International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3798–3803
DOI 10.1099/ijs.0.067033-0
Terrimonas arctica sp. nov., isolated from Arctic tundra soil Fan Jiang, Xia Qiu, Xulu Chang, Zhihao Qu, Lvzhi Ren, Wenjing Kan, Youhao Guo, Chengxiang Fang and Fang Peng Correspondence
College of Life Sciences, Wuhan University, Wuhan 430072, PR China
Youhao Guo [email protected] Fang Peng [email protected]
A novel, Gram-stain-negative, aerobic, non-motile and rod-shaped bacterium, designated R986T, was isolated from tundra soil collected near Ny-A˚lesund, Svalbard Archipelago, Norway (786 N). Growth occurred at 4–28 6C (optimum, 22–25 6C) and at pH 6.0–9.0 (optimum, pH 7.0). Flexirubin-type pigments were absent. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain R9-86T belonged to the genus Terrimonas in the family Chitinophagaceae. 16S rRNA gene sequence similarities between strain R9-86T and the type strains of species of the genus Terrimonas with validly published names ranged from 93.7 to 95.0 %. Strain R9-86T contained iso-C15 : 1-G (25.7 %), iso-C15 : 0 (24.5 %), iso-C17 : 0-3OH (18.3 %) and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c, 8.7 %) as its major cellular fatty acids; phosphatidylethanolamine and an unknown polar lipid as its main polar lipids, and MK-7 as its predominant respiratory quinone. The DNA G+C content was 48.4 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain R9-86T is considered to represent a novel species of the genus Terrimonas, for which the name Terrimonas arctica sp. nov. is proposed. The type strain is R9-86T (5CCTCC AB 2011004T5NRRL B-59114T).
The genus Terrimonas, belonging to the family Chitinophagaceae within the phylum Bacteroidetes, was first described by Xie & Yokota (2006) and its description was subsequently emended by Zhang et al. (2012) and Jin et al. (2013). The type strains of the recognized species of the genus are strictly aerobic, Gram-staining-negative, nonmotile, non-gliding bacteria containing menaquinone-7 (MK-7) as the major respiratory quinone (Xie & Yokota, 2006; Zhang et al., 2012; Jin et al., 2013). At the time of writing, the genus Terrimonas comprised five recognized species isolated from various environments, such as garden soil, a freshwater spring, polluted farmland soil and bulking sludge; namely Terrimonas ferruginea (the type species) and Terrimonas lutea (Xie & Yokota, 2006), Terrimonas aquatica (Sheu et al., 2010), Terrimonas rubra (Zhang et al., 2012) and Terrimonas pekingensis (Jin et al., 2013). During the course of an investigation into the culturable bacterial community in the soil from a high Arctic tundra near the settlement, Ny-A˚lesund (78u 589 N 12u 039 E), in the Svalbard Archipelago, Norway, a large number of bacteria were isolated. In this study, we report on the taxonomic characteriazation of one of these isolates, designated strain R9-86T. On the basis of its phenotypic characteristics, the
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain R9-86T is KJ631121. Four supplementary figures are available with the online version of this paper.
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chemotaxonomic data and phylogenetic analysis, the isolate represents a novel species of the genus Terrimonas. For strain isolation, the soil sample was diluted serially with a sterile 0.85 % (w/v) NaCl solution and these dilutions were plated onto 0.36R2A (BD) agar plates. Single colonies on these plates were purified by transferring them onto new plates followed by additional incubation for 7 days at 20 uC. The isolate was routinely cultivated on R2A agar at 25 uC and stored by lyophilization at 4 uC. For 16S rRNA gene sequencing and phylogenetic analysis, genomic DNA was extracted from a fresh culture of strain R9-86T following the methods of Sambrook et al. (1989). Primers 27f (59-GAGTTTGATCCTGGCTCAG-39) and 1527r (59-AGAAAGGAGGTGATCCAGCC-39) were used for amplification of the 16S rRNA gene (Lane, 1991). PCR and 16S rRNA gene sequencing were carried out as described by Lin et al. (2004). Sequence similarities were searched by NCBI BLAST and calculated by pairwise alignments obtained from the EzTaxon database (Kim et al., 2012). Phylogenetic analysis was performed by using the software package MEGA version 6.0 (Tamura et al., 2013) after multiple alignments of data via CLUSTAL W (Thompson et al., 1994). Phylogenetic trees were reconstructed by the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. Evolutionary distances for the neighbour-joining algorithm were calculated with Kimura’s two-parameter method (Kimura, 1980). Bootstrap 067033 G 2014 IUMS Printed in Great Britain
Terrimonas arctica sp. nov.
