Antonie van Leeuwenhoek (2014) 106:763–769 DOI 10.1007/s10482-014-0246-9
ORIGINAL PAPER
Siansivirga jejunensis sp. nov., isolated from seawater of Jeju Island in Korea and emendation of the genus Siansivirga Ji-Min Park • Sang-Keun Han • Dong-Geol Lee Heecheol Kang • Martha E. Trujillo • Dong-Kyu Lee
•
Received: 30 April 2014 / Accepted: 24 July 2014 / Published online: 2 August 2014 Ó Springer International Publishing Switzerland 2014
Abstract A Gram-stain negative, non-flagellated, non-gliding and rod-shaped bacterium, strain JHH-2T, was isolated from seawater collected in Jeju Island, Korea. The novel isolate was found to grow at 25–30 °C, at pH 6.5–7.0 and in the presence of 1–2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JHH-2T is closely related to Siansivirga zeaxanthinifaciens JCM 17682T, with 16S rRNA gene sequence similarity of 96.6 %. The predominant respiratory quinone
was identified as menaquinone-6 and the major cellular fatty acids were detected as iso-C15:0, C16:0 and iso-C17:0 3-OH. Polar lipid profiling revealed the presence of phosphatidylethanolamine, an unidentified glycolipid, four unidentified aminolipids and two unidentified lipids. The DNA G?C content of the chromosomal DNA of the type strain is 35.6 mol%. On the basis of several distinct phenotypic characteristics and phylogenetic analysis, a new species of the genus Siansivirga, Siansivirga jejunensis JHH-2T sp. nov. is proposed. The type strain is JHH-2T (= KCCM 92030T = JCM 19228T).
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0246-9) contains supplementary material, which is available to authorized users. J.-M. Park D.-G. Lee (&) Microbial Metabolite Division, GFC, 309 GFC, TowerDong, Heungdeok IT Valley, 13, Heungdeok 1-Ro, Giheung-gu, Yongin 446-908, Gyeonggi-do, Republic of Korea e-mail: [email protected] J.-M. Park e-mail: [email protected] S.-K. Han Skin Research Institute, Korea Kolmar Corporation, 1211, Dukgogae-gil, Sejong, Republic of Korea e-mail: [email protected]
M. E. Trujillo Departamento de Microbiologı´a y Gene´tica, Universidad de Salamanca, Edificio Departamental, Lab. 214, Campus Miguel de Unamuno, 37007 Salamanca, Spain e-mail: [email protected] D.-K. Lee (&) Department of Industrial Engineering Chemistry, College of Engineering, Chungbuk National University, Cheongju, Gaesin-dong, Republic of Korea e-mail: [email protected]
H. Kang GFC R&D center, GFC, 309 GFC, Tower-Dong, Heungdeok IT Valley, 13, Heungdeok 1-Ro, Giheung-gu, Yongin 446-908, Gyeonggi-do, Republic of Korea e-mail: [email protected]
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Keywords Siansivirga Flavobacteriaceae Seawater Jeju Island
Introduction The genus Siansivirga, a member of the family Flavobacteriaceae in the phylum Bacteroidetes, was recently described based on the single species Siansivirga zeaxanthinifaciens which was isolated from coastal seawater of the China Sea, Taiwan (Hameed et al. 2012). The type strain of S. zeaxanthinifaciens is characterized as strictly aerobic, Gram-stain negative and rod-shaped bacterium with gliding motility. This strain also produces zeaxanthin which is used as a nutraceutical and medicinal ingredient as well as a food and feed supplement (Hameed et al. 2012). In the present study, we describe a novel bacterial strain, JHH-2T, isolated from seawater of Jeju Island in Korea. Phylogenetic and phenotypic analyses suggest that strain JHH-2T represents a hitherto uncharacterized microorganism of the genus Siansivirga for which we propose the name Siansivirga jejunensis sp. nov.
Materials and methods Isolation procedure Strain JHH-2T was isolated from a seawater sample collected in Jeju Island in Korea (33°300 4600 N/ 126°310 2400 E) using a standard dilution-plating method on R2A agar (Becton, Dickinson and Company, BD) at 25 °C after 3 days incubation under aerobic conditions. 16S rRNA gene sequencing analysis Cell biomass for DNA extraction was obtained from cultures grown at 25 °C for 3 days in R2A broth. 16S rRNA gene amplification (Biorad C1000, USA) was performed using the universal primer pair, 27F-1492R as described previously (Lee et al. 2013). Direct sequencing was performed on an ABI PRISM 3730XL analyser (Applied Biosystems) using the BigDye terminator v3.0 cycle sequencing kit as supplied by the manufacturer (Applied Biosystems).Values of sequence similarity were calculated using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim
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et al. 2012). Alignment of sequences was carried out with the software package BioEdit (Hall 1999). A phylogenetic tree was constructed with the neighbourjoining, maximum parsimony and maximum likelihood methods using the software program MEGA 5.2 (Tamura et al. 2011) and bootstrap analysis was performed with 1,000 replications. Molar % G ? C determination and DNA–DNA hybridization The DNA G?C content was determined using HPLC according to the modified method of Tamaoka and Komagata (1984). DNA–DNA hybridization was carried out using a membrane filter technique (Seldin and Dubnau 1985) and a DIG High Prime DNA labelling and detection starter kit II (Roche Molecular Biochemicals). Chemotaxonomic analyses Biomass for chemotaxonomic analyses was obtained from cultures grown at 25 °C for 3 days in R2A broth. Polar lipids were extracted and identified