Antonie van Leeuwenhoek (2015) 107:1009–1015 DOI 10.1007/s10482-015-0393-7
ORIGINAL PAPER
Eudoraea chungangensis sp. nov., isolated from an aquafarm waste water sludge Chatuphon Siamphan • Young-Hyo Chang Wonyong Kim
•
Received: 12 October 2014 / Accepted: 20 January 2015 / Published online: 28 January 2015 Ó Springer International Publishing Switzerland 2015
Abstract A Gram-stain negative, non-spore-forming, non-motile, strictly aerobic bacterial strain, designated CAU 1296T, was isolated from an aquafarm waste water sludge and its taxonomic position was investigated using a polyphasic approach. Cells of strain CAU 1296T grew optimally at 30 °C, at pH 7.5 and in the presence of 4 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAU 1296T formed a distinct lineage within the genus Eudoraea and exhibited similarity to Eudoraea adriatica AS06/20aT (95.4 % similarity). The major cellular fatty acids of the isolate were isoC15:0, iso-C15:1 G, and summed feature 3 (C16:1 x6c and C16:1 x7c). The polar lipid pattern of strain CAU 1296T consisted of phosphatidylethanolamine, and unidentified lipids including a phosphoglycolipid, phospholipid, glycolipid, aminophospholipid, two aminolipids, and six further unidentified lipids. The strain contained menaquinone-6 (MK-6) as the major Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0393-7) contains supplementary material, which is available to authorized users. C. Siamphan W. Kim (&) Department of Microbiology, College of Medicine, Chung-Ang University (CAU), Seoul, South Korea e-mail: [email protected] Y.-H. Chang Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea
isoprenoid quinone. The G?C content of the genomic DNA was 38.7 mol%. On the basis of phenotypic, chemotaxonomic, and phylogenetic data, strain CAU 1296T should be classified as a novel species in the genus Eudoraea, for which the name Eudoraea chungangensis sp. nov. is proposed. The type strain is CAU 1296T (=KCTC 42048T, =CCUG 66239T, =CECT 8744T). Keywords Eudoraea chungangensis Flavobacteriaceae Fish farm sludge
Introduction The genus Eudoraea, a member of the family Flavobacteriaceae, was created by Alain et al. (2008) with the description of Eudoraea adriaca as the type species of the genus within the phylum Bacteroidetes. At the time of writing, this genus consists of only one recognized species, E. adriaca, which was isolated from coastal waters of the Adriatic Sea, Italy (http:// www.bacterio.net/eudoraea.html). Bacteroidetes represent a large fraction of the community degrading the high molecular weight fraction of marine dissolved organic material (DOM), known mainly from 16S metagenomic studies. For example Cottrell and Kirchman (2000) showed Bacteroidetes were a dominant fraction degrading chitin. Isolation and description of isolates such as Eudoraea remains the best approach to
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discovery of their physiological and genomic properties (Alain et al. 2008). The genus Eudoraea comprises aerobic, Gram-negative, non-motile, rod-shaped, mesophilic bacteria that are characterized chemotaxonomically by the presence of MK-6 as the major isoprenoid quinone and iso-C15:0, iso-C15:1 G, as the predominant cellular fatty acids. The only current member of the genus requires NaCl for growth. In the course of screening bacteria from marine environmental samples, a bacterial strain, designated CAU 1296T, was isolated from an aquafarm waste water sludge sample collected on Jeju Island (33.560905N, 126.81482E) in the Republic of Korea. The purpose of this study was to establish the taxonomic position of this bacterial strain using a polyphasic characterization that included the determination of phenotypic, chemotaxonomic properties, and 16S rRNA gene sequence analysis.
Materials and methods Isolation and maintenance of microorganism Strain CAU 1296T was isolated from a waste water sludge sample collected from a fish aquafarm of the eastern coast of Jeju Island following Gordon and Mihm (1962) using marine agar 2216 (MA; Difco Laboratories Detroit MI, USA), supplemented with cycloheximide (50 mg/l-1) and nalidixic acid (20 mg/l-1). The sample was diluted with sterilized saline solution so that appropriate dilutions could be spread on MA plates. The agar plates were incubated under aerobic conditions at 30 °C for 7 days. A pure single colony was purified by subculturing and preserved at -80 °C in marine broth (MB; Difco) supplemented with 25 % (v/v) glycerol for taxonomic analysis. Strain CAU 1296T has been deposited in the Korean Collection for Type Cultures (KCTC; Taejon, Korea) and the Culture Collection, University of Gothenburg (CCUG; Goteborg, Sweden) as KCTC 42048T and CCUG 66239, respectively. Phylogenetic analysis and genomic DNA analysis Genomic DNA of strain CAU 1296T was extracted by the method of Marmur (1961). PCR amplification and sequencing of the 16S rRNA gene of the strain was carried out following established procedures (Cho