values were determined based on 1000 replications (Felsenstein, 1985). The 16S rRNA gene sequence of strain R9-86T was a continuous stretch of 1419 bp. Sequence comparisons with 16S rRNA gene sequences from the EzTaxon database revealed that strain R9-86T had the highest similarity with T. pekingensis QHT (95.0 %), followed by T. rubra M-8T (95.0 %), T. lutea DYT (94.8 %), T. ferruginea DSM 30193T (94.7 %) and T. aquatica RIB1-6T (93.7 %). These values are well below the threshold for demarcating bacterial species (Stackebrandt & Goebel, 1994). The neighbourjoining tree showed that strain R9-86T was affiliated to the genus Terrimonas in the family Chitinophagaceae, and formed a robust clade with T. pekingensis QHT and T. lutea DYT (Fig. 1). The same relationship was also found in trees constructed using the maximum-parsimony (Fig. S1, available in the online Supplementary Material) and maximumlikelihood (Fig. S2) algorithms. These results suggest that strain R9-86T represents a novel species within the genus Terrimonas. Growth was evaluated at 25 uC on several standard bacteriological media (all from BD): R2A agar, 0.36R2A agar, nutrient broth (NB) agar, marine broth 2216 (MB) agar, tryptic soy broth (TSB) agar and MacConkey agar. Gram staining of cells was carried out according to the classical Gram procedure described by Doetsch (1981). Growth at different temperatures (0, 4, 10, 18, 20, 22, 25, 28, 37 and 42 uC) was investigated on R2A agar for up to 2 weeks. The pH range for growth was determined in R2A broth at pH 4.0–11.0 (in increments of 1.0 pH unit). Salt tolerance was tested on R2A agar supplemented with 0, 0.5, 1, 2, 3, 5 and 10 % NaCl (w/v) for 2 weeks at 25 uC. Cell morphology was examined by phase-contrast (Olympus BX51) and transmission electron (Hitachi 8100) microscopy using cells grown for 2 days at 25 uC on R2A. Gliding motility was investigated as described by Bowman (2000). Growth under anaerobic conditions was tested on R2A agar in a GasPak (BBL) jar at 25 uC for 21 days. Polyb-hydroxybutyrate (PHB) accumulation was observed by light microscopy after staining the cells with Sudan black (Smibert & Krieg, 1994). Catalase activity was determined by measurement of bubble production after the application of 3 % (v/v) hydrogen peroxide solution. Oxidase activity was evaluated by the oxidation of 1 % (w/v) tetramethyl-pphenylenediamine (Kova´cs, 1956). The presence of flexirubin-type pigments was tested using the KOH test, as described by Bernardet et al. (2002). Hydrolysis of chitin, starch, DNA, gelatin, agar, CM-cellulose and casein was investigated on R2A agar after one week of incubation, according to methods described by Smibert & Krieg (1994). The hydrolysis of Tween 20, Tween 40, Tween 60 and Tween 80 was measured using the formation of an opaque halo of precipitation around the colony (Barrow & Feltham, 1993). Antibiotic sensitivity was tested by using filter-paper discs (diameter, 6.35 mm) impregnated with the following antibiotics (per disc): amikacin (30 mg), vancomycin (30 mg), ciprofloxacin (5 mg), polymyxin B http://ijs.sgmjournals.org
(30 mg), ampicillin (10 mg), chloramphenicol (30 mg), gentamicin (10 mg), kanamycin (30 mg), penicillin G (10 mg), sulfamethoxazole (23.75 mg), trimethoprim (1.25 mg) and tetracycline (30 mg). Any sign of growth inhibition was scored as sensitivity to that antibiotic. Resistance to an antimicrobial drug was indicated if no inhibition zone was observed. Additional physiological and biochemical characteristics were determined by using the API 20NE and API ZYM kits (bioMe´rieux) and the GN2 MicroPlate (Biolog), according to the manufacturers’ instructions. Strain R9-86T grew well at 25 uC on R2A agar, 0.36R2A agar and NB agar, but not on MB agar, TSB agar and MacConkey agar. Cells were strictly aerobic, Gram-stainnegative, non-motile and rod-shaped (Fig. S3). Colonies were yellow, circular, convex and smooth after growing for 5 days at 25 uC on R2A agar. Growth occurred at 4–28 uC (optimum, 22–25 uC) and at pH 6.0–9.0 (optimum, pH 7.0). The range of NaCl concentrations for growth was 0–0.5 % (w/v); optimum growth occurred in the absence of NaCl. Other physiological characteristics of strain R9-86T are summarized in the species description. Selected characteristics that differentiate strain R9-86T from related species of the genus Terrimonas are shown in Table 1. To measure the G+C content of the chromosomal DNA, genomic DNA from the novel strain was extracted and purified, as described by Moore & Dowhan (1995), and degraded enzymically into nucleosides and the G+C content was then determined, as described by Mesbah et al. (1989) using reversed-phase HPLC (UltiMate 3000, Dionex). Respiratory quinones were extracted and identified by HPLC, as described by Xie & Yokota (2003). For analysis of cellular fatty acids, strain R9-86T and five related type strains, T. ferruginea DSM 30193T, T. lutea KACC 13047T, T. aquatica KACC 14981T, T. rubra CCTCC AB 2010401T and T. pekingensis CICC 10452T were grown on R2A agar plates at 25 uC and harvested at the lateexponential phase. The methods used for harvesting, saponification, methylation and extraction of cellular fatty acids were those according to the protocol of the Sherlock Microbial Identification System (MIDI) version 