using twodimensional TLC according to the methods described by Minnikin et al. (1984). Isoprenoid quinones were analysed according to the method of Komagata and Suzuki (1987) using reverse-phase HPLC. For cellular fatty acid methyl ester analysis, cells were cultivated for 3 days on R2A agar at 25 °C and collected from the third quadrant of an inoculated plate as explained in Sasser (1990). The cellular fatty acids were extracted using the standard protocol of the Sherlock Microbial Identification System, version 6.2 (MIDI), analysed by gas chromatography (Hewlett Packard 6890) and identified using the RTSBA6 database (Sasser 1990). The type strain zeaxanthinifaciens JCM 17682T was used for comparative fatty acid and polar lipid analyses. Morphological, cultural, physiological and biochemical studies Cell morphology was examined using an optical and a scanning electron microscopes (S4300 N; Hitachi; installed at the Korea Basic Science Institute) as described elsewhere (Lee et al. 2013). Observation of motility was carried out using wet mounts from cultures grown on R2A agar at 25 °C for 3 days. Gliding motility was determined as described by Bowman (2000). Gram reaction was performed using the
Antonie van Leeuwenhoek (2014) 106:763–769
bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Growth temperature range was determined on R2A agar at 4, 10, 15, 20, 25, 28, 30, 37, 40, and 45 °C. The pH range for growth was determined in R2A broth buffered with citrate phosphate or Tris/HCl buffers, adjusted to pH 3–12 (at 0.5 pH unit intervals) as described in Breznak and Costilow (1994). The NaCl concentration range for growth was investigated using R2A agar supplemented with 1–10 % NaCl (w/v, in increments of 1 %). Anaerobic growth was measured in an anaerobic chamber under the following conditions: 90 % N2, 5 % H2, 5 % CO2 (Anaerobic Chamber, Vision Scientific Co., Ltd.). Detection of flexirubin-type pigments was done according to the method of Bernardet et al. (2002). Catalase and oxidase activities were measured as described by Yang and Cho (2008). Hydrolysis of (w/v) casein (1 % skimmed milk), chitin (1 %), starch (1 %), CM cellulose (0.5 %), L-tyrosine (0.2 %) and Tween 80 (v/v, 5 %) were tested on R2A agar as described by Park et al. (2012). DNase activity was examined using DNase test agar (Difco). Carbon source utilization was performed in GN2 MicroPlates (Biolog) following the manufacturer’s instructions. API 20NE and API ZYM (bioMe´rieux) kits were used to record other biochemical characteristics following the manufacturer’s instructions. S. zeaxanthinifaciens JCM 17682T was used for comparative studies under the same conditions. Antibiotics susceptibility Susceptibility to antibiotics was tested on R2A agar plates using the disc diffusion method (Jorgensen et al. 1999). Amounts of antibiotics used per disc (Advantec) were as follows (lg): ampicillin (25), carbenicillin (100), cephalothin (30), chloramphenicol (30), gentamycin (30), kanamycin (30), lincomycin (15), neomycin (30), novobiocin (30), oleandomycin (15), penicillin G (10 U), polymyxin B (300 U), rifampicin (5), streptomysin (25), sulphamethoxazole (25), tetracycline (30).
Results and discussion The 16S rRNA gene sequence of strain JHH-2T comprised 1,423 nucleotides. Sequence similarity analysis in EzTaxon-e server shows moderate level of sequence similarity with S. zeaxanthinifaciens JCM 17682T (96.6 %), followed by Mariniflexile jejuense
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KCTC 23958T (95.9 %). A phylogenetic tree based on the maximum likelihood algorithm (Fig. 1) indicates that strain JHH-2T belongs to the family Flavobacteriaceae, forming a robust cluster with S. zeaxanthinifaciens, supported by a bootstrap value of 95 %. This topology was retrieved by the other two treeing methods used. The DNA G?C content of strain JHH-2T (35.6 mol%) is also similar to that of S. zeaxanthinifaciens JCM 17682T (33.7 mol %) supporting its inclusion in the genus Siansivirga. The DNA–DNA hybridization value of strain JHH-2T with S. zeaxanthinifaciens JCM 17682T was 23.2 %. These results confirmed that strain JHH-2T can be considered as a distinct species of the genus Siansivirga. Cells of strain JHH-2T are found to be facultatively anaerobic and Gram-stain negative. Colonies on R2A agar are circular, non-convex, smooth, yellow and 1.0–5.0 mm in diameter. In comparison to one Siansivirga species currently available, strain JHH-2T is distinct by its ability to degrade casein and gelatin; assimilation of maltose and mannose; and the production of b-glucuronidase, a- and b-glucosidase. Other differences between the two species are presented in Table 1. In addition, strain JHH-2T is susceptible to ampicillin, carbenicillin, chloramphenicol, gentamycin, kanamycin, lincomycin, neomycin, novobiocin, oleandomycin, penicillin G, polymyxin B, rifampicin, streptomysin, tetracycline, but not to cephalothin or sulphamethoxazole. The predominant isoprenoid quinone found in strain JHH-2T is MK-6, which was also reported for the species S. zeaxanthinifaciens (Hameed et al. 2012). The cellular fatty acid profiles of strain JHH-2T and S. zeaxanthinifaciens JCM 17682T are given in Table 2. The major fatty acids ([10 % of the total fatty acids) detected in strain JHH-2T are C16:0 (12.1 %) and isoC15:0 (14.5 %). While the cellular fatty acid profiles of the two strains were similar, several differences were found. The fatty acid iso-C15:1G is only found in S. zeaxanthinifaciens, the proportion of anteiso-C15:0 is differed by ten times between the two strains (Table 2). Strain JHH-2T contains phosphatidylethanolamine (PE), four unidentified aminolipids (AL1-4), an unidentified glycolipid (GL1) and two unidentified lipids (L1–L2) (Fig. 2). This composition is similar to that reported for the type strain of S. zeaxanthinifaciens but varies in the number of glycolipids detected. The polar lipid profile of S. zeaxanthinifaciens obtained in our laboratory differs slightly from the one reported by Hameed et al. (2012) by the number of
