported by the project on survey of indigenous species of Korea of the National Institute of Biological Resources (NIBR) under the Ministry of Environment (MOE) of the Republic of Korea and the Program for Collection, Management and Utilization of Biological Resources from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea (grant NRF2013M3A9A5075953).
References Barrow, G. I. & Feltham, R. K. A. (editors) (1993). Cowan and Steel’s
Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press. Baumann, P. & Baumann, L. (1981). The marine Gram-negative
eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes. In The Prokaryotes, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Tru¨per, A. Balows & H. G. Schlegel. Berlin: Springer. Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda
ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006. Cohen-Bazire, G., Sistrom, W. R. & Stanier, R. Y. (1957). Kinetic
studies of pigment synthesis by nonsulfur purple bacteria. J Cell Comp Physiol 49, 25–68. Embley, T. M. & Wait, R. (1994). Structural lipids of eubacteria. In
Chemical Methods in Prokaryotic Systematics, pp. 121–161. Edited by M. Goodfellow & A. G. O’Donnell. Chichester: Wiley. Euze´by, J. P. (1997). List of Bacterial Names with Standing in
Nomenclature: a folder available on the Internet. Int J Syst Bacteriol 47, 590–592. 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. 1677
S. Park and others
Fukui, Y., Abe, M., Kobayashi, M., Shimada, Y., Saito, H., Oikawa, H., Yano, Y. & Satomi, M. (2014). Sulfitobacter porphyrae sp. nov.,
isolated from the red alga Porphyra yezoensis. Int J Syst Evol Microbiol 64, 438–443.
Pukall, R., Buntefuss, D., Fru¨hling, A., Rohde, M., Kroppenstedt, R. M., Burghardt, J., Lebaron, P., Bernard, L. & Stackebrandt, E. (1999). Sulfitobacter mediterraneus sp. nov., a new sulfite-oxidizing member of the a-Proteobacteria. Int J Syst Bacteriol 49, 513–519.
Hong, Z., Lai, Q., Luo, Q., Jiang, S., Zhu, R., Liang, J. & Gao, Y. (2015).
Sasser, M. (1990). Identification of bacteria by gas chromatography
Sulfitobacter pseudonitzschiae sp. nov., isolated from the toxic marine diatom Pseudo-nitzschia multiseries. Int J Syst Evol Microbiol 65, 95– 100.
of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Sorokin, D. Y. (1995). [Sulfitobacter pontiacus gen. nov., sp. nov. – a new heterotrophic bacterium from the Black Sea, specialized on sulfite oxidation]. Mikrobiologiya 64, 354–365 (in Russian).
Hunter, I. S. (1985). Gene cloning in Streptomyces. In DNA Cloning: a Practical Approach, vol. 2, p. 19–44. Edited by D. M. Glover. Oxford: IRL Press.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place
Ivanova, E. P., Gorshkova, N. M., Sawabe, T., Zhukova, N. V., Hayashi, K., Kurilenko, V. V., Alexeeva, Y., Buljan, V., Nicolau, D. V. & other authors (2004). Sulfitobacter delicatus sp. nov. and Sulfitobacter
for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846– 849.
dubius sp. nov., respectively from a starfish (Stellaster equestris) and sea grass (Zostera marina). Int J Syst Evol Microbiol 54, 475–480.
Staley, J. T. (1968). Prosthecomicrobium and Ancalomicrobium: new prosthecate freshwater bacteria. J Bacteriol 95, 1921–1942.
Komagata, K. & Suzuki, K.-I. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
Kwak, M.-J., Lee, J.-S., Lee, K. C., Kim, K. K., Eom, M. K., Kim, B. K. & Kim, J. F. (2014). Sulfitobacter geojensis sp. nov., Sulfitobacter
composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
noctilucae sp. nov., and Sulfitobacter noctilucicola sp. nov., isolated from coastal seawater. Int J Syst Evol Microbiol 64, 3760–3767.
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
Labrenz, M., Tindall, B. J., Lawson, P. A., Collins, M. D., Schumann, P. & Hirsch, P. (2000). Staleya guttiformis gen. nov., sp. nov. and Sulfitobacter brevis sp. nov., a-3-Proteobacteria from hypersaline,
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific
Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
heliothermal and meromictic antarctic Ekho Lake. Int J Syst Evol Microbiol 50, 303–313.
phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194.
La´nyı´, B. (1987). Classical and rapid identification methods for
Yoon, J.-H., Kim, I.-G., Shin, D.-Y., Kang, K. H. & Park, Y.-H. (2003).
medically important bacteria. Methods Microbiol 19, 1–67. Leifson, E. (1963). Determination of carbohydrate metabolism of
marine bacteria. J Bacteriol 85, 1183–1184. Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
Microbulbifer salipaludis sp. nov., a moderate halophile isolated from a Korean salt marsh. Int J Syst Evol Microbiol 53, 53–57. Yoon, J.-H., Kang, S.-J. & Oh, T.-K. (2007a). Sulfitobacter marinus sp.
nov., isolated from seawater of the East Sea in Korea. Int J Syst Evol Microbiol 57, 302–305. Yoon, J.-H., Kang, S.-J., Lee, M.-H. & Oh, T.-K. (2007b). Description
aquaemixtae sp. nov., isolated from the junction between the ocean and a freshwater spring. Int J Syst Evol Microbiol 64, 1378–1383.
of Sulfitobacter donghicola sp. nov., isolated from seawater of the East Sea in Korea, transfer of Staleya guttiformis Labrenz et al. 2000 to the genus Sulfitobacter as Sulfitobacter guttiformis comb. nov. and emended description of the genus Sulfitobacter. Int J Syst Evol Microbiol 57, 1788–1792.
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International Journal of Systematic and Evolutionary Microbiology 65
Park, S., Park, D.-S., Bae, K. S. & Yoon, J.-H. (2014). Phaeobacter