6.0. Separation and identification of fatty acid methyl esters was performed using a Hewlett Packard 6890N gas chromatograph, with the MIDI Sherlock TSBA6 (version of the database) (Sasser, 1990). For polar lipid analysis, strain R986T and reference strains were grown on R2A broth at 25 uC. Polar lipids were extracted and analysed by twodimensional TLC (silica gel plates, layer thickness 0.2 mm, Merck) according tomethods described by Tindall (1990). The genomic DNA G+C content of strain R9-86T was 48.4 mol%, a value that fell within the range of those reported for the other species of the genus Terrimonas (Table 1). The major respiratory quinone of strain R9-86T was MK-7, which is consistent with other members of the genus Terrimonas. The cellular fatty acid profiles of strain R9-86T and five reference strains are presented in Table 2. The major cellular fatty acids of strain R9-86T were 3799
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99
100 80
100
92
100
99
82
96 100
100
100 100 100 100
75
100 99
73
82 96 71
71 100 99 96 100
Niabella hirudinis E96T (KC307771) Niabella drilacis E90T (KC307772) Niabella aurantiaca DSM 17617T (ARFF01000013) Niabella soli DSM 19437T (AGSA01000025) Niabella tibetensis 15-4T (GU291295) Niabella ginsengisoli GR10-1T (EU616816) Niabella yanshanensis CCBAU 05354T (FJ457040) Terrimonas rubra M-8T (JF803808) Terrimonas ferruginea DSM 30193T (AM230484) Terrimonas aquatica RIB1-6T (FJ347757) Terrimonas arctica R9-86T (KJ631121) Terrimonas pekingensis QHT (JF834159) Terrimonas lutea DYT (AB192292) Ferruginibacter alkalilentus HU1-GD23T (FJ177530) Ferruginibacter lapsinanis HU1-HG42T (FJ177532) Flavisolibacter ginsengisoli Gsoil 643T (AB267477) Flavisolibacter ginsengiterrae Gsoil 492T (AB267476) Flavitalea gansuensis JCN-23T (U295962) Flavitalea populi HY-50RT (HM130561) Niastella koreensis GR20-10T (CP003178) Niastella yeongjuensis GR20-13T (DQ244076) Niastella populi THYL-44T (EU877262) Segetibacter aerophilus 6424S-61T (GQ421847) Segetibacter koreensis DSM 18137T (ARFB01000010) Filimonas lacunae YT21T (AB362776) Parasegetibacter luojiensis RHYL-37T (EU877263) Flavihumibacter petaseus T41T (EU854577) Chitinophaga cymbidii R156-2T (JN680880) Chitinophaga japonensis IFO 16041T (AB078055) Chitinophaga rupis CS5-B1T (FM865977) Chitinophaga eiseniae YC6729T (FJ750951) Chitinophaga terrae KP01T (AB278570) Chitinophaga jiangningensis JN53T (KF150362) Chitinophaga polysaccharea MRP-15T (KC430923) Chitinophaga taiwanensis CC-ALB-1T (KC479802) Chitinophaga niastensis JS16-4T ( EU714260) Chitinophaga ginsengisegetis Gsoil 040T (AB264798) Chitinophaga arvensicola DSM 3695T (AM237311) Hydrotalea flava CCUG 51397T (FN665659) Hydrotalea sandarakina AF-51T (JF739858) Sediminibacterium salmoneum NJ-44T (EF407879) Sediminibacterium ginsengisoli DCY13T (EF067860) Lacibacter daechungensis H32-4T (KC759435) Lacibacter cauensis NJ-8T (EU521690) Flavobacterium aquatile DSM 1132T (AM230485)
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences, showing the relationship between strain R9-86T and related taxa. Percentage bootstrap values (1000 replications) greater than 70 % are shown at nodes. The sequence of Flavobacterium aquatile DSM 1132T was used as an outgroup.
iso-C15 : 1-G (25.7 %), iso-C15 : 0 (24.5 %), iso-C17 : 0-3OH (18.3 %) and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c, 8.7 %), which was consistent with data published for species of the genus Terrimonas with validly published names. The differences between strain R9-86T and other species of the genus Terrimonas were particularly marked with respect to the fatty acid profiles, as strain R9-86T
contained higher amounts of the major fatty acid isoC17 : 0-3OH. Additionally, strain R9-86T could be differentiated from other species of the genus Terrimonas due to the presence of C16 : 1v5c, which was not detected in the other species. The major polar lipids of strain R9-86T were phosphatidylethanolamine and an unknown polar lipid; six unknown aminolipids, five unknown polar lipids and one
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Terrimonas arctica sp. nov.
Table 1. Differential characteristics of strain R9-86T and related species of the genus Terrimonas Strains: 1, R9-86T; 2, T. pekingensis CICC 10452T; 3, T. lutea KACC 13047T; 4, T. rubra CCTCC AB 2010401T; 5, T. ferruginea DSM 30193T; 6, T. aquatica KACC 14981T. Data were obtained in this study unless otherwise indicated. +, Positive; 2, negative; W, weakly positive. Characteristic Growth at: 4 uC 37 uC 1 % NaCl Flexirubin-type pigments Hydrolysis of: Casein Agar Tween 80 CM-cellulose Enzyme activity (API ZYM) Trypsin a-Chymotrypsin a-Galactosidase b-Galactosidase b-Glucuronidase b-Glucosidase a-Mannosidase a-Fucosidase Assimilation of (API 20 NE) D-Glucose D-Mannose N-Acetylglucosamine Maltose Potassium gluconate Malate DNA G+C content (mol%)
1
2
3
4
5
6
+ 2 2 2
2 2 2 2
2 + + 2
2 + + +
2 + + +
2 + + +
2 2 2 +
2 + 2 2
+ + 2 2
+ 2 2 W
+ 2 + 2
+ 2 + 2
2 2 + 2 2 2 + 2
+ + + + 2 2 2 2
+ + + + 2 + + +
+ + + 2 2 2 2 +
2 2 2 2 2 2 2 2
+ 2 + + + + + +
2 2 2 2 2 2 48.4 %
+ + + + 2 2 41.0 %a
+ + + + + 2 47.2 %b
+ + + + 2 + 47.0 %c
+ + + + 2 2 48.9 %b
+ + + + 2 + 47.3 %d
Data taken from: (a) Jin et al. (2013), (b) Xie & Yokota (2006), (c) Zhang et al. (2012) and (d) Sheu et al. (2010).