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et al. 2008). The amplified 16S rRNA gene was sequenced directly by using a BigDye Terminator Cycle Sequnceing kit (Applied Biosystems Life Technologies Corporation, Carlsbad, CA, USA) and an automatic 3730 DNA sequencer (Applied Biosystems). Multiple alignments and calculation of the levels of 16S rRNA gene sequence similarity with sequences of the type strain of the Eudoraea species and a broad selection of the closely related genus were performed by using the EzTaxon-e server (Kim et al. 2012; http://eztaxon-e.ezbiocloud.net/) and CLUSTAL_X (Thompson et al. 1997). Evolutionary distance matrices were generated by the neighbor-joining method described by Jukes and Cantor (1969). Phylogenetic trees were generated using the neighbourjoining (Saitou and Nei 1987), least-squares (Fitch and Margoliash 1967), and maximum-likelihood (Felsenstein 1981) algorithms in the PHYLIP package (Felsenstein 1989). Branch support in the neighbourjoining tree was evaluated by the bootstrap resampling method, with 1,000 replicates (Felsenstein 1985). The mol% G?C content of the genomic DNA was determined following a modification of the method of Tamaoka and Komagata (1984), the DNA was hydrolyzed and the resultant nucleotides were analyzed by reverse-phase HPLC. Morphological, physiological and biochemical characterization For the investigation of morphological, physiological and biochemical characteristics, strain CAU 1296T was cultivated on MA at 30 °C. Cell morphology was observed under a DM 1,000 light microscope (Leica Microsystems AG, Wetzlar, Germany) using cells from an exponentially growing culture. Gram staining was carried out using the bioMe´rieux Gram staining kit (bioMe´rieux Inc., Durham, NC, USA) according to the manufacturer’s instructions. Motility was assessed on an MB culture using the hanging-drop method (Nedashkovskaya et al. 2010). Growth between 4 and 40 °C was determined in a MIR-253 aerobic incubator (Sanyo Electric Co. Ltd, Wood Dale, IL, USA) and in a Bactron anaerobic chamber (Sheldon Manufacturing, Inc., Cornelius, OR, USA) by measuring the turbidity of marine broth 2216 (MB, Difco) after 72 h incubation. The pH range for growth was investigated in MB that had been adjusted to pH 4.5–10.0 (at intervals of 0.5 pH unit) by using sodium acetate/
Antonie van Leeuwenhoek (2015) 107:1009–1015
1011 Maribacter orientalis KMM 3947T (AY271624) Maribacter arcticus KOPRI 20941T (AY771762) Maribacter forsetii KT02ds18-6T (AM712900)
94
Maribacter aquivivus KMM 3949T (AY271625) Maribacter stanieri KMM 6046T (EU246691) Maribacter dokdonensis DSW-8T (AY960749)
100
Maribacter polysiphoniae LMG 23671T (AM497875) Kriegella aquimaris KMM 3665T (AB084262)
66
Arenibacter palladensis LMG 21972T (AJ575643) 97 Arenibacter troitsensis KMM 3674T (AB080771)
100
Arenibacter echinorum KMM 6032T (EF536748)
100
Arenibacter nanhaiticus NH36AT ( EU999955) Cellulophaga pacifica KMM 3664T (AB100840) 99
Cellulophaga baltica NN015840T (AJ005972)
100
Cellulophaga tyrosinoxydans EM41T (EU443205) Eudoraea chungangensis CAU 1296T (KJ150297) 83
Eudoraea adriatica AS06/20aT (AM745437)
99
Muriicola jejuensis EM44T (EU443206) Zeaxanthinibacter enoshimensis TD-ZE3T (AB264057) Escherichia coli ATCC 11775T (X80725)
0.1
Fig. 1 Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships between strain CAU 1296T and the type strains of recognized Eudoraea species. Dots indicate that the corresponding nodes were also recovered in the trees generated with the maximumlikelihood and least squares algorithms. The numbers at the
nodes indicate levels of bootstrap support based on a neighbourjoining analysis of 1,000 resampled datasets; only values[70 % are given. Bar 0.1 substitutions per nucleotide position. Escherichia coli ATCC 11775T (X80725) is used as an outgroup organism
acetic acid and Na2CO3 buffers. Growth in the absence of NaCl and in the presence of 0–15.0 % (w/v) NaCl was investigated in Trypticase soy broth (TSB) prepared according to the formula of Difco medium except that NaCl was excluded and 0.45 % (w/v) MgCl26H2O or 0.06 % (w/v) KCl was added. Oxidase activity was evaluated from the oxidation of 0.1 % (w/v) tetramethyl-p-phenylenediamine (Cappuccino and Sherman 2002). Catalase activity was determined by bubble production in 3 % (v/v) H2O2 solution. Hydrolysis of casein, starch and urea were determined according to the methods of La´nyı´ (1987) and Smibert and Krieg (1994). Acid production from carbohydrates, enzyme activity, and other physiological and biochemical features were tested as described
by Leifson (1963), using the API 20NE, API ZYM strips (bioMe´rieux) and Biolog GN2 microplates (Omnilog system; Biolog,. Hayward, CA, USA). According to the manufacturer’s instructions with incubation time of up to 6 day at 30 °C. Chemotaxonomic characterization For the determination of fatty acid composition, the cell mass of strain CAU 1296T was harvested from MA (Difco) after cultivation for 6 days at 30 °C. The physiological age of the biomasses harvested for fatty acid analysis was standardized by observing growth development during incubation of the cultures and choosing the moment of harvesting according to the
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standard MIDI Sherlock Microbial Identification System (MIDI, Inc., Newark, DE, USA) method. Cellular fatty acid methyl esters (FAMEs) were obtained by using the method of Minnikin et al. (1980) and separated in a 6,890 N gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) fitted with a 7,683 autosampler (Agilent). Peaks were identified by using the Microbial Identification software package (MOORE library ver. 5.0; MIDI database TSBA6). The polar lipids of strain CAU 1296T were identified using twodimensional thin-layer chromatography (TLC) by the method of Minnikin et al. (1984). The plate was sprayed with 10 % ethanolic molybdophosphoric acid (for the total lipids), molybdenum blue (for phospholipids), ninhydrin (for aminolipids) and a-naphthol/sulphuric acid reagent (for glycolipids). Menaquinones were separated on HPLC using an isocratic solvent system [methanol/isopropyl ether (3:1, v/v)] and the flow rate (1 ml/min) (Komagata and Suzuki 1987).