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Antonie van Leeuwenhoek (2014) 106:763–769 100 100
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Mariniflexile gromovii KMM6038T (DQ312294) Mariniflexile fucanivornas SW5 T (AJ628046)
Mariniflexile aquimaris HWR-17T (HQ144198) Siansivirga jejunensis JHH-2T (KF023518)
95
94
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Siansivirga jzeathinifaciens CC-SAMT-1-2T (HM179539) Olleya marilimosa CAM030T (EF660466)
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Algibacter lectus KMM 3902T (AY187689) Meridianimaribacter flavus NH57NT (FJ360684)
63 66
Yeosuana aromativorans GW1-1T (AY682382)
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Snuella lapsa JC2132T (HM475133)
Gaetbulibacter saemankumensis SMK-12T (AY883937) Aquibacter zeaxanthinifaciens CC-AMZ-304T (JX235670) 78
Aestuariibaculum suncheonense SC-17T (JF751043)
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Tamlana crocina HST1-43T (AM286230)
0.01
Fig. 1 Maximum likelihood tree based on 16S rRNA gene sequences of strain JHH-2T and related taxa. Evolutionary distances were computed using the Kimura 2-parameter method. Bootstrap values greater than 50 % are indicated. Bar 0.01
substitutions per nucleotide position. Filled dots indicate branches that were also recovered with the neighbour-joining and maximum parsimony methods
unidentified glycolipids and lipids reported by these authors. Therefore, on the basis of 16S rRNA gene sequence similarity, phylogenetic analyses and DDH hybridization, together with the chemotaxonomic and physiological results, strain JHH-2T should be classified as a novel species of the genus Siansivirga, and the name Siansivirga jejunensis sp. nov. is proposed. The genus description has also been emended to accommodate variable physiological characteristics such as facultative anaerobic growth, the lack of gliding motility and oxidase.
yellow, circular, non-convex, entirely smooth and approximately 1.0–1.5 mm in diameter. Growth occurs between 15 and 37 °C, but not at 10 or 40 °C; optimal growth temperature is 25–30 °C. Growth occurs at pH 5.0–11.5, but not at pH 4.5 or 12; optimal growth at pH 6.5–7.0. Growth occurs in the presence of 1–2 % (w/v). Flexirubin-type pigments are absent. Casein, gelatin, and starch are hydrolysed, but not chitin, tween 80 or L-tyrosine. In the Biolog GN2 microplate, a-cyclodextrin, dextrin, Larabinose, D-galactose, gentiobiose, a-D-glucose, a-Dlactose, lactulose, maltose, D-mannose, D-melibiose, D-raffinose, L-rhamnose, sucrose, methyl pyruvate, Dgalacturonic acid, D-gluconic acid, D-glucuronic acid, a-keto-butyric acid, D,L-lactic acid, malonic acid, Dsaccharic acid, glucuronamide, L-alanyl-glycine, Laspartic acid, L-glutamic acid, glycyl-L-glutamic acid, L-ornithine, L-proline, and L-serine are used as carbon sources. In API 20NE, cells are positive for hydrolysis of esculin, b-galactosidase, assimilation of D-glucose L-arabinose, D-mannose, and D-maltose. In API ZYM, alkaline and acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, b-glucuronidase, a-glucosidase, and b-glucosidase are positive. The major isoprenoid quinone is MK-6 and the predominant cellular fatty acids ([10 %) are iso-C15:0 and C16:0. Phospholipid profile contains PE, one
Emended description of the genus Siansivirga Hameed et al. 2012 The description is based on that given by Hameed et al. (2012) and emended as follows: strict aerobic or facultatively anaerobic, gliding motility is variable, oxidase variable. Description of Siansivirga jejunensis sp. nov. Siansivirga jejunensis (jejunensis: je.ju.en’sis. N.L. fem.adj. jejuensis, of or belonging to Jeju-do). Cells are Gram-stain negative rods 0.2–0.3 lm in diameter and 0.8–1.0 lm in length facultatively anaerobic, and non-motile. After 3 days of incubation on R2A agar at 25 °C, colonies on R2A agar are
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Antonie van Leeuwenhoek (2014) 106:763–769 Table 1 Differential phenotypic characteristics of strain JHH2T and Siansivirga zeaxanthinifaciens JCM 17682T Characteristic
JHH-2T
S. zeaxanthinifaciens JCM 17682T
Optimal growth temperature (°C)
25–30
28–30
Facultatively anaerobic growth pH growth range
?
–
5.0–11.5
5.5–9.0
NaCl tolerance
2%
4%
Gliding motility
–
?
Oxidase
–
?
767 Table 2 Cellular fatty acid profiles of strain JHH-2T (1) and the type strain Siansivirga zeaxanthinifaciens JCM 17682T (2) obtained in the present study Fatty acids
?
–
Gelatin
?
–
Chitin
–
?
Tween 80
–
?
W
–
L-arginine
–
?
D-mannose
?
–
D-maltose
?
–
Cellobiose
–
?
–
?
Trypsin
–
W
a-Chymotrypsin
–
W
b-Glucuronidase
?
