Antonie van Leeuwenhoek (2015) 107:723–729 DOI 10.1007/s10482-014-0366-2
ORIGINAL PAPER
Colwellia arctica sp. nov., isolated from Arctic marine sediment Feng-Qing Wang • Xue-Zheng Lin Guan-Jun Chen • Zong-Jun Du
•
Received: 12 October 2014 / Accepted: 18 December 2014 / Published online: 20 January 2015 Ó Springer International Publishing Switzerland 2015
Abstract Strain 435T, a catalase- and oxidasepositive, beige-pigmented, facultatively anaerobic and Gram-stain-negative marine bacterium, was isolated from marine sediment collected in the Arctic (8°210 62900 E 72°80 82700 N). The cells of the type strain are short- to curve-rods and able to grow at 4–25 °C, pH 6.0–9.0 and in the presence of 0.5–6.0 % (w/v) NaCl. It can reduce nitrate to nitrite. The predominant isoprenoid quinone was identified as Q-8 and the polar lipids are comprised of phosphatidylethanolamine and phosphatidylglycerol. The DNA G?C content is 38.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 435T belongs to the
genus Colwellia. Strain 435T was found to exhibit 92.1–95.6 % 16S rRNA gene sequence similarities with other species of the genus Colwellia. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness indicated that strain 435T can be considered to represent a novel species, Colwellia arctica sp. nov., of the genus Colwellia. The type strain is 435T (=CICC 10860T = ATCC BAA-2609T). Keywords Colwellia arctica sp. nov. 16S rRNA gene Polyphasic taxonomy Marine sediment
Introduction The GenBank accession number for the 16S rRNA gene sequence of Colwellia arctica 435T is KM108776.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0366-2) contains supplementary material, which is available to authorized users. F.-Q. Wang G.-J. Chen Z.-J. Du (&) College of Marine Science, Shandong University at Weihai, Weihai 264209, China e-mail: [email protected] X.-Z. Lin (&) Key Laboratory of Marine Bioactive Substances, First Institute of Oceanography, SOA, Qingdao, China e-mail: [email protected] G.-J. Chen Z.-J. Du State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
Bacteria of the genus Colwellia belong to the family Colwelliaceae of the order Alteromonadales and the class Gammaproteobacteria (Ivanova et al. 2004). The genus Colwellia was created in 1988 to accommodate ‘‘Vibrio’’ psychroerythrus (Deming et al. 1988), which was initially classified as one of the Vibrio species (D’Aoust and Kushner 1972). Bacteria of the genus Colwellia are Gram-stain-negative and most of them are motile. Colwellia spp. have been isolated from different ecological habitats, e.g., Colwellia psychrerythraea was recovered from flounder eggs (D’Aoust and Kushner 1972; Deming et al. 1988); Colwellia asteriadis was associated with Amur starfish (Choi et al. 2010); several species were isolated from the Antarctic, including Colwellia demingiae, Colwellia hornerae, Colwellia
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psychrotropica and Colwellia rossensis (Bowman et al. 1998). Some of the members of the genus Colwellia, such as C. psychrerythraea and C. asteriadis are psychrophilic organisms that grow optimally at temperatures below 20 °C and do not grow at temperatures above 30 °C (Bowman et al. 1998; Zhang et al. 2008; Jung et al. 2006). Some other bacteria, such as Colwellia aquaemaris (Liu et al. 2014b) and Colwellia meonggei (Kim et al. 2014), grow optimally at 25 °C. During Chinese National Arctic Research Expeditions, several Colwellia-like marine bacteria were isolated. For example, Colwellia chukchiensis isolated from seawater collected at the time of the Second Chinese National Arctic Research Expedition of the icebreaker ‘Xue Long’ was recently described (Yu et al. 2011). In this study, we aimed at investigating the taxonomic status of strain 435T, isolated during the Fifth Chinese National Arctic Research Expedition, based on its phenotypic and chemotaxonomic properties, and a detailed phylogenetic analysis of the 16S rRNA gene sequences.