Result and discussion The nearly complete 16S rRNA gene sequence of strain CAU 1296T (1,540 bp) was determined and Table 1 Differential properties of strain CAU 1296T and the type strains of the genus Eudoraea, E. adriatica AS06/20aT (data from Alain et al. 2008)
compared with the corresponding sequences of other bacterial strains in the public database (GenBank/ EMBL/DDBJ accession number KJ150297). Phylogenetic analysis indicated that the strain fell into the genus Eudoraea. The neighbour-joining tree is shown in Fig. 1. The trees obtained with the maximumlikelihood and least-square algorithms showed basically the same topology (data not shown). Pairwise analysis showed that the most closely related strain is E. adriatica AS06/20aT (16S rRNA gene similarity, 95.4 %). The G?C content of the DNA of strain CAU 1296T was 38.7 mol%, which is slightly lower than that of the type species in the genus, E. adriatica AS06/20aT (Alain et al. 2008), which is distinctive. The detailed morphological, cultural, physiological and biochemical characteristics are presented in Table 1 and the species description. Strain CAU 1296T is Gram-stain negative, non-spore forming, strictly aerobic, and non-motile. Colonies were yellow in color, entire, circular, and convex with diameters of 0.3–0.5 mm after 5 days of cultivation on MA at 30 °C and at pH 6.5–8.5 (optimum, pH 7.5). No colonies were formed under anaerobic growth condition. Cells are rod-shaped approximately 0.3–0.5 lm in diameter and 1.3–2.0 lm in length. Because CAU
Characteristic
Strain CAU 1296T
E. adriatica AS06/20aT
Temperature for growth (°C) (optimum)
20–30 (30)
15–33 (30)
NaCl for growth (%, w/v) (optimum)
2–6 (4)
2–6 (2)
pH for growth (optimum)
6.5–8.5 (7.5)
6.5–8.5 (7.5–8.0)
Catalase
?
?
Oxidase
?
?
Hydrolysis of: gelatin
?
-
urease
-
-
aesculin
?
?
D-Glucose
-
?
L-Arabinose
-
?
Tween 40
?
-
Alkaline phosphatase
?
?
esterase (C4)
?
?
a-Chymotrypsin
-
?
b-Glucosidasse
-
?
a-Mannosidase
-
?
DNA G?C content (mol%)
38.7
38.9
Substrate assimilation:
Enzymic activities:
? Positive, - negative
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Antonie van Leeuwenhoek (2015) 107:1009–1015 Table 2 Characteristic and cellular fatty acid contents (%) of strain CAU 1296T and the type strains of the genus Eudoraea, E. adriatica AS06/20aT (data from Alain et al. 2008)
Fatty acid
1013 Strain CAU 1296T
E. adriatica AS06/20aT
Hydroxy C15:0 2-OH
0.8
0.3
C15:0 3-OH
1.2
1.3
iso-C15:0 3-OH
4.8
7.3
C16:0 3-OH
1.0
0.4
iso-C16:0 3-OH
0.8
1.4
C17:0 3-OH
–
0.2
iso-C17:0 3-OH Straight-chain
5.7
14.1
C14:0
0.4
0.2
C16:0
1.0
0.7
iso-C13:0
0.6
0.5
iso-C15:0
22.4
29.4
anteiso-C15:0
3.2
0.7
iso-C15:1 G
24.1
16.0
iso-C16:0
1.1
0.3
iso-C17:0
–
0.2
Branched
Unsaturated – not detected a
Summed features consist of two or more fatty acids that could not be separated by GLC using the MIDI system. Summed feature 3 comprised C16:1 x7c/C16:1 x6c
C15:1 x6c
6.1
1.1
C17:1 x6c
2.0
0.8
C17:1 x8c
1.2
0.5
iso-C17:1 x9c C18:1 x5c
6.7 0.3
9.0 0.7
Summed feature 3a
14.5
11.6
1296T was isolated from a saline environment, it require NaCl for growth ranging from 2 to 6 % (w/v) NaCl [optimum, 4 % (w/v) NaCl]. Strain CAU 1296T hydrolysed gelatin and aesulin, assimilated Tween 40, and had enzyme activities for alkaline phosphatase, and esterase (C4). However, strain CAU 1296T differed from its closest relative, E. adriatica AS06/ 20aT (Alain et al. 2008) by its ability to hydrolyze gelatin, assimilate tween 40, inability to assimilate Dglucose and L-arabinose and lack of a-chymotrypsin, b-glucosidase, and a-mannosidase enzymatic activity. Overall, the chemotaxonomy was in agreement with previously published data for the genus Eudoraea. The predominant isoprenoid quinone detected in strain CAU 1296T was MK-6, in line with all members of the family Flavobacteriaceae (Bernardet et al. 2002), which is consistent with data for the type species of the genus, E. adriatica AS06/20aT (Alain et al. 2008). A minor amount of MK-7 was also detected. The fatty acid profile of strain CAU 1296T is