–
a-Glucosidase
?
–
b-Glucosidase
?
–
PNPG
?
–
35.6
33.7*
Enzyme activities Cystine arylamidase
DNA G?C content (mol%)
Both strains are positive for catalase activity, hydrolysis of esculin, starch, naphthol-AS-BI-phosphohydrolase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and sucrose assimilation. The strains are negative for hydrolysis of L-tyrosine, urea, carboxymethaylcellulose, and DNA, production of indole, a-galactosidase, b-galactosidase, Nacetyl-b-glucosaminidase, a-fucosidase, a-mannosidase and nitrate reduction, activity of lipase (C14), D-glucose fermentation and assimilation of D-mannitol. All data from the present study except for G?C content for the type strain S. zeaxanthinifaciens taken from Hameed et al. 2012 PNPG paranitrophenyl-b-D-galactopyranosidase, ? positive, negative, w weakly positive
C14:0
5.8
–
C16:0
12.1
8.4
C18:0
6.5
tr
Iso-C12:0
2.5
–
Iso-C13:0
3.9
1.2
Iso-C14:0
5.4
1.1
Iso-C15:0
14.5
19.3
Iso-C16:0
tr
1.4
1.1
10.3
Branched saturated
Anteiso-C15:0 Monounsaturated
Assimilation tests L-arabinose
2
Saturated
Hydrolysis of Casein
1
C15:1x5c
–
2.6
C15:1x6c
tr
2.7
C17:1x6c
tr
1.4
C18:1x9c
4.4
tr
Iso-C15:0 3-OH
6.2
6.3
C15:0 2-OH
tr
3.8
Hydroxy acids
C15:0 3-OH
1.9
1.9
Iso-C16:0 3-OH
5.3
1.8
C16:0 3-OH
1.1
1.1
Iso-C17:0 3-OH
8.0
4.4
Branched monounsaturated Iso-C15:1 G
–
16.4
Anteiso-C15:1 A Anteiso-C17:1 x9c
– –
2.4 1.8
Summed feature 1a
6.2
–
Summed feature 3b
4.8
6.7
c
2.0
tr
tr
1.0
Unknown
Summed feature 8
Summed feature 9d
Values are expressed as percentages of total fatty acids - Not detected, tr trace amount (\1.0 %) a
Summed feature 1 consists of C12:0 3-OH and/or iso-C15:1 H
b
Summed feature 3 consists of C16:1x6c and/or C16:1x7c
c
Summed feature 8 consists of C18:1x6c or C18:1x7c
d
Summed feature 9 consists of C16:0 10-methyl or isoC17:1x9c
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(A)
(B)
L1 GL1
L1
GL1 AL3
AL3
L2 AL2
PE
AL2
AL4
1st Dimension
L3
AL1
2nd Dimension
2nd Dimension
PE AL1
GL2 AL4
1st Dimension
Fig. 2 Thin-layer chromatograms of total polar lipids of S. jejunensis sp. nov. JHH-2T (a), S. zeaxanthinifaciens JCM 17682T (b). Spots of total lipids, phospholipids, aminolipids and glycolipids were identified using molybdatophosphoric acid,
molybdenum blue, ninhydrin and a-naphthol reagents. PE, phosphatidylethanolamine; L1-3, unidentified lipids; AL1-4, unidentified aminolipids; GL1-GL2, unidentified glycolipids
unidentified glycolipid, four unidentified aminolipids and two unidentified lipids. DNA G?C content of the type strain is 35.6 mol%. The type strain, JHH-2T (= KCCM 92030T = JCM 19228T), was isolated from seawater of Jeju island in Korea.
Hameed A, Shahina M, Lin SY, Sridhar KR, Young LS, Lee MR, Chen WM, Chou JH, Young CC (2012) Siansivirga zeaxanthinifaciens gen. nov., sp. nov., a novel zeaxanthinproducing member of the family Flavobacteriaceae isolated from coastal seawater of Taiwan. FEMS Microbiol Lett 333:37–45 Jorgensen JH, Turnidge JD, Washington JA (1999) Antibacterial susceptibility tests: dilution and disk diffusion methods. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (eds) Manual of clinical microbiology. American Society for Microbiology, Washington, DC, pp 1526–1543 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721 Komagata K, Suzuki K (1987) Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19:161–207 Lee DG, Park JM, Kang H, Hong SY, Lee KR, Chang HB, Trujillo ME (2013) Asinibacterium lactis gen. nov., sp. nov., a member of the family Chitinophagaceae, isolated from donkey (Equus asinus) milk. Int J Syst Evol Microbiol 63:3180–3185 Minnikin DE, O’onnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, Parlett JH (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2:233–241 Park SC, Choe HN, Baik KS, Lee KH, Seong CN (2012) Gaetbulibacter aestuarii sp. nov., isolated from shallow coastal seawater, and emended description of the genus Gaetbulibacter. Int J Syst Evol Microbiol 62:150–154 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. MIDI Inc, Newark Seldin L, Dubnau D (1985) Deoxyribonucleic acid homology among Bacillus polymyxa, Bacillus macerans, Bacillus
Acknowledgments This work was supported by project National Coordinating Center for Global Cosmetics R&D (A103017) of the Foundation of Korea Cosmetic Industry Institute, Republic of Korea.