unknown aminophospholipid were also detected. The polar lipid profile of strain R9-86T was similar to that of the four reference strains of the genus Terrimonas (Fig. S4). On the basis of the data presented, isolate R9-86T represents a novel species of the genus Terrimonas, for which the name Terrimonas arctica sp. nov. is proposed. Description of Terrimonas arctica sp. nov. Terrimonas arctica (arc9ti.ca. L. fem. adj. arctica northern, from the Arctic, referring to the site where the type strain was isolated). Cells are strictly aerobic, Gram-stain-negative, non-motile and rod-shaped. Colonies are yellow, circular, convex and smooth after growing for 5 days at 25 uC on R2A agar. Growth occurs at 25 uC on R2A agar, 0.36R2A agar and NB agar, but not on MB agar, TSB agar and MacConkey agar. Growth occurs at 4–28 uC (optimum, 22–25 uC) and at pH 6.0–9.0 (optimum, pH 7.0). The range of NaCl for growth is 0–0.5 % (w/v); optimum growth occurs without NaCl. Tests for catalase and oxidase are positive. http://ijs.sgmjournals.org
Flexirubin-type pigments are absent. Poly-b-hydroxybutyrate granules are not accumulated. The strain hydrolyses CM-cellulose and Tween 60. Starch and Tween 20 are weakly hydrolysed, but chitin, DNA, gelatin, agar, casein, Tween 40 and Tween 80 are not hydrolysed. In the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, a-glucosidase, N-acetyl-b-glucosaminidase and a-mannosidase activities are present, but lipase(C14), trypsin, a-chymotrypsin, b-galactosidase, bglucuronidase, b-glucosidase and a-fucosidase activities are absent. In API 20 NE strips, reactions are positive for the hydrolysis of aesculin, b-galactosidase activity and the assimilation of L-arabinose. Negative for reduction of nitrate, production of indole, fermentation of glucose, arginine dihydrolase and urease activities and the assimilation of D-glucose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Positive for the Biolog GN2 MicroPlate substrates: dextrin, N-acetyl-D-glucosamine, cellobiose, D-fructose, D-galactose, 3801
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Table 2. Cellular fatty acid composition of strain R9-86T and type strains of phylogenetically related species Strains: 1, R9-86T; 2, T. pekingensis CICC 10452T; 3, T. lutea KACC 13047T; 4, T. rubra CCTCC AB 2010401T; 5, T. ferruginea DSM 30193T; 6, T. aquatica KACC 14981T. Data were obtained in this study. Values are percentages of total fatty acids; fatty acids amounting to ,1 % of the total fatty acids in all strains listed are omitted. Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c. 2, None detected; tr, trace amounts (,1 %). Fatty acid iso-C14 : 0 C14 : 0 iso-C15 : 1-G anteiso-C15 : 1-A iso-C15 : 0 anteiso-C15 : 0 iso-C16 : 0 C16 : 1v5c C16 : 0 iso-C15 : 0-3OH C17 : 1v6c C15 : 0-2OH iso-C16 : 0-3OH C16 : 0 23OH C18 : 0 iso-C17 : 0-3OH C17 : 0-2OH C17 : 0-3OH Summed feature 3
1
2
3
4
5
6
tr 1.4 25.7 tr 24.5 1.9 tr 1.5 5.0 2.6 2 tr tr 4.0 tr 18.3 1.1 tr 8.7
1.2 1.2 18.0 tr 26.0 2.6 1.4 2 7.1 1.5 1.6 tr 2.3 5.4 tr 12.0 tr 2.7 12.2
tr tr 26.7 1.3 32.2 3.7 tr 2 3.0 2.1 2 tr tr 2.4 tr 14.5 1.0 tr 6.2
tr 1.5 26.3 tr 17.9 1.2 tr 2 2.8 2.6 2 tr tr 4.0 1.0 14.9 tr tr 21.0
1.3 1.5 19.9 tr 35.1 1.7 1.3 2 4.6 2.7 tr 1.0 tr 1.9 tr 14.1 tr tr 9.4
tr 1.3 23.2 tr 26.9 1.5 tr 2 5.4 1.3 tr tr tr 5.0 tr 12.8 tr 1.7 14.7
polymyxin B, gentamicin, kanamycin, penicillin G, sulfamethoxazole, trimethoprim and tetracycline, but resistant to ampicillin and chloramphenicol. The major respiratory quinone is MK-7 and the major cellular fatty acids are isoC15 : 1-G, iso-C15 : 0, iso-C17 : 0-3OH and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c). The major polar lipids of strain R9-86T are phosphatidylethanolamine and an unknown polar lipid; six unknown aminolipids, five unknown polar lipids and one unknown aminophospholipid are also detected. The type strain, R9-86T (5CCTCC AB 2011004T5NRRL B-59114T), was isolated from tundra soil near Ny-A˚lesund, Svalbard Archipelago, Norway. The DNA G+C content of the type strain is 48.4 mol%.
Acknowledgements This work was supported by National Basic Research Program of China (973 Program) (2011CB808800), the State Oceanic Administration, PR. China (project no. 10/11YR06), the National Infrastructure of Natural Resources for Science and Technology Program of China (no. NIMR-2014-8), National Natural Science Foundation of China (no. 31200038) and Key Project of Chinese Ministry of Education (V201308).
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Xie, C. H. & Yokota, A. (2006). Reclassification of [Flavobacterium]
hyalina based on the 16S rRNA gene sequence. J Gen Appl Microbiol 49, 345–349.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning:
ferrugineum as Terrimonas ferruginea gen. nov., comb. nov., and description of Terrimonas lutea sp. nov., isolated from soil. Int J Syst Evol Microbiol 56, 1117–1121.
a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Zhang, J., Gu, T., Zhou, Y., He, J., Zheng, L. Q., Li, W. J., Huang, X. & Li, S. P. (2012). Terrimonas rubra sp. nov., isolated from a polluted
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
farmland soil and emended description of the genus Terrimonas. Int J Syst Evol Microbiol 62, 2593–2597.