shown in Table 2, together with E. adriatica AS06/ 20aT data obtained from the previous study (Alain et al. 2008). The strain contained unsaturated, straightchain, branched, and hydroxy fatty acids. The major fatty acids ([10 %) were iso-C15:1 G (24.1 %), isoC15:0 (22.4 %), and C16:1 x6c and C16:1 x7c (summed feature 3; groups of two or three fatty acids could not be separated with the MIDI system, 14.5 %). The following fatty acids ([1 %) C15:0 3-OH, iso-C15:0 3-OH, C16:0 3-OH, iso-C17:0 3-OH, anteiso-C15:0, iso-C16:0, C15:1 x6c, C17:1 x6c, C17:1 x8c and iso-C17:1 x9c were also present. This fatty acid profile is very similar to the closely related E. adriatica AS06/ 20aT in that iso-C15:1 G and iso-C15:0 were predominant, although there were differences in the proportions of some fatty acids. However, strain CAU 1296T could be distinguished from E. adriatica AS06/20aT (Alain et al. 2008) by absence of C17:0 3-OH and iso-C17:0. Strain CAU 1296T contained phosphatidylethanolamine as the major polar lipid identified, as
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in E. adriatica AS06/20aT. In addition, cells of CAU 1296T contained unidentified lipids: a phosphoglycolipid; phospholipid: glycolipid; aminophospholipid; two aminolipids, and six other unidentified lipids (Suppl. Fig. S1.). By contrast, the polar lipid composition of E. adriatica AS06/20aT clearly differs from this, largely unidentified lipid composition. In conclusion, it is evident from the phenotypic, chemotaxonomic and genotypic data that strain CAU 1296T represents a novel species of the genus Eudoraea, for which the name Eudoraea chungangensis sp. nov. is proposed. Description of Eudoraea chungangensis sp. nov Eudoraea chungangensis sp. nov. (chung.ang.en’sis. N.L. fem. adj. chungangensis referring to Chung-Ang University where the first taxonomic studies on this species were performed). Cells are Gram-stain negative, non-motile, strictly aerobic rods approximately 0.3–0.5 lm in diameter and 1.3–2.0 lm in length. Colonies on MA are yellow colored, circular, convex and 0.3–0.5 mm in diameter after 5 days of incubation at 30 °C. Growth occurs at 20–30 °C (optimum, 30 °C) and at pH 6.5–8.5 (optimum, pH 7.50). Growth occurs in the presence of 2–6 % (w/v) NaCl [optimum, 4 % (w/v) NaCl]. Catalase and oxidase positive, does not reduce nitrate to nitrite. Gelatin and aesculin are hydrolyzed. Citrate is not utilized. b-galactosidase (PNPG) is positive (API20NE). Substrate assimilation positive for Tween 40, D-galactonic acid lactone and a-D-glucose-1phosphate. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naptholAS-BI-phosphohydrolase and N-acetyl-b-glucosaminidase are detected. The predominant respiratory quinone is MK-6. The polar lipid pattern consists of phosphatidylethanolamine, and unidentified phosphoglycolipid, phospholipid, glycolipid, aminophospholipid, two aminolipids, and six other unidentified lipids. The predominant cellular fatty acids ([10 % of the total fatty acids) are iso-C15:1 G, iso-C15:0 and summed feature 3 (C16:1 x6c and C16:1 x7c). The DNA G?C content of the type strain is 38.7 mol%. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CAU 1296T is KJ150297. The type strain is CAU 1296T (=KCTC 42048T, =CCUG 66239T, =CECT 8744T), isolated
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from an aquafarm waste water sludge collected on Jeju Island in the Republic of Korea.