Materials and methods Organism, maintenance and cultural conditions During the Fifth Chinese National Arctic Expedition, a sample of marine sediment was collected at the depth of 2,619 m in Arctic (8°210 62900 E 72°80 82700 N). The sediment sample (10 g) was mixed with 100 mL sterile seawater. One mL of this homogenate was added to 9 mL sterile seawater. An aliquot (0.1 mL) from this dilution was spread on marine agar 2216 (MA, Hopebio, China). After incubation at 20 °C for 5 days, a beige-pigmented bacterium, designated 435T, was selected and subcultured on MA at 20 °C to confirm its purity. The strain 435T was routinely cultured on MA at 20 °C and stored at -80 °C in sterile 1 % (w/v) saline supplemented with 15 % (v/v) glycerol. C. asteriadis JCM 15608T obtained from the JCM was used as reference strain for physiological tests and chemotaxonomic characteristics (except polar lipids analysis). Morphological, physiological and biochemical analysis Colony morphology examination of strain 435T was performed on MA after 12, 24, 36, 48 and 60 h of
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incubation. Bacterial cell size, morphology, motility and the presence of flagella were observed using light microscopy (E600; Nikon) and the transmission electron microscope after incubation on MA at 20 °C for 48 h. Gliding motility was examined according to the method described by Bowman (2000). The Gram reaction was determined as described elsewhere (Smibert and Krieg 1994). Strain 435T was grown on MA and in marine broth 2216 (MB, Hopebio, China) for 2–14 days at various temperatures (4, 10, 15, 20, 28, 37 and 42 °C) for determination of the optimal growth temperature. The tolerance of the NaCl was tested on MA-NaCl media (MA-NaCl medium: Agar 18 g/L, Peptone 5 g/L, MgSO4 3.2 g/L, MgCl2 2.2 g/L, CaCl2 1.2 g/L, Yeast Extract 1 g/L, KCl 0.7 g/L, NaHCO3 0.2 g/L) and in MB-NaCl media (MB-NaCl medium was MA-NaCl medium without agar) with the NaCl concentrations ranging from 0 to 9.0 % (w/v) at intervals of 1 %. The cells of strain 435T were grown on plates (20 °C) and in MB (120 rpm, 20 °C) for 2–14 days. The effect of pH on growth was investigated between pH 5.5 and 10.0 (in increments of 0.5 pH unit) in MB at 20 °C containing the following buffers (Sangon, China): MES (pH 5.5 and 6.0), PIPES (pH 6.5 and 7.0), HEPES (pH 7.5 and 8.0), Tricine (pH 8.5) and CAPSO (pH 9.0, 9.5 and 10.0), at the concentration of 20 mM. Growth under strictly anaerobic condition was tested on MA with or without 0.1 % NaNO3 for 2–7 days at 20 °C. Oxidase activity was evaluated using the oxidase reagent (bioMe´rieux). Catalase activity was confirmed by the bubble production after applying 3 % (v/v) hydrogen peroxide solution. The procedures for hydrolysis of starch, casein, gelatin and Tween 80 were carried out according to the methods described elsewhere (Smibert and Krieg 1994). Since strain 435T showed poor growth on Iso-Sensitest agar (Oxoid) and Mueller–Hinton agar, the antibiotic sensitivity profile of the novel isolate was determined using the MA plates. The test was performed as described by Du et al. (2014) and according to Clinical and Laboratory Standards Institute (2012) using streptomycin (10 lg), chloramphenicol (30 lg), erythromycin (15 lg), ofloxacin (5 lg), neomycin (30 lg), sulfamethoxydiazine (5 lg), kanamycin (30 lg), tobramycin (10 lg), vancomycin (30 lg), gentamycin (10 lg) and clindamycin (30 lg). The acid production and utilization of different compounds as sole sources of carbon and energy were determined using the API 50CH system (bioMe´rieux).
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Additional physiological and biochemical properties were investigated by using the API 20E, API 20NE and API ZYM kits (bioMe´rieux). All the API tests were carried out according to the manufacturer’s instructions except the salinity was adjusted to 3 %.
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.
Molecular studies and phylogenetic analysis
Morphological, physiological and biochemical analysis
The genomic DNA of strain 435T was extracted using a genomic DNA extraction kit (Takara, Japan). The DNA G?C content was detected using the high-performance liquid chromatography (HPLC) as described by Tamaoka and Komagata (1984) and Mesbah et al. (1989). kDNA (Takara, Japan) was used as a standard. 16S rRNA gene was amplified and cloned as described by Liu et al. (2014a). PCR products were purified by using a PCR purification kit (Tiangen, China) and ligated into vector pGM-T (Tiangen, China). Sequencing reactions were carried out using an ABI BigDye 3.1 Sequencing Kit (Applied BioSystems) and an automated DNA sequencer (model ABI3730; Applied BioSystems). To confirm the taxonomic status of strain 435T, the 16S rDNA sequence (1,481 bp) was submitted to GenBank to search for similar sequences using the BLAST algorithm. EzTaxon server was used to obtain the sequences of the type strains (http://eztaxone.ezbiocloud.net/; Kim et al. 2012). Alignment of sequences was carried out using CLUSTAL_X software (version 1.81) (Thompson et al. 1997). Phylogenetic tree of strain 435T and several closely related species based on 16S rRNA gene sequence similarities was constructed using the neighbor-joining (NJ) method implemented in the computer program MEGA (version 6.0) (Tamura et al. 2013). The maximum-likelihood (ML) algorithm was applied as well to estimate and verify the taxonomic positions of the novel isolate and reference strains on the tree. Chemotaxonomic characterization The fatty acid compositions were determined as described by Sasser (1990) using the microbial identification system (MIDI; Microbial ID). For extraction of respiratory isoprenoid quinone and fatty acids, cells cultured in MB (120 rpm, 20 °C, 48 h) were harvested and subjected to freeze drying. Respiratory isoprenoid quinones were isolated and separated according to the method described by Tindall (1990a, b). Analysis of polar lipids was carried out by the Identification Service, Leibniz-Institut DSMZ-