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DOI 10.1099/ijs.0.067033-0
Terrimonas arctica sp. nov., isolated from Arctic tundra soil Fan Jiang, Xia Qiu, Xulu Chang, Zhihao Qu, Lvzhi Ren, Wenjing Kan, Youhao Guo, Chengxiang Fang and Fang Peng Correspondence
College of Life Sciences, Wuhan University, Wuhan 430072, PR China
Youhao Guo [email protected] Fang Peng [email protected]
A novel, Gram-stain-negative, aerobic, non-motile and rod-shaped bacterium, designated R986T, was isolated from tundra soil collected near Ny-A˚lesund, Svalbard Archipelago, Norway (786 N). Growth occurred at 4–28 6C (optimum, 22–25 6C) and at pH 6.0–9.0 (optimum, pH 7.0). Flexirubin-type pigments were absent. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain R9-86T belonged to the genus Terrimonas in the family Chitinophagaceae. 16S rRNA gene sequence similarities between strain R9-86T and the type strains of species of the genus Terrimonas with validly published names ranged from 93.7 to 95.0 %. Strain R9-86T contained iso-C15 : 1-G (25.7 %), iso-C15 : 0 (24.5 %), iso-C17 : 0-3OH (18.3 %) and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c, 8.7 %) as its major cellular fatty acids; phosphatidylethanolamine and an unknown polar lipid as its main polar lipids, and MK-7 as its predominant respiratory quinone. The DNA G+C content was 48.4 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain R9-86T is considered to represent a novel species of the genus Terrimonas, for which the name Terrimonas arctica sp. nov. is proposed. The type strain is R9-86T (5CCTCC AB 2011004T5NRRL B-59114T).
The genus Terrimonas, belonging to the family Chitinophagaceae within the phylum Bacteroidetes, was first described by Xie & Yokota (2006) and its description was subsequently emended by Zhang et al. (2012) and Jin et al. (2013). The type strains of the recognized species of the genus are strictly aerobic, Gram-staining-negative, nonmotile, non-gliding bacteria containing menaquinone-7 (MK-7) as the major respiratory quinone (Xie & Yokota, 2006; Zhang et al., 2012; Jin et al., 2013). At the time of writing, the genus Terrimonas comprised five recognized species isolated from various environments, such as garden soil, a freshwater spring, polluted farmland soil and bulking sludge; namely Terrimonas ferruginea (the type species) and Terrimonas lutea (Xie & Yokota, 2006), Terrimonas aquatica (Sheu et al., 2010), Terrimonas rubra (Zhang et al., 2012) and Terrimonas pekingensis (Jin et al., 2013). During the course of an investigation into the culturable bacterial community in the soil from a high Arctic tundra near the settlement, Ny-A˚lesund (78u 589 N 12u 039 E), in the Svalbard Archipelago, Norway, a large number of bacteria were isolated. In this study, we report on the taxonomic characteriazation of one of these isolates, designated strain R9-86T. On the basis of its phenotypic characteristics, the
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain R9-86T is KJ631121. Four supplementary figures are available with the online version of this paper.
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chemotaxonomic data and phylogenetic analysis, the isolate represents a novel species of the genus Terrimonas. For strain isolation, the soil sample was diluted serially with a sterile 0.85 % (w/v) NaCl solution and these dilutions were plated onto 0.36R2A (BD) agar plates. Single colonies on these plates were purified by transferring them onto new plates followed by additional incubation for 7 days at 20 uC. The isolate was routinely cultivated on R2A agar at 25 uC and stored by lyophilization at 4 uC. For 16S rRNA gene sequencing and phylogenetic analysis, genomic DNA was extracted from a fresh culture of strain R9-86T following the methods of Sambrook et al. (1989). Primers 27f (59-GAGTTTGATCCTGGCTCAG-39) and 1527r (59-AGAAAGGAGGTGATCCAGCC-39) were used for amplification of the 16S rRNA gene (Lane, 1991). PCR and 16S rRNA gene sequencing were carried out as described by Lin et al. (2004). Sequence similarities were searched by NCBI BLAST and calculated by pairwise alignments obtained from the EzTaxon database (Kim et al., 2012). Phylogenetic analysis was performed by using the software package MEGA version 6.0 (Tamura et al., 2013) after multiple alignments of data via CLUSTAL W (Thompson et al., 1994). Phylogenetic trees were reconstructed by the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. Evolutionary distances for the neighbour-joining algorithm were calculated with Kimura’s two-parameter method (Kimura, 1980). Bootstrap 067033 G 2014 IUMS Printed in Great Britain
Terrimonas arctica sp. nov.