References Alain K, Intertaglia L, Catala P, Lebaron P (2008) Eudoraea adriatica gen. nov., sp. nov., a novel marine bacterium of the family Flavobacteriaceae. Int J Syst Evol Microbiol 58:2275–2281 Bernardet J-F, Nakagawa Y, Holmes B for the 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 Syst Evol Microbiol 52:1049–1070 Cappuccino JG, Sherman N (2002) Microbiology: a laboratory manual, 6th edn. Benjamin/Cummings, Menlo Park Cho SL, Nam SW, Yoon JH, Lee JS, Sukhoom A, Kim W (2008) Lactococcus chungangensis sp. nov., a lactic acid bacterium isolated from activated sludge foam. Int J Syst Evol Microbiol 58:1844–1849 Cottrell MT, Kirchman DL (2000) Natural assemblages of marine proteobacteria and members of the CytophagaFlavobacter cluster consuming low- and high-molecularweight dissolved organic matter. Appl Environ Microbiol 66:1692–1697 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evolut 17:368–376 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791 Felsenstein J (1989) PHYLIP—phylogeny inference package (version3.2). Cladistics 5:164–166 Fitch WM, Margoliash E (1967) Construction of phylogenetic trees. Science 155:279–284 Gordon RE, Mihm JM (1962) Identification of Nocardia caviae (Erikson) nov. comb. Ann N Y Acad Sci 98:628–636 Jukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HH (ed) Mammalian protein metabolism. Academic Press, New York, pp 21–132 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 19:161–208 La´nyı´ B (1987) Classical and rapid identification methods for medically important bacteria. Methods Microbiol 19:1–67 Leifson E (1963) Determination of carbohydrate metabolism of marine bacteria. J Bacteriol 85:1183–1184 Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218 Minnikin DE, Hutchinson IG, Caldicott AB, Goodfellow M (1980) Thin-layer chromatography of methanolysates of mycolic acid-containing bacteria. J Chromatogr 188:221–233
Antonie van Leeuwenhoek (2015) 107:1009–1015 Minnikin DE, O’Donnell 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 Nedashkovskaya OI, Kim SB, Mikhailov VV (2010) Maribacter stanieri sp. nov., a marine bacterium of the family Flavobacteriaceae. Int J Syst Evol Microbiol 60:214–218 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds)
1015 Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 607–654 Tamaoka J, Komagata K (1984) Determination of DNA base composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25:125–128 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882
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ORIGINAL PAPER
Eudoraea chungangensis sp. nov., isolated from an aquafarm waste water sludge Chatuphon Siamphan • Young-Hyo Chang Wonyong Kim
•
Received: 12 October 2014 / Accepted: 20 January 2015 / Published online: 28 January 2015 Ó Springer International Publishing Switzerland 2015
Abstract A Gram-stain negative, non-spore-forming, non-motile, strictly aerobic bacterial strain, designated CAU 1296T, was isolated from an aquafarm waste water sludge and its taxonomic position was investigated using a polyphasic approach. Cells of strain CAU 1296T grew optimally at 30 °C, at pH 7.5 and in the presence of 4 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAU 1296T formed a distinct lineage within the genus Eudoraea and exhibited similarity to Eudoraea adriatica AS06/20aT (95.4 % similarity). The major cellular fatty acids of the isolate were isoC15:0, iso-C15:1 G, and summed feature 3 (C16:1 x6c and C16:1 x7c). The polar lipid pattern of strain CAU 1296T consisted of phosphatidylethanolamine, and unidentified lipids including a phosphoglycolipid, phospholipid, glycolipid, aminophospholipid, two aminolipids, and six further unidentified lipids. The strain contained menaquinone-6 (MK-6) as the major Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0393-7) contains supplementary material, which is available to authorized users. C. Siamphan W. Kim (&) Department of Microbiology, College of Medicine, Chung-Ang University (CAU), Seoul, South Korea e-mail: [email protected] Y.-H. Chang Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea
isoprenoid quinone. The G?C content of the genomic DNA was 38.7 mol%. On the basis of phenotypic, chemotaxonomic, and phylogenetic data, strain CAU 1296T should be classified as a novel species in the genus Eudoraea, for which the name Eudoraea chungangensis sp. nov. is proposed. The type strain is CAU 1296T (=KCTC 42048T, =CCUG 66239T, =CECT 8744T). Keywords Eudoraea chungangensis Flavobacteriaceae Fish farm sludge
Introduction The genus Eudoraea, a member of the family Flavobacteriaceae, was created by Alain et al. (2008) with the description of Eudoraea adriaca as the type species of the genus within the phylum Bacteroidetes. At the time of writing, this genus consists of only one recognized species, E. adriaca, which was isolated from coastal waters of the Adriatic Sea, Italy (http:// www.bacterio.net/eudoraea.html). Bacteroidetes represent a large fraction of the community degrading the high molecular weight fraction of marine dissolved organic material (DOM), known mainly from 16S metagenomic studies. For example Cottrell and Kirchman (2000) showed Bacteroidetes were a dominant fraction degrading chitin. Isolation and description of isolates such as Eudoraea remains the best approach to
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discovery of their physiological and genomic properties (Alain et al. 2008). The genus Eudoraea comprises aerobic, Gram-negative, non-motile, rod-shaped, mesophilic bacteria that are characterized chemotaxonomically by the presence of MK-6 as the major isoprenoid quinone and iso-C15:0, iso-C15:1 G, as the predominant cellular fatty acids. The only current member of the genus requires NaCl for growth. In the course of screening bacteria from marine environmental samples, a bacterial strain, designated CAU 1296T, was isolated from an aquafarm waste water sludge sample collected on Jeju Island (33.560905N, 126.81482E) in the Republic of Korea. The purpose of this study was to establish the taxonomic position of this bacterial strain using a polyphasic characterization that included the determination of phenotypic, chemotaxonomic properties, and 16S rRNA gene sequence analysis.