Results and discussion
Strain 435T was found to form beige-pigmented colonies after incubation at 20 °C for 48 h. Cells are rod-like, short-rods and curved-rods, motile by single polar flagellum, as are most species of the genus Colwellia. Growth was found to occur between 4 and 25 °C (optimum 18–20 °C) both on plates and in liquid medium, pH 6.0–9.0 (optimum pH 6.5–7.0) and with 0.5–4.0 % (w/v) NaCl (optimum 2.0–3.0 %) on MA but with 0.5–6.0 % (w/v) NaCl (optimum 2.0–3.0 %) in MB. Strain 435T can be distinguished from C. psychrerythraea, C. rossensis and C. demingiae by ability of growing at 25 °C. It can be differentiated from C. rossensis which is unable to grow in the presence of 6.0 % (w/v) NaCl, or C. asteriadis, which grows in the presence of 7.0–11.0 % (w/v) NaCl. Hydrolysis of gelatin, starch and Tween 80 are detected, but hydrolysis of casein and urease were not detected. The strain 435T was found to be able to utilize D-glucose, D-fructose, Dcellobiose, D-maltose, inulin, D-raffinose, starch and glycogen as sole sources of carbon and energy. The strain 435T was susceptible to streptomycin (10 lg), chloramphenicol (30 lg), erythromycin (15 lg), ofloxacin (5 lg), neomycin (30 lg) and sulfamethoxydiazine (5 lg); moderately susceptible to kanamycin (30 lg) and tobramycin (10 lg); weakly susceptible to vancomycin (30 lg) and gentamycin (10 lg); resistant to clindamycin (30 lg). Further data on the morphological, physiological and biochemical characteristics of strain 435T are given in the species description and Table 1. Molecular studies and phylogenetic analysis The 16S rRNA gene sequence (1,481 bp) of strain 435T was used to determine its phylogenetic position. Phylogenetic trees were constructed using the neighbor-joining and maximum-likelihood algorithms (data not shown) and had similar topology. Strain 435T forms a cluster with species of the genus Colwellia as shown in the phylogenetic tree (Fig. 1). Phylogenetic
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Table 1 Differential phenotypic characteristics of strain 435T and some of other Colwellia species Characteristic
1
2
3
4
5
Colony colour
Beige
Beige
Red
Chalky-white
Greyish yellow
Motility
?
?
?
-
?
Growth At 25 °C
?
?
-
-
?
?
?
ND
-
?
-
-
V(?)
?
-
-
-
?
-
? -
With 6.0 % (w/v) NaCl Urease Hydrolysis of Casein Starch
?
?
V(?)
?
Gelatin
?
?
?
-
-
Tween 80
?
-
?
NG
?
L-Arabinose
-
-
-
-
?
D-Glucose
?
?
V(?)
?
-
D-Fructose
?
?
-
-
?
D-Cellobiose
?
-
-
-
?
D-Galactose
?
?
-
?
-
Sucrose
?
-
-
-
?
N-acetylglucosamine Melibiose
-
? ?
-
? -
?
Xylose
-
?
-
-
-
Maltose
-
?
?
-
-
DNA G?C content (mol %)
38.7
35–38
38
39.3
37
Acid production from
Strains 1 435T (this study), 2 C. asteriadis JCM 15608T (data from this study except DNA G?C content), 3 C. psychrerythraea (Bowman et al. 1998; D’Aoust and Kushner 1972), 4 C. rossensis (Bowman et al. 1998), 5 C. aesturii (Jung et al. 2006). All the strains grow at 4 °C and reduce nitrate to nitrite ? positive, - negative, NG no growth occurred on test medium, ND no data available, V (?) variable (data in parentheses are for the type strain)
analysis based on 16S rRNA gene sequence similarities revealed that strain 435T had 16S rRNA gene sequence similarity with the species of the genus Colwellia (92.1–95.6 %). The highest degree of 16S rRNA gene sequence similarity was found to be with C. psychrerythraea (95.56 %) and C. asteriadis (95.28 %), which is a lower value than the borderline used for defination of bacterial species (i.e. 97 %) as proposed by Stackebrandt and Goebel (1994). Chemotaxonomic characterization The predominant isoprenoid quinone present in strain 435T was identified as ubiquinone-8 (Q-8),
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which is characteristic for the genus Colwellia. Major polar lipids identified in strain 435T are phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Six unknown phospholipids (PL1, PL2, PL3, PL4, PL5 and PL6) and aminophospholipid are present in moderate to minor amounts in the polar lipid profile (see Supplementary Material Fig. S1). Major cellular fatty acids were identified as C15:1x8c, sum in feature 3 (comprising C16:1x7c/15 iso 2-OH) and C17:1x8c (Table S1). Overall, the chemotaxonomic characteristics of strain 435T are typical for bacteria of this genus, although there were some differences in the proportions and minor fatty acids.
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67 78 78
727 Colwellia psychrotropica ACAM 179T (U85846) Colwellia rossensis ACAM 608T (U14581)
Colwellia maris JCM 10085T (AB002630)
73
Colwellia piezophila JCM 11831T (AB094412)
67
Colwellia hornerae ACAM 607T (JN175346) Colwellia demingiae ACAM 459T (U85845)
76
Colwellia asteriadis JCM 15608T (NR116385) 86
Colwellia psychrerythraea ATCC 27364T (AB011364)
50
Colwellia arctica 435T (KM108776)
51 98
Colwellia aquaemaris JCM 18479T (JQ948043) Colwellia meonggei KCTC 32380T (KF193862)
99
Colwellia aestuarii DSM 17314T (DQ055844)
94
Colwellia chukchiensis DSM 22576T (FJ889599)
93 97
Colwellia polaris JCM 13952T (DQ007434) Thalassomonas agarivorans JCM 13379T (DQ212914)
91
Thalassomonas eurytherma JCM 18482T (JQ288724) Psychromonas antarctica DSM 10704T (Y14697) Pseudoalteromonas haloplanktis ATCC 14293T (X67024)
94 58
Alteromonas macleodii ATCC 27126T (Y18228) Pseudomonas aeruginosa ATCC 10145T (HE978271)
0.01
Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain 435T, Colwellia species and some other phylogenetically related taxa.