values were determined based on 1000 replications (Felsenstein, 1985). The 16S rRNA gene sequence of strain R9-86T was a continuous stretch of 1419 bp. Sequence comparisons with 16S rRNA gene sequences from the EzTaxon database revealed that strain R9-86T had the highest similarity with T. pekingensis QHT (95.0 %), followed by T. rubra M-8T (95.0 %), T. lutea DYT (94.8 %), T. ferruginea DSM 30193T (94.7 %) and T. aquatica RIB1-6T (93.7 %). These values are well below the threshold for demarcating bacterial species (Stackebrandt & Goebel, 1994). The neighbourjoining tree showed that strain R9-86T was affiliated to the genus Terrimonas in the family Chitinophagaceae, and formed a robust clade with T. pekingensis QHT and T. lutea DYT (Fig. 1). The same relationship was also found in trees constructed using the maximum-parsimony (Fig. S1, available in the online Supplementary Material) and maximumlikelihood (Fig. S2) algorithms. These results suggest that strain R9-86T represents a novel species within the genus Terrimonas. Growth was evaluated at 25 uC on several standard bacteriological media (all from BD): R2A agar, 0.36R2A agar, nutrient broth (NB) agar, marine broth 2216 (MB) agar, tryptic soy broth (TSB) agar and MacConkey agar. Gram staining of cells was carried out according to the classical Gram procedure described by Doetsch (1981). Growth at different temperatures (0, 4, 10, 18, 20, 22, 25, 28, 37 and 42 uC) was investigated on R2A agar for up to 2 weeks. The pH range for growth was determined in R2A broth at pH 4.0–11.0 (in increments of 1.0 pH unit). Salt tolerance was tested on R2A agar supplemented with 0, 0.5, 1, 2, 3, 5 and 10 % NaCl (w/v) for 2 weeks at 25 uC. Cell morphology was examined by phase-contrast (Olympus BX51) and transmission electron (Hitachi 8100) microscopy using cells grown for 2 days at 25 uC on R2A. Gliding motility was investigated as described by Bowman (2000). Growth under anaerobic conditions was tested on R2A agar in a GasPak (BBL) jar at 25 uC for 21 days. Polyb-hydroxybutyrate (PHB) accumulation was observed by light microscopy after staining the cells with Sudan black (Smibert & Krieg, 1994). Catalase activity was determined by measurement of bubble production after the application of 3 % (v/v) hydrogen peroxide solution. Oxidase activity was evaluated by the oxidation of 1 % (w/v) tetramethyl-pphenylenediamine (Kova´cs, 1956). The presence of flexirubin-type pigments was tested using the KOH test, as described by Bernardet et al. (2002). Hydrolysis of chitin, starch, DNA, gelatin, agar, CM-cellulose and casein was investigated on R2A agar after one week of incubation, according to methods described by Smibert & Krieg (1994). The hydrolysis of Tween 20, Tween 40, Tween 60 and Tween 80 was measured using the formation of an opaque halo of precipitation around the colony (Barrow & Feltham, 1993). Antibiotic sensitivity was tested by using filter-paper discs (diameter, 6.35 mm) impregnated with the following antibiotics (per disc): amikacin (30 mg), vancomycin (30 mg), ciprofloxacin (5 mg), polymyxin B http://ijs.sgmjournals.org
(30 mg), ampicillin (10 mg), chloramphenicol (30 mg), gentamicin (10 mg), kanamycin (30 mg), penicillin G (10 mg), sulfamethoxazole (23.75 mg), trimethoprim (1.25 mg) and tetracycline (30 mg). Any sign of growth inhibition was scored as sensitivity to that antibiotic. Resistance to an antimicrobial drug was indicated if no inhibition zone was observed. Additional physiological and biochemical characteristics were determined by using the API 20NE and API ZYM kits (bioMe´rieux) and the GN2 MicroPlate (Biolog), according to the manufacturers’ instructions. Strain R9-86T grew well at 25 uC on R2A agar, 0.36R2A agar and NB agar, but not on MB agar, TSB agar and MacConkey agar. Cells were strictly aerobic, Gram-stainnegative, non-motile and rod-shaped (Fig. S3). Colonies were yellow, circular, convex and smooth after growing for 5 days at 25 uC on R2A agar. Growth occurred at 4–28 uC (optimum, 22–25 uC) and at pH 6.0–9.0 (optimum, pH 7.0). The range of NaCl concentrations for growth was 0–0.5 % (w/v); optimum growth occurred in the absence of NaCl. Other physiological characteristics of strain R9-86T are summarized in the species description. Selected characteristics that differentiate strain R9-86T from related species of the genus Terrimonas are shown in Table 1. To measure the G+C content of the chromosomal DNA, genomic DNA from the novel strain was extracted and purified, as described by Moore & Dowhan (1995), and degraded enzymically into nucleosides and the G+C content was then determined, as described by Mesbah et al. (1989) using reversed-phase HPLC (UltiMate 3000, Dionex). Respiratory quinones were extracted and identified by HPLC, as described by Xie & Yokota (2003). For analysis of cellular fatty acids, strain R9-86T and five related type strains, T. ferruginea DSM 30193T, T. lutea KACC 13047T, T. aquatica KACC 14981T, T. rubra CCTCC AB 2010401T and T. pekingensis CICC 10452T were grown on R2A agar plates at 25 uC and harvested at the lateexponential phase. The methods used for harvesting, saponification, methylation and extraction of cellular fatty acids were those according to the protocol of the Sherlock Microbial Identification System (MIDI) version 