Materials and methods Isolation and maintenance of microorganism Strain CAU 1296T was isolated from a waste water sludge sample collected from a fish aquafarm of the eastern coast of Jeju Island following Gordon and Mihm (1962) using marine agar 2216 (MA; Difco Laboratories Detroit MI, USA), supplemented with cycloheximide (50 mg/l-1) and nalidixic acid (20 mg/l-1). The sample was diluted with sterilized saline solution so that appropriate dilutions could be spread on MA plates. The agar plates were incubated under aerobic conditions at 30 °C for 7 days. A pure single colony was purified by subculturing and preserved at -80 °C in marine broth (MB; Difco) supplemented with 25 % (v/v) glycerol for taxonomic analysis. Strain CAU 1296T has been deposited in the Korean Collection for Type Cultures (KCTC; Taejon, Korea) and the Culture Collection, University of Gothenburg (CCUG; Goteborg, Sweden) as KCTC 42048T and CCUG 66239, respectively. Phylogenetic analysis and genomic DNA analysis Genomic DNA of strain CAU 1296T was extracted by the method of Marmur (1961). PCR amplification and sequencing of the 16S rRNA gene of the strain was carried out following established procedures (Cho
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et al. 2008). The amplified 16S rRNA gene was sequenced directly by using a BigDye Terminator Cycle Sequnceing kit (Applied Biosystems Life Technologies Corporation, Carlsbad, CA, USA) and an automatic 3730 DNA sequencer (Applied Biosystems). Multiple alignments and calculation of the levels of 16S rRNA gene sequence similarity with sequences of the type strain of the Eudoraea species and a broad selection of the closely related genus were performed by using the EzTaxon-e server (Kim et al. 2012; http://eztaxon-e.ezbiocloud.net/) and CLUSTAL_X (Thompson et al. 1997). Evolutionary distance matrices were generated by the neighbor-joining method described by Jukes and Cantor (1969). Phylogenetic trees were generated using the neighbourjoining (Saitou and Nei 1987), least-squares (Fitch and Margoliash 1967), and maximum-likelihood (Felsenstein 1981) algorithms in the PHYLIP package (Felsenstein 1989). Branch support in the neighbourjoining tree was evaluated by the bootstrap resampling method, with 1,000 replicates (Felsenstein 1985). The mol% G?C content of the genomic DNA was determined following a modification of the method of Tamaoka and Komagata (1984), the DNA was hydrolyzed and the resultant nucleotides were analyzed by reverse-phase HPLC. Morphological, physiological and biochemical characterization For the investigation of morphological, physiological and biochemical characteristics, strain CAU 1296T was cultivated on MA at 30 °C. Cell morphology was observed under a DM 1,000 light microscope (Leica Microsystems AG, Wetzlar, Germany) using cells from an exponentially growing culture. Gram staining was carried out using the bioMe´rieux Gram staining kit (bioMe´rieux Inc., Durham, NC, USA) according to the manufacturer’s instructions. Motility was assessed on an MB culture using the hanging-drop method (Nedashkovskaya et al. 2010). Growth between 4 and 40 °C was determined in a MIR-253 aerobic incubator (Sanyo Electric Co. Ltd, Wood Dale, IL, USA) and in a Bactron anaerobic chamber (Sheldon Manufacturing, Inc., Cornelius, OR, USA) by measuring the turbidity of marine broth 2216 (MB, Difco) after 72 h incubation. The pH range for growth was investigated in MB that had been adjusted to pH 4.5–10.0 (at intervals of 0.5 pH unit) by using sodium acetate/
Antonie van Leeuwenhoek (2015) 107:1009–1015
1011 Maribacter orientalis KMM 3947T (AY271624) Maribacter arcticus KOPRI 20941T (AY771762) Maribacter forsetii KT02ds18-6T (AM712900)
94
Maribacter aquivivus KMM 3949T (AY271625) Maribacter stanieri KMM 6046T (EU246691) Maribacter dokdonensis DSW-8T (AY960749)
100
Maribacter polysiphoniae LMG 23671T (AM497875) Kriegella aquimaris KMM 3665T (AB084262)
66
Arenibacter palladensis LMG 21972T (AJ575643) 97 Arenibacter troitsensis KMM 3674T (AB080771)
100
Arenibacter echinorum KMM 6032T (EF536748)
100
Arenibacter nanhaiticus NH36AT ( EU999955) Cellulophaga pacifica KMM 3664T (AB100840) 99
Cellulophaga baltica NN015840T (AJ005972)
100
Cellulophaga tyrosinoxydans EM41T (EU443205) Eudoraea chungangensis CAU 1296T (KJ150297) 83
Eudoraea adriatica AS06/20aT (AM745437)
99
Muriicola jejuensis EM44T (EU443206) Zeaxanthinibacter enoshimensis TD-ZE3T (AB264057) Escherichia coli ATCC 11775T (X80725)
0.1
Fig. 1 Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships between strain CAU 1296T and the type strains of recognized Eudoraea species. Dots indicate that the corresponding nodes were also recovered in the trees generated with the maximumlikelihood and least squares algorithms. The numbers at the