Bootstrap values (expressed as percentages of 1,000 replications) of [50 % are shown at branching nodes. Bar 0.01 substitutions per nucleotide position
Conclusion
Description of Colwellia arctica sp. nov.
The results of the phenotypic, chemotaxonomic and phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 435T is a member of the genus Colwellia and represents a genomic species that is separate from validly named Colwellia species. Further, the novel isolate can be readily distinguished from other Colwellia species by the combination of phenotypic, genotypic and phylogenetic features, e.g., range of salinity and temperature for growth, hydrolysis of Tween 80, starch and gelatin (Table 1). In particular, the new species can be differentiated from C. asteriadis by its inability to grow in the presence of 7.0–11.0 % (w/v) NaCl and Tween 80 hydrolysis. Strain 435T can be distinguished from C. psychrerythraea by its ability to grow at 25 °C and urea utilization; from C. rossensis by its motility; from C. aesturii by starch, gelatin and Tween 80 hydrolysis. Thus, the results obtained in this study provide evidence that strain 435T constitutes a novel species of the genus Colwellia for which the name Colwellia arctica sp. nov. is proposed.
Colwellia arctica (arc’ti.ca. L. fem. adj. arctica, pertaining to the Arctic, where the type strain was isolated). Cells are Gram-stain-negative, 0.6–1.0 lm wide, 1.6–2.8 lm long and motile by means of a single polar flagellum. Facultatively anaerobic, catalase- and oxidase-positive. Colonies on MA are circular, smooth, beige-pigmented and approximately 1.0–1.5 mm in diameter after incubation for 48 h at 20 °C. Growth occurs at 4–25 °C (optimum 18–20 °C), pH 6.0–9.0 (optimum 6.5–7.0) and in the presence of 0.5–6.0 % (w/v) NaCl (optimum 2.0–3.0 %). Reduces nitrate to nitrite. In API ZYM tests, alkaline phosphatase, esterase (C4), acid phosphatase, esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, valine arylamidase and a-glucosidase are detected, but lipase (C14), cystine arylamidase, trypsin, a-galactosidase, b-galactosidase, b-glucuronidase, b-galactosidase, bglucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and b-afucosidase are not. According to API 20E, gelatin hydrolase is positive
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and acetoin (Voges–Proskauer reaction) is produced. Arginine dihydrolase, citrate utilization, lysine decarboxylase, ornithine decarboxylase, urease and tryptophan deaminase are negative. Indole and H2S are not produced. According to API 50CH, acid can be produced from D-glucose, D-fructose, esculin ferric citrate, D-cellobiose, D-maltose, D-sucrose, inulin, Draffinose, starch, glycogen, D-gentiobiose without oxygen. D-glucose, D-fructose, D-cellobiose, D-maltose, inulin, D-raffinose, starch and glycogen are utilized as sole sources of carbon and energy. The predominant cellular fatty acids are C15:1x8c, sum in feature 3 (comprising C16:1x7c/15 iso 2-OH), C17:1x8c and C15:0. The major polar lipids are PE and PG. The main respiratory quinone is Q-8 and the DNA G?C content of the type strain is 38.7 mol%. The type strain 435T (= CICC 10860T = ATCC BAA2609T) was isolated from marine sediment in the Arctic. Acknowledgments This work was financially supported by the Chinese Polar Environment Comprehensive Investigation and Assessment Program (CHINARE2012-03-05, CHINARE201303-05), Public Science and Technology Research Funds Projects of Ocean (201005032-2) and the National Natural Science Foundation of China (31370057).
References 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 Bowman JP, Gosink JJ, Mccammon SA, Lewis TE, Nichols DS, Nichols PD, Skerratt JH, Staley JT, Mcmeekin TA (1998) Colwellia demingiae sp.nov., Colwellia hornerae sp.nov., Colwellia rossensis and Colwellia psychrotropica sp. nov.: psychrophilic Antarctic species with the ability to synthesize docosahexaenoic acid (22:6x3). Int J Syst Bacteriol 48:1171–1180 Choi EJ, Kwon HC, Kon HY, Kim YS, Yang HO (2010) Colwellia asteriadis sp. nov., a marine bacterium isolated from the starfish Asterias amurensis. Int J Syst Evol Microbiol 60:1952–1957 CLSI (2012) Performance standards for antimicrobial susceptibility testing; 22nd informational supplement M100-S22. Clinical and Laboratory Standards Institute, Wayne D’Aoust JY, Kushner DJ (1972) Vibrio psychroerythrus sp. n.: classification of the psychrophilic marine bacterium, NRC 1004. J Bacteriol 111:340–342 Deming JW, Somers LK, Straube WL, Swartz DG, Macdonell MT (1988) Isolation of an obligately barophilic bacterium