References Bernardet JF, Nakagawa Y, Holmes B and Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes (2002). Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J SystEvol Microbiol 52:1049–1070 Bowman JP (2000) Description of Cellulophaga algicola sp. nov., isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50:1861–1868 Breznak JA, Costilow RN (1994) Physicochemical factors in growth. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 137–154 Hall TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis135 program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
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769 using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Yang SJ, Cho JC (2008) Gaetbulibacter marinus sp. nov., isolated from coastal seawater, and emended description of the genus Gaetbulibacter. Int J Syst Evol Microbiol 58:315–318
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ORIGINAL PAPER
Siansivirga jejunensis sp. nov., isolated from seawater of Jeju Island in Korea and emendation of the genus Siansivirga Ji-Min Park • Sang-Keun Han • Dong-Geol Lee Heecheol Kang • Martha E. Trujillo • Dong-Kyu Lee
•
Received: 30 April 2014 / Accepted: 24 July 2014 / Published online: 2 August 2014 Ó Springer International Publishing Switzerland 2014
Abstract A Gram-stain negative, non-flagellated, non-gliding and rod-shaped bacterium, strain JHH-2T, was isolated from seawater collected in Jeju Island, Korea. The novel isolate was found to grow at 25–30 °C, at pH 6.5–7.0 and in the presence of 1–2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JHH-2T is closely related to Siansivirga zeaxanthinifaciens JCM 17682T, with 16S rRNA gene sequence similarity of 96.6 %. The predominant respiratory quinone
was identified as menaquinone-6 and the major cellular fatty acids were detected as iso-C15:0, C16:0 and iso-C17:0 3-OH. Polar lipid profiling revealed the presence of phosphatidylethanolamine, an unidentified glycolipid, four unidentified aminolipids and two unidentified lipids. The DNA G?C content of the chromosomal DNA of the type strain is 35.6 mol%. On the basis of several distinct phenotypic characteristics and phylogenetic analysis, a new species of the genus Siansivirga, Siansivirga jejunensis JHH-2T sp. nov. is proposed. The type strain is JHH-2T (= KCCM 92030T = JCM 19228T).
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0246-9) contains supplementary material, which is available to authorized users. J.-M. Park D.-G. Lee (&) Microbial Metabolite Division, GFC, 309 GFC, TowerDong, Heungdeok IT Valley, 13, Heungdeok 1-Ro, Giheung-gu, Yongin 446-908, Gyeonggi-do, Republic of Korea e-mail: [email protected] J.-M. Park e-mail: [email protected] S.-K. Han Skin Research Institute, Korea Kolmar Corporation, 1211, Dukgogae-gil, Sejong, Republic of Korea e-mail: [email protected]
M. E. Trujillo Departamento de Microbiologı´a y Gene´tica, Universidad de Salamanca, Edificio Departamental, Lab. 214, Campus Miguel de Unamuno, 37007 Salamanca, Spain e-mail: [email protected] D.-K. Lee (&) Department of Industrial Engineering Chemistry, College of Engineering, Chungbuk National University, Cheongju, Gaesin-dong, Republic of Korea e-mail: [email protected]
H. Kang GFC R&D center, GFC, 309 GFC, Tower-Dong, Heungdeok IT Valley, 13, Heungdeok 1-Ro, Giheung-gu, Yongin 446-908, Gyeonggi-do, Republic of Korea e-mail: [email protected]
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Keywords Siansivirga Flavobacteriaceae Seawater Jeju Island
Introduction The genus Siansivirga, a member of the family Flavobacteriaceae in the phylum Bacteroidetes, was recently described based on the single species Siansivirga zeaxanthinifaciens which was isolated from coastal seawater of the China Sea, Taiwan (Hameed et al. 2012). The type strain of S. zeaxanthinifaciens is characterized as strictly aerobic, Gram-stain negative and rod-shaped bacterium with gliding motility. This strain also produces zeaxanthin which is used as a nutraceutical and medicinal ingredient as well as a food and feed supplement (Hameed et al. 2012). In the present study, we describe a novel bacterial strain, JHH-2T, isolated from seawater of Jeju Island in Korea. Phylogenetic and phenotypic analyses suggest that strain JHH-2T represents a hitherto uncharacterized microorganism of the genus Siansivirga for which we propose the name Siansivirga jejunensis sp. nov.