6.0. Separation and identification of fatty acid methyl esters was performed using a Hewlett Packard 6890N gas chromatograph, with the MIDI Sherlock TSBA6 (version of the database) (Sasser, 1990). For polar lipid analysis, strain R986T and reference strains were grown on R2A broth at 25 uC. Polar lipids were extracted and analysed by twodimensional TLC (silica gel plates, layer thickness 0.2 mm, Merck) according tomethods described by Tindall (1990). The genomic DNA G+C content of strain R9-86T was 48.4 mol%, a value that fell within the range of those reported for the other species of the genus Terrimonas (Table 1). The major respiratory quinone of strain R9-86T was MK-7, which is consistent with other members of the genus Terrimonas. The cellular fatty acid profiles of strain R9-86T and five reference strains are presented in Table 2. The major cellular fatty acids of strain R9-86T were 3799
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99
100 80
100
92
100
99
82
96 100
100
100 100 100 100
75
100 99
73
82 96 71
71 100 99 96 100
Niabella hirudinis E96T (KC307771) Niabella drilacis E90T (KC307772) Niabella aurantiaca DSM 17617T (ARFF01000013) Niabella soli DSM 19437T (AGSA01000025) Niabella tibetensis 15-4T (GU291295) Niabella ginsengisoli GR10-1T (EU616816) Niabella yanshanensis CCBAU 05354T (FJ457040) Terrimonas rubra M-8T (JF803808) Terrimonas ferruginea DSM 30193T (AM230484) Terrimonas aquatica RIB1-6T (FJ347757) Terrimonas arctica R9-86T (KJ631121) Terrimonas pekingensis QHT (JF834159) Terrimonas lutea DYT (AB192292) Ferruginibacter alkalilentus HU1-GD23T (FJ177530) Ferruginibacter lapsinanis HU1-HG42T (FJ177532) Flavisolibacter ginsengisoli Gsoil 643T (AB267477) Flavisolibacter ginsengiterrae Gsoil 492T (AB267476) Flavitalea gansuensis JCN-23T (U295962) Flavitalea populi HY-50RT (HM130561) Niastella koreensis GR20-10T (CP003178) Niastella yeongjuensis GR20-13T (DQ244076) Niastella populi THYL-44T (EU877262) Segetibacter aerophilus 6424S-61T (GQ421847) Segetibacter koreensis DSM 18137T (ARFB01000010) Filimonas lacunae YT21T (AB362776) Parasegetibacter luojiensis RHYL-37T (EU877263) Flavihumibacter petaseus T41T (EU854577) Chitinophaga cymbidii R156-2T (JN680880) Chitinophaga japonensis IFO 16041T (AB078055) Chitinophaga rupis CS5-B1T (FM865977) Chitinophaga eiseniae YC6729T (FJ750951) Chitinophaga terrae KP01T (AB278570) Chitinophaga jiangningensis JN53T (KF150362) Chitinophaga polysaccharea MRP-15T (KC430923) Chitinophaga taiwanensis CC-ALB-1T (KC479802) Chitinophaga niastensis JS16-4T ( EU714260) Chitinophaga ginsengisegetis Gsoil 040T (AB264798) Chitinophaga arvensicola DSM 3695T (AM237311) Hydrotalea flava CCUG 51397T (FN665659) Hydrotalea sandarakina AF-51T (JF739858) Sediminibacterium salmoneum NJ-44T (EF407879) Sediminibacterium ginsengisoli DCY13T (EF067860) Lacibacter daechungensis H32-4T (KC759435) Lacibacter cauensis NJ-8T (EU521690) Flavobacterium aquatile DSM 1132T (AM230485)
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences, showing the relationship between strain R9-86T and related taxa. Percentage bootstrap values (1000 replications) greater than 70 % are shown at nodes. The sequence of Flavobacterium aquatile DSM 1132T was used as an outgroup.
iso-C15 : 1-G (25.7 %), iso-C15 : 0 (24.5 %), iso-C17 : 0-3OH (18.3 %) and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c, 8.7 %), which was consistent with data published for species of the genus Terrimonas with validly published names. The differences between strain R9-86T and other species of the genus Terrimonas were particularly marked with respect to the fatty acid profiles, as strain R9-86T
contained higher amounts of the major fatty acid isoC17 : 0-3OH. Additionally, strain R9-86T could be differentiated from other species of the genus Terrimonas due to the presence of C16 : 1v5c, which was not detected in the other species. The major polar lipids of strain R9-86T were phosphatidylethanolamine and an unknown polar lipid; six unknown aminolipids, five unknown polar lipids and one
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Terrimonas arctica sp. nov.
Table 1. Differential characteristics of strain R9-86T and related species of the genus Terrimonas Strains: 1, R9-86T; 2, T. pekingensis CICC 10452T; 3, T. lutea KACC 13047T; 4, T. rubra CCTCC AB 2010401T; 5, T. ferruginea DSM 30193T; 6, T. aquatica KACC 14981T. Data were obtained in this study unless otherwise indicated. +, Positive; 2, negative; W, weakly positive. Characteristic Growth at: 4 uC 37 uC 1 % NaCl Flexirubin-type pigments Hydrolysis of: Casein Agar Tween 80 CM-cellulose Enzyme activity (API ZYM) Trypsin a-Chymotrypsin a-Galactosidase b-Galactosidase b-Glucuronidase b-Glucosidase a-Mannosidase a-Fucosidase Assimilation of (API 20 NE) D-Glucose D-Mannose N-Acetylglucosamine Maltose Potassium gluconate Malate DNA G+C content (mol%)
1
2
3
4
5
6
+ 2 2 2
2 2 2 2
2 + + 2
2 + + +
2 + + +
2 + + +
2 2 2 +
2 + 2 2
+ + 2 2
+ 2 2 W
+ 2 + 2
+ 2 + 2
2 2 + 2 2 2 + 2
+ + + + 2 2 2 2
+ + + + 2 + + +
+ + + 2 2 2 2 +
2 2 2 2 2 2 2 2
+ 2 + + + + + +
2 2 2 2 2 2 48.4 %
+ + + + 2 2 41.0 %a
+ + + + + 2 47.2 %b
+ + + + 2 + 47.0 %c
+ + + + 2 2 48.9 %b
+ + + + 2 + 47.3 %d
Data taken from: (a) Jin et al. (2013), (b) Xie & Yokota (2006), (c) Zhang et al. (2012) and (d) Sheu et al. (2010).