nodes indicate levels of bootstrap support based on a neighbourjoining analysis of 1,000 resampled datasets; only values[70 % are given. Bar 0.1 substitutions per nucleotide position. Escherichia coli ATCC 11775T (X80725) is used as an outgroup organism
acetic acid and Na2CO3 buffers. Growth in the absence of NaCl and in the presence of 0–15.0 % (w/v) NaCl was investigated in Trypticase soy broth (TSB) prepared according to the formula of Difco medium except that NaCl was excluded and 0.45 % (w/v) MgCl26H2O or 0.06 % (w/v) KCl was added. Oxidase activity was evaluated from the oxidation of 0.1 % (w/v) tetramethyl-p-phenylenediamine (Cappuccino and Sherman 2002). Catalase activity was determined by bubble production in 3 % (v/v) H2O2 solution. Hydrolysis of casein, starch and urea were determined according to the methods of La´nyı´ (1987) and Smibert and Krieg (1994). Acid production from carbohydrates, enzyme activity, and other physiological and biochemical features were tested as described
by Leifson (1963), using the API 20NE, API ZYM strips (bioMe´rieux) and Biolog GN2 microplates (Omnilog system; Biolog,. Hayward, CA, USA). According to the manufacturer’s instructions with incubation time of up to 6 day at 30 °C. Chemotaxonomic characterization For the determination of fatty acid composition, the cell mass of strain CAU 1296T was harvested from MA (Difco) after cultivation for 6 days at 30 °C. The physiological age of the biomasses harvested for fatty acid analysis was standardized by observing growth development during incubation of the cultures and choosing the moment of harvesting according to the
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standard MIDI Sherlock Microbial Identification System (MIDI, Inc., Newark, DE, USA) method. Cellular fatty acid methyl esters (FAMEs) were obtained by using the method of Minnikin et al. (1980) and separated in a 6,890 N gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) fitted with a 7,683 autosampler (Agilent). Peaks were identified by using the Microbial Identification software package (MOORE library ver. 5.0; MIDI database TSBA6). The polar lipids of strain CAU 1296T were identified using twodimensional thin-layer chromatography (TLC) by the method of Minnikin et al. (1984). The plate was sprayed with 10 % ethanolic molybdophosphoric acid (for the total lipids), molybdenum blue (for phospholipids), ninhydrin (for aminolipids) and a-naphthol/sulphuric acid reagent (for glycolipids). Menaquinones were separated on HPLC using an isocratic solvent system [methanol/isopropyl ether (3:1, v/v)] and the flow rate (1 ml/min) (Komagata and Suzuki 1987).
Result and discussion The nearly complete 16S rRNA gene sequence of strain CAU 1296T (1,540 bp) was determined and Table 1 Differential properties of strain CAU 1296T and the type strains of the genus Eudoraea, E. adriatica AS06/20aT (data from Alain et al. 2008)
compared with the corresponding sequences of other bacterial strains in the public database (GenBank/ EMBL/DDBJ accession number KJ150297). Phylogenetic analysis indicated that the strain fell into the genus Eudoraea. The neighbour-joining tree is shown in Fig. 1. The trees obtained with the maximumlikelihood and least-square algorithms showed basically the same topology (data not shown). Pairwise analysis showed that the most closely related strain is E. adriatica AS06/20aT (16S rRNA gene similarity, 95.4 %). The G?C content of the DNA of strain CAU 1296T was 38.7 mol%, which is slightly lower than that of the type species in the genus, E. adriatica AS06/20aT (Alain et al. 2008), which is distinctive. The detailed morphological, cultural, physiological and biochemical characteristics are presented in Table 1 and the species description. Strain CAU 1296T is Gram-stain negative, non-spore forming, strictly aerobic, and non-motile. Colonies were yellow in color, entire, circular, and convex with diameters of 0.3–0.5 mm after 5 days of cultivation on MA at 30 °C and at pH 6.5–8.5 (optimum, pH 7.5). No colonies were formed under anaerobic growth condition. Cells are rod-shaped approximately 0.3–0.5 lm in diameter and 1.3–2.0 lm in length. Because CAU
Characteristic
Strain CAU 1296T
E. adriatica AS06/20aT
Temperature for growth (°C) (optimum)
20–30 (30)
15–33 (30)
NaCl for growth (%, w/v) (optimum)
2–6 (4)
2–6 (2)
pH for growth (optimum)
6.5–8.5 (7.5)
6.5–8.5 (7.5–8.0)
Catalase
?
?
Oxidase
?
?
Hydrolysis of: gelatin
?
-
urease
-
-
aesculin
?
?
D-Glucose
-
?
L-Arabinose
-
?
Tween 40
?
-
Alkaline phosphatase
?
?
esterase (C4)
?
?
a-Chymotrypsin
-
?
b-Glucosidasse
-
?
a-Mannosidase
-
?