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Antonie van Leeuwenhoek (2015) 107:723–729 and description of a new genus, Colwellia gen. nov. Syst Appl Microbiol 10:152–160 Du ZJ, Wang Y, Dunlap C, Rooney AP, Chen GJ (2014) Draconibacterium orientale gen. nov., sp. nov., isolated from two distinct marine environments, and proposal of Draconibacteriaceae fam. nov. Int J Syst Evol Microbiol 64:1690–1696 Ivanova EP, Flavier S, Christen R (2004) Phylogenetic relationships among marine Alteromonas-like proteobacteria: emended description of the family Alteromonadaceae and proposal of Pseudoalteromonadaceae fam. nov., Colwelliaceae fam. nov., Shewanellaceae fam. nov., Moritellaceae fam. nov., Ferrimonadaceae fam. nov., Idiomarinaceae fam. nov. and Psychromonadaceae fam. nov. Int J Syst Evol Microbiol 54:1773–1788 Jung SY, Oh TK, Yoon JH (2006) Colwellia aestuarii sp. nov., isolated from a tidal flat sediment in Korea. Int J Syst Evol Microbiol 56:33–37 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 Kim YO, Park S, Nam BH, Jung YT, Kim DG, Yoon JH (2014) Colwellia meonggei sp. nov., a novel gammaproteobacterium isolated from sea squirt Halocynthia roretzi. Antonie Van Leeuwenhoek 104:1021–1027 Liu QQ, Wang Y, Li J, Du ZJ, Chen GJ (2014a) Saccharicrinis carchari sp. nov., isolated from a shark, and emended descriptions of the genus Saccharicrinis and Saccharicrinis fermentans. Int J Syst Evol Microbiol 64:2204–2209 Liu Y, Liu LZ, Zhong ZP, Zhou YG, Liu Y, Liu ZP (2014b) Colwellia aquaemaris sp. nov., isolated from the Cynoglossus semilaevis culture tank in a recirculating mariculture system. Int J Syst Evol Microbiol 64:3926–3930 Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G?C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159–167 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI technical note 101. MIDI Inc, Newark Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general, molecular bacteriology. American Society for Microbiology, Washington, DC, pp 607–654 Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849 Tamaoka J, Komagata K (1984) Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25:125–128 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729 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
Colwellia arctica sp. nov., isolated from Arctic marine sediment Feng-Qing Wang • Xue-Zheng Lin Guan-Jun Chen • Zong-Jun Du
•
Received: 12 October 2014 / Accepted: 18 December 2014 / Published online: 20 January 2015 Ó Springer International Publishing Switzerland 2015
Abstract Strain 435T, a catalase- and oxidasepositive, beige-pigmented, facultatively anaerobic and Gram-stain-negative marine bacterium, was isolated from marine sediment collected in the Arctic (8°210 62900 E 72°80 82700 N). The cells of the type strain are short- to curve-rods and able to grow at 4–25 °C, pH 6.0–9.0 and in the presence of 0.5–6.0 % (w/v) NaCl. It can reduce nitrate to nitrite. The predominant isoprenoid quinone was identified as Q-8 and the polar lipids are comprised of phosphatidylethanolamine and phosphatidylglycerol. The DNA G?C content is 38.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 435T belongs to the
genus Colwellia. Strain 435T was found to exhibit 92.1–95.6 % 16S rRNA gene sequence similarities with other species of the genus Colwellia. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness indicated that strain 435T can be considered to represent a novel species, Colwellia arctica sp. nov., of the genus Colwellia. The type strain is 435T (=CICC 10860T = ATCC BAA-2609T). Keywords Colwellia arctica sp. nov. 16S rRNA gene Polyphasic taxonomy Marine sediment
Introduction The GenBank accession number for the 16S rRNA gene sequence of Colwellia arctica 435T is KM108776.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0366-2) contains supplementary material, which is available to authorized users. F.-Q. Wang G.-J. Chen Z.-J. Du (&) College of Marine Science, Shandong University at Weihai, Weihai 264209, China e-mail: [email protected] X.-Z. Lin (&) Key Laboratory of Marine Bioactive Substances, First Institute of Oceanography, SOA, Qingdao, China e-mail: [email protected] G.-J. Chen Z.-J. Du State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
Bacteria of the genus Colwellia belong to the family Colwelliaceae of the order Alteromonadales and the class Gammaproteobacteria (Ivanova et al. 2004). The genus Colwellia was created in 1988 to accommodate ‘‘Vibrio’’ psychroerythrus (Deming et al. 1988), which was initially classified as one of the Vibrio species (D’Aoust and Kushner 1972). Bacteria of the genus Colwellia are Gram-stain-negative and most of them are motile. Colwellia spp. have been isolated from different ecological habitats, e.g., Colwellia psychrerythraea was recovered from flounder eggs (D’Aoust and Kushner 1972; Deming et al. 1988); Colwellia asteriadis was associated with Amur starfish (Choi et al. 2010); several species were isolated from the Antarctic, including Colwellia demingiae, Colwellia hornerae, Colwellia
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psychrotropica and Colwellia rossensis (Bowman et al. 1998). Some of the members of the genus Colwellia, such as C. psychrerythraea and C. asteriadis are psychrophilic organisms that grow optimally at temperatures below 20 °C and do not grow at temperatures above 30 °C (Bowman et al. 1998; Zhang et al. 2008; Jung et al. 2006). Some other bacteria, such as Colwellia aquaemaris (Liu et al. 2014b) and Colwellia meonggei (Kim et al. 2014), grow optimally at 25 °C. During Chinese National Arctic Research Expeditions, several Colwellia-like marine bacteria were isolated. For example, Colwellia chukchiensis isolated from seawater collected at the time of the Second Chinese National Arctic Research Expedition of the icebreaker ‘Xue Long’ was recently described (Yu et al. 2011). In this study, we aimed at investigating the taxonomic status of strain 435T, isolated during the Fifth Chinese National Arctic Research Expedition, based on its phenotypic and chemotaxonomic properties, and a detailed phylogenetic analysis of the 16S rRNA gene sequences.