unknown aminophospholipid were also detected. The polar lipid profile of strain R9-86T was similar to that of the four reference strains of the genus Terrimonas (Fig. S4). On the basis of the data presented, isolate R9-86T represents a novel species of the genus Terrimonas, for which the name Terrimonas arctica sp. nov. is proposed. Description of Terrimonas arctica sp. nov. Terrimonas arctica (arc9ti.ca. L. fem. adj. arctica northern, from the Arctic, referring to the site where the type strain was isolated). Cells are strictly aerobic, Gram-stain-negative, non-motile and rod-shaped. Colonies are yellow, circular, convex and smooth after growing for 5 days at 25 uC on R2A agar. Growth occurs at 25 uC on R2A agar, 0.36R2A agar and NB agar, but not on MB agar, TSB agar and MacConkey agar. Growth occurs at 4–28 uC (optimum, 22–25 uC) and at pH 6.0–9.0 (optimum, pH 7.0). The range of NaCl for growth is 0–0.5 % (w/v); optimum growth occurs without NaCl. Tests for catalase and oxidase are positive. http://ijs.sgmjournals.org
Flexirubin-type pigments are absent. Poly-b-hydroxybutyrate granules are not accumulated. The strain hydrolyses CM-cellulose and Tween 60. Starch and Tween 20 are weakly hydrolysed, but chitin, DNA, gelatin, agar, casein, Tween 40 and Tween 80 are not hydrolysed. In the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, a-glucosidase, N-acetyl-b-glucosaminidase and a-mannosidase activities are present, but lipase(C14), trypsin, a-chymotrypsin, b-galactosidase, bglucuronidase, b-glucosidase and a-fucosidase activities are absent. In API 20 NE strips, reactions are positive for the hydrolysis of aesculin, b-galactosidase activity and the assimilation of L-arabinose. Negative for reduction of nitrate, production of indole, fermentation of glucose, arginine dihydrolase and urease activities and the assimilation of D-glucose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Positive for the Biolog GN2 MicroPlate substrates: dextrin, N-acetyl-D-glucosamine, cellobiose, D-fructose, D-galactose, 3801
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Table 2. Cellular fatty acid composition of strain R9-86T and type strains of phylogenetically related species Strains: 1, R9-86T; 2, T. pekingensis CICC 10452T; 3, T. lutea KACC 13047T; 4, T. rubra CCTCC AB 2010401T; 5, T. ferruginea DSM 30193T; 6, T. aquatica KACC 14981T. Data were obtained in this study. Values are percentages of total fatty acids; fatty acids amounting to ,1 % of the total fatty acids in all strains listed are omitted. Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c. 2, None detected; tr, trace amounts (,1 %). Fatty acid iso-C14 : 0 C14 : 0 iso-C15 : 1-G anteiso-C15 : 1-A iso-C15 : 0 anteiso-C15 : 0 iso-C16 : 0 C16 : 1v5c C16 : 0 iso-C15 : 0-3OH C17 : 1v6c C15 : 0-2OH iso-C16 : 0-3OH C16 : 0 23OH C18 : 0 iso-C17 : 0-3OH C17 : 0-2OH C17 : 0-3OH Summed feature 3
1
2
3
4
5
6
tr 1.4 25.7 tr 24.5 1.9 tr 1.5 5.0 2.6 2 tr tr 4.0 tr 18.3 1.1 tr 8.7
1.2 1.2 18.0 tr 26.0 2.6 1.4 2 7.1 1.5 1.6 tr 2.3 5.4 tr 12.0 tr 2.7 12.2
tr tr 26.7 1.3 32.2 3.7 tr 2 3.0 2.1 2 tr tr 2.4 tr 14.5 1.0 tr 6.2
tr 1.5 26.3 tr 17.9 1.2 tr 2 2.8 2.6 2 tr tr 4.0 1.0 14.9 tr tr 21.0
1.3 1.5 19.9 tr 35.1 1.7 1.3 2 4.6 2.7 tr 1.0 tr 1.9 tr 14.1 tr tr 9.4
tr 1.3 23.2 tr 26.9 1.5 tr 2 5.4 1.3 tr tr tr 5.0 tr 12.8 tr 1.7 14.7
polymyxin B, gentamicin, kanamycin, penicillin G, sulfamethoxazole, trimethoprim and tetracycline, but resistant to ampicillin and chloramphenicol. The major respiratory quinone is MK-7 and the major cellular fatty acids are isoC15 : 1-G, iso-C15 : 0, iso-C17 : 0-3OH and summed feature 3 (C16 : 1v7c and/or C16 : 1v6c). The major polar lipids of strain R9-86T are phosphatidylethanolamine and an unknown polar lipid; six unknown aminolipids, five unknown polar lipids and one unknown aminophospholipid are also detected. The type strain, R9-86T (5CCTCC AB 2011004T5NRRL B-59114T), was isolated from tundra soil near Ny-A˚lesund, Svalbard Archipelago, Norway. The DNA G+C content of the type strain is 48.4 mol%.
Acknowledgements This work was supported by National Basic Research Program of China (973 Program) (2011CB808800), the State Oceanic Administration, PR. China (project no. 10/11YR06), the National Infrastructure of Natural Resources for Science and Technology Program of China (no. NIMR-2014-8), National Natural Science Foundation of China (no. 31200038) and Key Project of Chinese Ministry of Education (V201308).
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