DNA G?C content (mol%)
38.7
38.9
Substrate assimilation:
Enzymic activities:
? Positive, - negative
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Antonie van Leeuwenhoek (2015) 107:1009–1015 Table 2 Characteristic and cellular fatty acid contents (%) of strain CAU 1296T and the type strains of the genus Eudoraea, E. adriatica AS06/20aT (data from Alain et al. 2008)
Fatty acid
1013 Strain CAU 1296T
E. adriatica AS06/20aT
Hydroxy C15:0 2-OH
0.8
0.3
C15:0 3-OH
1.2
1.3
iso-C15:0 3-OH
4.8
7.3
C16:0 3-OH
1.0
0.4
iso-C16:0 3-OH
0.8
1.4
C17:0 3-OH
–
0.2
iso-C17:0 3-OH Straight-chain
5.7
14.1
C14:0
0.4
0.2
C16:0
1.0
0.7
iso-C13:0
0.6
0.5
iso-C15:0
22.4
29.4
anteiso-C15:0
3.2
0.7
iso-C15:1 G
24.1
16.0
iso-C16:0
1.1
0.3
iso-C17:0
–
0.2
Branched
Unsaturated – not detected a
Summed features consist of two or more fatty acids that could not be separated by GLC using the MIDI system. Summed feature 3 comprised C16:1 x7c/C16:1 x6c
C15:1 x6c
6.1
1.1
C17:1 x6c
2.0
0.8
C17:1 x8c
1.2
0.5
iso-C17:1 x9c C18:1 x5c
6.7 0.3
9.0 0.7
Summed feature 3a
14.5
11.6
1296T was isolated from a saline environment, it require NaCl for growth ranging from 2 to 6 % (w/v) NaCl [optimum, 4 % (w/v) NaCl]. Strain CAU 1296T hydrolysed gelatin and aesulin, assimilated Tween 40, and had enzyme activities for alkaline phosphatase, and esterase (C4). However, strain CAU 1296T differed from its closest relative, E. adriatica AS06/ 20aT (Alain et al. 2008) by its ability to hydrolyze gelatin, assimilate tween 40, inability to assimilate Dglucose and L-arabinose and lack of a-chymotrypsin, b-glucosidase, and a-mannosidase enzymatic activity. Overall, the chemotaxonomy was in agreement with previously published data for the genus Eudoraea. The predominant isoprenoid quinone detected in strain CAU 1296T was MK-6, in line with all members of the family Flavobacteriaceae (Bernardet et al. 2002), which is consistent with data for the type species of the genus, E. adriatica AS06/20aT (Alain et al. 2008). A minor amount of MK-7 was also detected. The fatty acid profile of strain CAU 1296T is
shown in Table 2, together with E. adriatica AS06/ 20aT data obtained from the previous study (Alain et al. 2008). The strain contained unsaturated, straightchain, branched, and hydroxy fatty acids. The major fatty acids ([10 %) were iso-C15:1 G (24.1 %), isoC15:0 (22.4 %), and C16:1 x6c and C16:1 x7c (summed feature 3; groups of two or three fatty acids could not be separated with the MIDI system, 14.5 %). The following fatty acids ([1 %) C15:0 3-OH, iso-C15:0 3-OH, C16:0 3-OH, iso-C17:0 3-OH, anteiso-C15:0, iso-C16:0, C15:1 x6c, C17:1 x6c, C17:1 x8c and iso-C17:1 x9c were also present. This fatty acid profile is very similar to the closely related E. adriatica AS06/ 20aT in that iso-C15:1 G and iso-C15:0 were predominant, although there were differences in the proportions of some fatty acids. However, strain CAU 1296T could be distinguished from E. adriatica AS06/20aT (Alain et al. 2008) by absence of C17:0 3-OH and iso-C17:0. Strain CAU 1296T contained phosphatidylethanolamine as the major polar lipid identified, as
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1014
in E. adriatica AS06/20aT. In addition, cells of CAU 1296T contained unidentified lipids: a phosphoglycolipid; phospholipid: glycolipid; aminophospholipid; two aminolipids, and six other unidentified lipids (Suppl. Fig. S1.). By contrast, the polar lipid composition of E. adriatica AS06/20aT clearly differs from this, largely unidentified lipid composition. In conclusion, it is evident from the phenotypic, chemotaxonomic and genotypic data that strain CAU 1296T represents a novel species of the genus Eudoraea, for which the name Eudoraea chungangensis sp. nov. is proposed. Description of Eudoraea chungangensis sp. nov Eudoraea chungangensis sp. nov. (chung.ang.en’sis. N.L. fem. adj. chungangensis referring to Chung-Ang University where the first taxonomic studies on this species were performed). Cells are Gram-stain negative, non-motile, strictly aerobic rods approximately 0.3–0.5 lm in diameter and 1.3–2.0 lm in length. Colonies on MA are yellow colored, circular, convex and 0.3–0.5 mm in diameter after 5 days of incubation at 30 °C. Growth occurs at 20–30 °C (optimum, 30 °C) and at pH 6.5–8.5 (optimum, pH 7.50). Growth occurs in the presence of 2–6 % (w/v) NaCl [optimum, 4 % (w/v) NaCl]. Catalase and oxidase positive, does not reduce nitrate to nitrite. Gelatin and aesculin are hydrolyzed. Citrate is not utilized. b-galactosidase (PNPG) is positive (API20NE). Substrate assimilation positive for Tween 40, D-galactonic acid lactone and a-D-glucose-1phosphate. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naptholAS-BI-phosphohydrolase and N-acetyl-b-glucosaminidase are detected. The predominant respiratory quinone is MK-6. The polar lipid pattern consists of phosphatidylethanolamine, and unidentified phosphoglycolipid, phospholipid, glycolipid, aminophospholipid, two aminolipids, and six other unidentified lipids. The predominant cellular fatty acids ([10 % of the total fatty acids) are iso-C15:1 G, iso-C15:0 and summed feature 3 (C16:1 x6c and C16:1 x7c). The DNA G?C content of the type strain is 38.7 mol%. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CAU 1296T is KJ150297. The type strain is CAU 1296T (=KCTC 42048T, =CCUG 66239T, =CECT 8744T), isolated
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from an aquafarm waste water sludge collected on Jeju Island in the Republic of Korea.
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