Materials and methods Organism, maintenance and cultural conditions During the Fifth Chinese National Arctic Expedition, a sample of marine sediment was collected at the depth of 2,619 m in Arctic (8°210 62900 E 72°80 82700 N). The sediment sample (10 g) was mixed with 100 mL sterile seawater. One mL of this homogenate was added to 9 mL sterile seawater. An aliquot (0.1 mL) from this dilution was spread on marine agar 2216 (MA, Hopebio, China). After incubation at 20 °C for 5 days, a beige-pigmented bacterium, designated 435T, was selected and subcultured on MA at 20 °C to confirm its purity. The strain 435T was routinely cultured on MA at 20 °C and stored at -80 °C in sterile 1 % (w/v) saline supplemented with 15 % (v/v) glycerol. C. asteriadis JCM 15608T obtained from the JCM was used as reference strain for physiological tests and chemotaxonomic characteristics (except polar lipids analysis). Morphological, physiological and biochemical analysis Colony morphology examination of strain 435T was performed on MA after 12, 24, 36, 48 and 60 h of
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incubation. Bacterial cell size, morphology, motility and the presence of flagella were observed using light microscopy (E600; Nikon) and the transmission electron microscope after incubation on MA at 20 °C for 48 h. Gliding motility was examined according to the method described by Bowman (2000). The Gram reaction was determined as described elsewhere (Smibert and Krieg 1994). Strain 435T was grown on MA and in marine broth 2216 (MB, Hopebio, China) for 2–14 days at various temperatures (4, 10, 15, 20, 28, 37 and 42 °C) for determination of the optimal growth temperature. The tolerance of the NaCl was tested on MA-NaCl media (MA-NaCl medium: Agar 18 g/L, Peptone 5 g/L, MgSO4 3.2 g/L, MgCl2 2.2 g/L, CaCl2 1.2 g/L, Yeast Extract 1 g/L, KCl 0.7 g/L, NaHCO3 0.2 g/L) and in MB-NaCl media (MB-NaCl medium was MA-NaCl medium without agar) with the NaCl concentrations ranging from 0 to 9.0 % (w/v) at intervals of 1 %. The cells of strain 435T were grown on plates (20 °C) and in MB (120 rpm, 20 °C) for 2–14 days. The effect of pH on growth was investigated between pH 5.5 and 10.0 (in increments of 0.5 pH unit) in MB at 20 °C containing the following buffers (Sangon, China): MES (pH 5.5 and 6.0), PIPES (pH 6.5 and 7.0), HEPES (pH 7.5 and 8.0), Tricine (pH 8.5) and CAPSO (pH 9.0, 9.5 and 10.0), at the concentration of 20 mM. Growth under strictly anaerobic condition was tested on MA with or without 0.1 % NaNO3 for 2–7 days at 20 °C. Oxidase activity was evaluated using the oxidase reagent (bioMe´rieux). Catalase activity was confirmed by the bubble production after applying 3 % (v/v) hydrogen peroxide solution. The procedures for hydrolysis of starch, casein, gelatin and Tween 80 were carried out according to the methods described elsewhere (Smibert and Krieg 1994). Since strain 435T showed poor growth on Iso-Sensitest agar (Oxoid) and Mueller–Hinton agar, the antibiotic sensitivity profile of the novel isolate was determined using the MA plates. The test was performed as described by Du et al. (2014) and according to Clinical and Laboratory Standards Institute (2012) using streptomycin (10 lg), chloramphenicol (30 lg), erythromycin (15 lg), ofloxacin (5 lg), neomycin (30 lg), sulfamethoxydiazine (5 lg), kanamycin (30 lg), tobramycin (10 lg), vancomycin (30 lg), gentamycin (10 lg) and clindamycin (30 lg). The acid production and utilization of different compounds as sole sources of carbon and energy were determined using the API 50CH system (bioMe´rieux).
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Additional physiological and biochemical properties were investigated by using the API 20E, API 20NE and API ZYM kits (bioMe´rieux). All the API tests were carried out according to the manufacturer’s instructions except the salinity was adjusted to 3 %.
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.