Materials and methods Isolation procedure Strain JHH-2T was isolated from a seawater sample collected in Jeju Island in Korea (33°300 4600 N/ 126°310 2400 E) using a standard dilution-plating method on R2A agar (Becton, Dickinson and Company, BD) at 25 °C after 3 days incubation under aerobic conditions. 16S rRNA gene sequencing analysis Cell biomass for DNA extraction was obtained from cultures grown at 25 °C for 3 days in R2A broth. 16S rRNA gene amplification (Biorad C1000, USA) was performed using the universal primer pair, 27F-1492R as described previously (Lee et al. 2013). Direct sequencing was performed on an ABI PRISM 3730XL analyser (Applied Biosystems) using the BigDye terminator v3.0 cycle sequencing kit as supplied by the manufacturer (Applied Biosystems).Values of sequence similarity were calculated using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim
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et al. 2012). Alignment of sequences was carried out with the software package BioEdit (Hall 1999). A phylogenetic tree was constructed with the neighbourjoining, maximum parsimony and maximum likelihood methods using the software program MEGA 5.2 (Tamura et al. 2011) and bootstrap analysis was performed with 1,000 replications. Molar % G ? C determination and DNA–DNA hybridization The DNA G?C content was determined using HPLC according to the modified method of Tamaoka and Komagata (1984). DNA–DNA hybridization was carried out using a membrane filter technique (Seldin and Dubnau 1985) and a DIG High Prime DNA labelling and detection starter kit II (Roche Molecular Biochemicals). Chemotaxonomic analyses Biomass for chemotaxonomic analyses was obtained from cultures grown at 25 °C for 3 days in R2A broth. Polar lipids were extracted and identified using twodimensional TLC according to the methods described by Minnikin et al. (1984). Isoprenoid quinones were analysed according to the method of Komagata and Suzuki (1987) using reverse-phase HPLC. For cellular fatty acid methyl ester analysis, cells were cultivated for 3 days on R2A agar at 25 °C and collected from the third quadrant of an inoculated plate as explained in Sasser (1990). The cellular fatty acids were extracted using the standard protocol of the Sherlock Microbial Identification System, version 6.2 (MIDI), analysed by gas chromatography (Hewlett Packard 6890) and identified using the RTSBA6 database (Sasser 1990). The type strain zeaxanthinifaciens JCM 17682T was used for comparative fatty acid and polar lipid analyses. Morphological, cultural, physiological and biochemical studies Cell morphology was examined using an optical and a scanning electron microscopes (S4300 N; Hitachi; installed at the Korea Basic Science Institute) as described elsewhere (Lee et al. 2013). Observation of motility was carried out using wet mounts from cultures grown on R2A agar at 25 °C for 3 days. Gliding motility was determined as described by Bowman (2000). Gram reaction was performed using the
Antonie van Leeuwenhoek (2014) 106:763–769
bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Growth temperature range was determined on R2A agar at 4, 10, 15, 20, 25, 28, 30, 37, 40, and 45 °C. The pH range for growth was determined in R2A broth buffered with citrate phosphate or Tris/HCl buffers, adjusted to pH 3–12 (at 0.5 pH unit intervals) as described in Breznak and Costilow (1994). The NaCl concentration range for growth was investigated using R2A agar supplemented with 1–10 % NaCl (w/v, in increments of 1 %). Anaerobic growth was measured in an anaerobic chamber under the following conditions: 90 % N2, 5 % H2, 5 % CO2 (Anaerobic Chamber, Vision Scientific Co., Ltd.). Detection of flexirubin-type pigments was done according to the method of Bernardet et al. (2002). Catalase and oxidase activities were measured as described by Yang and Cho (2008). Hydrolysis of (w/v) casein (1 % skimmed milk), chitin (1 %), starch (1 %), CM cellulose (0.5 %), L-tyrosine (0.2 %) and Tween 80 (v/v, 5 %) were tested on R2A agar as described by Park et al. (2012). DNase activity was examined using DNase test agar (Difco). Carbon source utilization was performed in GN2 MicroPlates (Biolog) following the manufacturer’s instructions. API 20NE and API ZYM (bioMe´rieux) kits were used to record other biochemical characteristics following the manufacturer’s instructions. S. zeaxanthinifaciens JCM 17682T was used for comparative studies under the same conditions. Antibiotics susceptibility Susceptibility to antibiotics was tested on R2A agar plates using the disc diffusion method (Jorgensen et al. 1999). Amounts of antibiotics used per disc (Advantec) were as follows (lg): ampicillin (25), carbenicillin (100), cephalothin (30), chloramphenicol (30), gentamycin (30), kanamycin (30), lincomycin (15), neomycin (30), novobiocin (30), oleandomycin (15), penicillin G (10 U), polymyxin B (300 U), rifampicin (5), streptomysin (25), sulphamethoxazole (25), tetracycline (30).
Results and discussion The 16S rRNA gene sequence of strain JHH-2T comprised 1,423 nucleotides. Sequence similarity analysis in EzTaxon-e server shows moderate level of sequence similarity with S. zeaxanthinifaciens JCM 17682T (96.6 %), followed by Mariniflexile jejuense
765