Molecular studies and phylogenetic analysis
Morphological, physiological and biochemical analysis
The genomic DNA of strain 435T was extracted using a genomic DNA extraction kit (Takara, Japan). The DNA G?C content was detected using the high-performance liquid chromatography (HPLC) as described by Tamaoka and Komagata (1984) and Mesbah et al. (1989). kDNA (Takara, Japan) was used as a standard. 16S rRNA gene was amplified and cloned as described by Liu et al. (2014a). PCR products were purified by using a PCR purification kit (Tiangen, China) and ligated into vector pGM-T (Tiangen, China). Sequencing reactions were carried out using an ABI BigDye 3.1 Sequencing Kit (Applied BioSystems) and an automated DNA sequencer (model ABI3730; Applied BioSystems). To confirm the taxonomic status of strain 435T, the 16S rDNA sequence (1,481 bp) was submitted to GenBank to search for similar sequences using the BLAST algorithm. EzTaxon server was used to obtain the sequences of the type strains (http://eztaxone.ezbiocloud.net/; Kim et al. 2012). Alignment of sequences was carried out using CLUSTAL_X software (version 1.81) (Thompson et al. 1997). Phylogenetic tree of strain 435T and several closely related species based on 16S rRNA gene sequence similarities was constructed using the neighbor-joining (NJ) method implemented in the computer program MEGA (version 6.0) (Tamura et al. 2013). The maximum-likelihood (ML) algorithm was applied as well to estimate and verify the taxonomic positions of the novel isolate and reference strains on the tree. Chemotaxonomic characterization The fatty acid compositions were determined as described by Sasser (1990) using the microbial identification system (MIDI; Microbial ID). For extraction of respiratory isoprenoid quinone and fatty acids, cells cultured in MB (120 rpm, 20 °C, 48 h) were harvested and subjected to freeze drying. Respiratory isoprenoid quinones were isolated and separated according to the method described by Tindall (1990a, b). Analysis of polar lipids was carried out by the Identification Service, Leibniz-Institut DSMZ-
Results and discussion
Strain 435T was found to form beige-pigmented colonies after incubation at 20 °C for 48 h. Cells are rod-like, short-rods and curved-rods, motile by single polar flagellum, as are most species of the genus Colwellia. Growth was found to occur between 4 and 25 °C (optimum 18–20 °C) both on plates and in liquid medium, pH 6.0–9.0 (optimum pH 6.5–7.0) and with 0.5–4.0 % (w/v) NaCl (optimum 2.0–3.0 %) on MA but with 0.5–6.0 % (w/v) NaCl (optimum 2.0–3.0 %) in MB. Strain 435T can be distinguished from C. psychrerythraea, C. rossensis and C. demingiae by ability of growing at 25 °C. It can be differentiated from C. rossensis which is unable to grow in the presence of 6.0 % (w/v) NaCl, or C. asteriadis, which grows in the presence of 7.0–11.0 % (w/v) NaCl. Hydrolysis of gelatin, starch and Tween 80 are detected, but hydrolysis of casein and urease were not detected. The strain 435T was found to be able to utilize D-glucose, D-fructose, Dcellobiose, D-maltose, inulin, D-raffinose, starch and glycogen as sole sources of carbon and energy. The strain 435T was susceptible to streptomycin (10 lg), chloramphenicol (30 lg), erythromycin (15 lg), ofloxacin (5 lg), neomycin (30 lg) and sulfamethoxydiazine (5 lg); moderately susceptible to kanamycin (30 lg) and tobramycin (10 lg); weakly susceptible to vancomycin (30 lg) and gentamycin (10 lg); resistant to clindamycin (30 lg). Further data on the morphological, physiological and biochemical characteristics of strain 435T are given in the species description and Table 1. Molecular studies and phylogenetic analysis The 16S rRNA gene sequence (1,481 bp) of strain 435T was used to determine its phylogenetic position. Phylogenetic trees were constructed using the neighbor-joining and maximum-likelihood algorithms (data not shown) and had similar topology. Strain 435T forms a cluster with species of the genus Colwellia as shown in the phylogenetic tree (Fig. 1). Phylogenetic
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Table 1 Differential phenotypic characteristics of strain 435T and some of other Colwellia species Characteristic
1
2
3
4
5
Colony colour
Beige
Beige
Red
Chalky-white
Greyish yellow
Motility
?
?
?
-
?
Growth At 25 °C
?
?
-
-
?
?
?
ND
-
?
-
-
V(?)
?
-
-
-
?
-
? -
With 6.0 % (w/v) NaCl Urease Hydrolysis of Casein Starch
?
?
V(?)
?
Gelatin
?
?
?
-
-
Tween 80
?
-
?
NG
?
L-Arabinose
-
-
-
-
?
D-Glucose
?
?
V(?)
?
-
D-Fructose
?
?
-
-
?
D-Cellobiose
?
-
-
-
?
D-Galactose
?
?
-
?
-
Sucrose
?
-
-
-
?
N-acetylglucosamine Melibiose
-
? ?
-
? -
?
Xylose
-
?
-
-
-
Maltose
-
?
?
-
-
DNA G?C content (mol %)
38.7
35–38
38
39.3
37
Acid production from
Strains 1 435T (this study), 2 C. asteriadis JCM 15608T (data from this study except DNA G?C content), 3 C. psychrerythraea (Bowman et al. 1998; D’Aoust and Kushner 1972), 4 C. rossensis (Bowman et al. 1998), 5 C. aesturii (Jung et al. 2006). All the strains grow at 4 °C and reduce nitrate to nitrite ? positive, - negative, NG no growth occurred on test medium, ND no data available, V (?) variable (data in parentheses are for the type strain)
analysis based on 16S rRNA gene sequence similarities revealed that strain 435T had 16S rRNA gene sequence similarity with the species of the genus Colwellia (92.1–95.6 %). The highest degree of 16S rRNA gene sequence similarity was found to be with C. psychrerythraea (95.56 %) and C. asteriadis (95.28 %), which is a lower value than the borderline used for defination of bacterial species (i.e. 97 %) as proposed by Stackebrandt and Goebel (1994). Chemotaxonomic characterization The predominant isoprenoid quinone present in strain 435T was identified as ubiquinone-8 (Q-8),
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which is characteristic for the genus Colwellia. Major polar lipids identified in strain 435T are phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Six unknown phospholipids (PL1, PL2, PL3, PL4, PL5 and PL6) and aminophospholipid are present in moderate to minor amounts in the polar lipid profile (see Supplementary Material Fig. S1). Major cellular fatty acids were identified as C15:1x8c, sum in feature 3 (comprising C16:1x7c/15 iso 2-OH) and C17:1x8c (Table S1). Overall, the chemotaxonomic characteristics of strain 435T are typical for bacteria of this genus, although there were some differences in the proportions and minor fatty acids.