KCTC 23958T (95.9 %). A phylogenetic tree based on the maximum likelihood algorithm (Fig. 1) indicates that strain JHH-2T belongs to the family Flavobacteriaceae, forming a robust cluster with S. zeaxanthinifaciens, supported by a bootstrap value of 95 %. This topology was retrieved by the other two treeing methods used. The DNA G?C content of strain JHH-2T (35.6 mol%) is also similar to that of S. zeaxanthinifaciens JCM 17682T (33.7 mol %) supporting its inclusion in the genus Siansivirga. The DNA–DNA hybridization value of strain JHH-2T with S. zeaxanthinifaciens JCM 17682T was 23.2 %. These results confirmed that strain JHH-2T can be considered as a distinct species of the genus Siansivirga. Cells of strain JHH-2T are found to be facultatively anaerobic and Gram-stain negative. Colonies on R2A agar are circular, non-convex, smooth, yellow and 1.0–5.0 mm in diameter. In comparison to one Siansivirga species currently available, strain JHH-2T is distinct by its ability to degrade casein and gelatin; assimilation of maltose and mannose; and the production of b-glucuronidase, a- and b-glucosidase. Other differences between the two species are presented in Table 1. In addition, strain JHH-2T is susceptible to ampicillin, carbenicillin, chloramphenicol, gentamycin, kanamycin, lincomycin, neomycin, novobiocin, oleandomycin, penicillin G, polymyxin B, rifampicin, streptomysin, tetracycline, but not to cephalothin or sulphamethoxazole. The predominant isoprenoid quinone found in strain JHH-2T is MK-6, which was also reported for the species S. zeaxanthinifaciens (Hameed et al. 2012). The cellular fatty acid profiles of strain JHH-2T and S. zeaxanthinifaciens JCM 17682T are given in Table 2. The major fatty acids ([10 % of the total fatty acids) detected in strain JHH-2T are C16:0 (12.1 %) and isoC15:0 (14.5 %). While the cellular fatty acid profiles of the two strains were similar, several differences were found. The fatty acid iso-C15:1G is only found in S. zeaxanthinifaciens, the proportion of anteiso-C15:0 is differed by ten times between the two strains (Table 2). Strain JHH-2T contains phosphatidylethanolamine (PE), four unidentified aminolipids (AL1-4), an unidentified glycolipid (GL1) and two unidentified lipids (L1–L2) (Fig. 2). This composition is similar to that reported for the type strain of S. zeaxanthinifaciens but varies in the number of glycolipids detected. The polar lipid profile of S. zeaxanthinifaciens obtained in our laboratory differs slightly from the one reported by Hameed et al. (2012) by the number of
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Antonie van Leeuwenhoek (2014) 106:763–769 100 100
•
•
Mariniflexile gromovii KMM6038T (DQ312294) Mariniflexile fucanivornas SW5 T (AJ628046)
Mariniflexile aquimaris HWR-17T (HQ144198) Siansivirga jejunensis JHH-2T (KF023518)
95
94
•
Siansivirga jzeathinifaciens CC-SAMT-1-2T (HM179539) Olleya marilimosa CAM030T (EF660466)
•
Algibacter lectus KMM 3902T (AY187689) Meridianimaribacter flavus NH57NT (FJ360684)
63 66
Yeosuana aromativorans GW1-1T (AY682382)
•
Snuella lapsa JC2132T (HM475133)
Gaetbulibacter saemankumensis SMK-12T (AY883937) Aquibacter zeaxanthinifaciens CC-AMZ-304T (JX235670) 78
Aestuariibaculum suncheonense SC-17T (JF751043)
•
Tamlana crocina HST1-43T (AM286230)
0.01
Fig. 1 Maximum likelihood tree based on 16S rRNA gene sequences of strain JHH-2T and related taxa. Evolutionary distances were computed using the Kimura 2-parameter method. Bootstrap values greater than 50 % are indicated. Bar 0.01
substitutions per nucleotide position. Filled dots indicate branches that were also recovered with the neighbour-joining and maximum parsimony methods
unidentified glycolipids and lipids reported by these authors. Therefore, on the basis of 16S rRNA gene sequence similarity, phylogenetic analyses and DDH hybridization, together with the chemotaxonomic and physiological results, strain JHH-2T should be classified as a novel species of the genus Siansivirga, and the name Siansivirga jejunensis sp. nov. is proposed. The genus description has also been emended to accommodate variable physiological characteristics such as facultative anaerobic growth, the lack of gliding motility and oxidase.
yellow, circular, non-convex, entirely smooth and approximately 1.0–1.5 mm in diameter. Growth occurs between 15 and 37 °C, but not at 10 or 40 °C; optimal growth temperature is 25–30 °C. Growth occurs at pH 5.0–11.5, but not at pH 4.5 or 12; optimal growth at pH 6.5–7.0. Growth occurs in the presence of 1–2 % (w/v). Flexirubin-type pigments are absent. Casein, gelatin, and starch are hydrolysed, but not chitin, tween 80 or L-tyrosine. In the Biolog GN2 microplate, a-cyclodextrin, dextrin, Larabinose, D-galactose, gentiobiose, a-D-glucose, a-Dlactose, lactulose, maltose, D-mannose, D-melibiose, D-raffinose, L-rhamnose, sucrose, methyl pyruvate, Dgalacturonic acid, D-gluconic acid, D-glucuronic acid, a-keto-butyric acid, D,L-lactic acid, malonic acid, Dsaccharic acid, glucuronamide, L-alanyl-glycine, Laspartic acid, L-glutamic acid, glycyl-L-glutamic acid, L-ornithine, L-proline, and L-serine are used as carbon sources. In API 20NE, cells are positive for hydrolysis of esculin, b-galactosidase, assimilation of D-glucose L-arabinose, D-mannose, and D-maltose. In API ZYM, alkaline and acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, b-glucuronidase, a-glucosidase, and b-glucosidase are positive. The major isoprenoid quinone is MK-6 and the predominant cellular fatty acids ([10 %) are iso-C15:0 and C16:0. Phospholipid profile contains PE, one
Emended description of the genus Siansivirga Hameed et al. 2012 The description is based on that given by Hameed et al. (2012) and emended as follows: strict aerobic or facultatively anaerobic, gliding motility is variable, oxidase variable. Description of Siansivirga jejunensis sp. nov. Siansivirga jejunensis (jejunensis: je.ju.en’sis. N.L. fem.adj. jejuensis, of or belonging to Jeju-do). Cells are Gram-stain negative rods 0.2–0.3 lm in diameter and 0.8–1.0 lm in length facultatively anaerobic, and non-motile. After 3 days of incubation on R2A agar at 25 °C, colonies on R2A agar are
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Antonie van Leeuwenhoek (2014) 106:763–769 Table 1 Differential phenotypic characteristics of strain JHH2T and Siansivirga zeaxanthinifaciens JCM 17682T Characteristic
JHH-2T
S. zeaxanthinifaciens JCM 17682T
Optimal growth temperature (°C)
25–30
28–30
Facultatively anaerobic growth pH growth range
?
–
5.0–11.5
5.5–9.0
NaCl tolerance
2%
4%
Gliding motility
–
?
Oxidase
–
?
767 Table 2 Cellular fatty acid profiles of strain JHH-2T (1) and the type strain Siansivirga zeaxanthinifaciens JCM 17682T (2) obtained in the present study Fatty acids
?
–
Gelatin
?
–
Chitin
–
?