Antonie van Leeuwenhoek (2015) 107:723–729
67 78 78
727 Colwellia psychrotropica ACAM 179T (U85846) Colwellia rossensis ACAM 608T (U14581)
Colwellia maris JCM 10085T (AB002630)
73
Colwellia piezophila JCM 11831T (AB094412)
67
Colwellia hornerae ACAM 607T (JN175346) Colwellia demingiae ACAM 459T (U85845)
76
Colwellia asteriadis JCM 15608T (NR116385) 86
Colwellia psychrerythraea ATCC 27364T (AB011364)
50
Colwellia arctica 435T (KM108776)
51 98
Colwellia aquaemaris JCM 18479T (JQ948043) Colwellia meonggei KCTC 32380T (KF193862)
99
Colwellia aestuarii DSM 17314T (DQ055844)
94
Colwellia chukchiensis DSM 22576T (FJ889599)
93 97
Colwellia polaris JCM 13952T (DQ007434) Thalassomonas agarivorans JCM 13379T (DQ212914)
91
Thalassomonas eurytherma JCM 18482T (JQ288724) Psychromonas antarctica DSM 10704T (Y14697) Pseudoalteromonas haloplanktis ATCC 14293T (X67024)
94 58
Alteromonas macleodii ATCC 27126T (Y18228) Pseudomonas aeruginosa ATCC 10145T (HE978271)
0.01
Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain 435T, Colwellia species and some other phylogenetically related taxa.
Bootstrap values (expressed as percentages of 1,000 replications) of [50 % are shown at branching nodes. Bar 0.01 substitutions per nucleotide position
Conclusion
Description of Colwellia arctica sp. nov.
The results of the phenotypic, chemotaxonomic and phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 435T is a member of the genus Colwellia and represents a genomic species that is separate from validly named Colwellia species. Further, the novel isolate can be readily distinguished from other Colwellia species by the combination of phenotypic, genotypic and phylogenetic features, e.g., range of salinity and temperature for growth, hydrolysis of Tween 80, starch and gelatin (Table 1). In particular, the new species can be differentiated from C. asteriadis by its inability to grow in the presence of 7.0–11.0 % (w/v) NaCl and Tween 80 hydrolysis. Strain 435T can be distinguished from C. psychrerythraea by its ability to grow at 25 °C and urea utilization; from C. rossensis by its motility; from C. aesturii by starch, gelatin and Tween 80 hydrolysis. Thus, the results obtained in this study provide evidence that strain 435T constitutes a novel species of the genus Colwellia for which the name Colwellia arctica sp. nov. is proposed.
Colwellia arctica (arc’ti.ca. L. fem. adj. arctica, pertaining to the Arctic, where the type strain was isolated). Cells are Gram-stain-negative, 0.6–1.0 lm wide, 1.6–2.8 lm long and motile by means of a single polar flagellum. Facultatively anaerobic, catalase- and oxidase-positive. Colonies on MA are circular, smooth, beige-pigmented and approximately 1.0–1.5 mm in diameter after incubation for 48 h at 20 °C. Growth occurs at 4–25 °C (optimum 18–20 °C), pH 6.0–9.0 (optimum 6.5–7.0) and in the presence of 0.5–6.0 % (w/v) NaCl (optimum 2.0–3.0 %). Reduces nitrate to nitrite. In API ZYM tests, alkaline phosphatase, esterase (C4), acid phosphatase, esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, valine arylamidase and a-glucosidase are detected, but lipase (C14), cystine arylamidase, trypsin, a-galactosidase, b-galactosidase, b-glucuronidase, b-galactosidase, bglucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and b-afucosidase are not. According to API 20E, gelatin hydrolase is positive
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and acetoin (Voges–Proskauer reaction) is produced. Arginine dihydrolase, citrate utilization, lysine decarboxylase, ornithine decarboxylase, urease and tryptophan deaminase are negative. Indole and H2S are not produced. According to API 50CH, acid can be produced from D-glucose, D-fructose, esculin ferric citrate, D-cellobiose, D-maltose, D-sucrose, inulin, Draffinose, starch, glycogen, D-gentiobiose without oxygen. D-glucose, D-fructose, D-cellobiose, D-maltose, inulin, D-raffinose, starch and glycogen are utilized as sole sources of carbon and energy. The predominant cellular fatty acids are C15:1x8c, sum in feature 3 (comprising C16:1x7c/15 iso 2-OH), C17:1x8c and C15:0. The major polar lipids are PE and PG. The main respiratory quinone is Q-8 and the DNA G?C content of the type strain is 38.7 mol%. The type strain 435T (= CICC 10860T = ATCC BAA2609T) was isolated from marine sediment in the Arctic. Acknowledgments This work was financially supported by the Chinese Polar Environment Comprehensive Investigation and Assessment Program (CHINARE2012-03-05, CHINARE201303-05), Public Science and Technology Research Funds Projects of Ocean (201005032-2) and the National Natural Science Foundation of China (31370057).
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