Tween 80
–
?
W
–
L-arginine
–
?
D-mannose
?
–
D-maltose
?
–
Cellobiose
–
?
–
?
Trypsin
–
W
a-Chymotrypsin
–
W
b-Glucuronidase
?
–
a-Glucosidase
?
–
b-Glucosidase
?
–
PNPG
?
–
35.6
33.7*
Enzyme activities Cystine arylamidase
DNA G?C content (mol%)
Both strains are positive for catalase activity, hydrolysis of esculin, starch, naphthol-AS-BI-phosphohydrolase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and sucrose assimilation. The strains are negative for hydrolysis of L-tyrosine, urea, carboxymethaylcellulose, and DNA, production of indole, a-galactosidase, b-galactosidase, Nacetyl-b-glucosaminidase, a-fucosidase, a-mannosidase and nitrate reduction, activity of lipase (C14), D-glucose fermentation and assimilation of D-mannitol. All data from the present study except for G?C content for the type strain S. zeaxanthinifaciens taken from Hameed et al. 2012 PNPG paranitrophenyl-b-D-galactopyranosidase, ? positive, negative, w weakly positive
C14:0
5.8
–
C16:0
12.1
8.4
C18:0
6.5
tr
Iso-C12:0
2.5
–
Iso-C13:0
3.9
1.2
Iso-C14:0
5.4
1.1
Iso-C15:0
14.5
19.3
Iso-C16:0
tr
1.4
1.1
10.3
Branched saturated
Anteiso-C15:0 Monounsaturated
Assimilation tests L-arabinose
2
Saturated
Hydrolysis of Casein
1
C15:1x5c
–
2.6
C15:1x6c
tr
2.7
C17:1x6c
tr
1.4
C18:1x9c
4.4
tr
Iso-C15:0 3-OH
6.2
6.3
C15:0 2-OH
tr
3.8
Hydroxy acids
C15:0 3-OH
1.9
1.9
Iso-C16:0 3-OH
5.3
1.8
C16:0 3-OH
1.1
1.1
Iso-C17:0 3-OH
8.0
4.4
Branched monounsaturated Iso-C15:1 G
–
16.4
Anteiso-C15:1 A Anteiso-C17:1 x9c
– –
2.4 1.8
Summed feature 1a
6.2
–
Summed feature 3b
4.8
6.7
c
2.0
tr
tr
1.0
Unknown
Summed feature 8
Summed feature 9d
Values are expressed as percentages of total fatty acids - Not detected, tr trace amount (\1.0 %) a
Summed feature 1 consists of C12:0 3-OH and/or iso-C15:1 H
b
Summed feature 3 consists of C16:1x6c and/or C16:1x7c
c
Summed feature 8 consists of C18:1x6c or C18:1x7c
d
Summed feature 9 consists of C16:0 10-methyl or isoC17:1x9c
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Antonie van Leeuwenhoek (2014) 106:763–769
(A)
(B)
L1 GL1
L1
GL1 AL3
AL3
L2 AL2
PE
AL2
AL4
1st Dimension
L3
AL1
2nd Dimension
2nd Dimension
PE AL1
GL2 AL4
1st Dimension
Fig. 2 Thin-layer chromatograms of total polar lipids of S. jejunensis sp. nov. JHH-2T (a), S. zeaxanthinifaciens JCM 17682T (b). Spots of total lipids, phospholipids, aminolipids and glycolipids were identified using molybdatophosphoric acid,
molybdenum blue, ninhydrin and a-naphthol reagents. PE, phosphatidylethanolamine; L1-3, unidentified lipids; AL1-4, unidentified aminolipids; GL1-GL2, unidentified glycolipids
unidentified glycolipid, four unidentified aminolipids and two unidentified lipids. DNA G?C content of the type strain is 35.6 mol%. The type strain, JHH-2T (= KCCM 92030T = JCM 19228T), was isolated from seawater of Jeju island in Korea.
Hameed A, Shahina M, Lin SY, Sridhar KR, Young LS, Lee MR, Chen WM, Chou JH, Young CC (2012) Siansivirga zeaxanthinifaciens gen. nov., sp. nov., a novel zeaxanthinproducing member of the family Flavobacteriaceae isolated from coastal seawater of Taiwan. FEMS Microbiol Lett 333:37–45 Jorgensen JH, Turnidge JD, Washington JA (1999) Antibacterial susceptibility tests: dilution and disk diffusion methods. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (eds) Manual of clinical microbiology. American Society for Microbiology, Washington, DC, pp 1526–1543 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721 Komagata K, Suzuki K (1987) Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19:161–207 Lee DG, Park JM, Kang H, Hong SY, Lee KR, Chang HB, Trujillo ME (2013) Asinibacterium lactis gen. nov., sp. nov., a member of the family Chitinophagaceae, isolated from donkey (Equus asinus) milk. Int J Syst Evol Microbiol 63:3180–3185 Minnikin DE, O’onnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, Parlett JH (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2:233–241 Park SC, Choe HN, Baik KS, Lee KH, Seong CN (2012) Gaetbulibacter aestuarii sp. nov., isolated from shallow coastal seawater, and emended description of the genus Gaetbulibacter. Int J Syst Evol Microbiol 62:150–154 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. MIDI Inc, Newark Seldin L, Dubnau D (1985) Deoxyribonucleic acid homology among Bacillus polymyxa, Bacillus macerans, Bacillus
Acknowledgments This work was supported by project National Coordinating Center for Global Cosmetics R&D (A103017) of the Foundation of Korea Cosmetic Industry Institute, Republic of Korea.
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