International Journal of Systematic and Evolutionary Microbiology (2014), 64, 621–624
DOI 10.1099/ijs.0.056739-0
Myroides xuanwuensis sp. nov., a mineral-weathering bacterium isolated from forest soil Zhen-Dong Zhang, Lin-Yan He, Zhi Huang and Xia-Fang Sheng Correspondence Xia-Fang Sheng
Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
[email protected]
A Gram-reaction-negative, aerobic, non-motile, yellow-pigmented, rod-shaped bacterium, designated strain TH-19T, was isolated from a forest soil sample in Jiangsu province, China. On the basis of 16S rRNA gene sequence similarity, strain TH-19T was shown to belong to the genus Myroides, a member of the phylum Bacteroidetes, and was related to Myroides odoratimimus LMG 4029T (98.7 % similarity), Myroides profundi D25T (98.2 %) and Myroides marinus JS-08T (97.5 %). Strain TH-19T contained menaquinone-6 (MK-6) as the predominant menaquinone, and the dominant fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH. The DNA G+C content of strain TH-19T was 37.2 mol%. The DNA–DNA relatedness values of strain TH-19T with Myroides odoratimimus JCM 7460T, Myroides profundi D25T and Myroides marinus JS-08T were below 70 %. Based on phenotypic, genotypic and phylogenetic evidence, it is suggested that strain TH19T represents a novel species of the genus Myroides, for which the name Myroides xuanwuensis sp. nov. is proposed. The type strain is TH-19T (5CCTCC AB 2013145T5JCM 19200T).
The genus Myroides, belonging to the family Flavobacteriaceae, was proposed in 1996 to reclassify strains of the species Flavobacterium odoratum (Vancanneyt et al., 1996). At the time of writing, the genus Myroides comprised six recognized species, namely Myroides odoratimimus, Myroides profundi, Myroides marinus, Myroides phaeus, Myroides pelagicus and Myroides odoratus (Cho et al., 2011; Vancanneyt et al., 1996; Yan et al., 2012; Yoon et al., 2006; Zhang et al., 2008). Most strains of M. odoratus, M. odoratimimus and M. phaeus originate from clinical specimens (Yan et al., 2012), whereas those of the other species come from the marine environment. Species of the genus Myroides also occur in freshwater fish (Maull et al., 2012) and the insect gut (Spiteller et al., 2000). Here, we isolated a strain from a forest soil. Preliminary analysis of the 16S rRNA gene sequence indicated that strain TH-19T belongs to the genus Myroides. The exact taxonomic status of strain TH-19T was determined by using a polyphasic approach. Strain TH-19T was isolated from a forest soil sample collected from Nanjing, Jiangsu Province, China by using the standard dilution plating technique on 1/5-strength Luria–Bertani (LB) agar. Strain TH-19T was able to weather biotite (a silicate mineral) and release Si, Al and Fe from the mineral. Mineral dissolution experiments [based on the method of Zhao et al. (2013)] showed that The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain TH-19T is HF947513. Three supplementary figures and one supplementary table are available with the online version of this paper.
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the Si, Al and Fe released from the mineral by strain TH19T were increased by 3.2-fold, 4.2-fold and 40-fold, respectively, compared to the uninoculated controls. Cellular morphology and the presence of flagella were examined using light microscopy (CX21; Olympus) and transmission electron microscopy (H-7650; Hitachi) as described by Bogan et al. (2003) and Zhang et al. (2008). Examination of the biochemical properties of strain TH-19T followed procedures previously described by Cho et al. (2011) and Yan et al. (2012). The following phenotypic tests were performed: Gram-reaction testing, cell morphology, catalase and oxidase activities, and hydrolysis of casein, cellulose, DNA, starch, Tweens 40, 60 and 80 and urea. Growth in Marine broth 2216 (Difco) at different temperatures (6, 10, 18, 28, 37, 42 and 45 uC) and at various pH values (pH 4.0–11.0, at intervals of 1.0 pH unit, adjusted with 1M HCl or 1M NaOH solution before autoclaving) was assayed after 5 days of incubation. The buffers used to keep the pH of the marine broth stable (each at a final concentration of 100 mM) were acetate (for pH 4.0–5.0), phosphate (for pH 6.0–8.0) and Tris (for pH 9.0–11.0). The requirement for and tolerance of NaCl (0, 3, 5, 10, 15 and 20 % NaCl) were investigated by using a basal medium containing10 g peptone and 5 g yeast extract per litre. Other biochemical and physiological properties were examined by using API 20E and API ZYM strips (bioMe´rieux) according to the manufacturer’s instructions. The profile of carbon substrate oxidation was obtained using Biolog GN2 microplates according to the manufacturer’s instructions. 621
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Respiratory quinone, fatty acid methyl ester and DNA G+C content analysis were performed. Briefly, cell mass of strain TH-19T was grown on Marine agar 2216 (Difco) after incubation for 2 days at 30 uC and harvested. Analysis of fatty acid methyl esters was carried out as described by Ka¨mpfer & Kroppenstedt (1996). The DNA G+C content was determined by using the HPLC method (Mesbah et al., 1989). Determination of the respiratory quinone was performed via reversed-phase HPLC as described by Collins et al. (1977) and Xie & Yokota (2003). DNA–DNA hybridization between TH-19T and type strains of members of the genus Myroides was carried out as described by De Ley et al. (1970) using a UV/VIS spectrophotometer (UV1201; Rayleigh). The 16S rRNA gene of strain TH-19T was amplified by PCR from genomic DNA and sequenced as described by Park et al. (2005). The 16S rRNA gene sequence of strain TH-19T was aligned with corresponding sequences of the type strains of recognized species of the genus Myroides from GenBank with CLUSTAL X software. Phylogenetic trees were reconstructed by using the neighbour-joining, maximum-likelihood and minimum-evolution methods following procedures described by Kim et al. (2003) and Park et al. (2006). The neighbour-joining tree is shown in Fig. 1. The maximum-likelihood and minimum-evolution
96 100
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trees are available as Figs S2 and S3 (available in IJSEM Online). Strain TH-19T formed yellow, circular, convex colonies with entire margins on 2216 agar at 30 uC within 48h. Growth occurred between 10 and 42 uC, at pH 4–10 and with 0–6 % NaCl. Cells were aerobic, Gram-reaction-negative, nonspore-forming, non-motile rods. Other phenotypic characteristics are given in the species description and in Table 1. The major quinone system of strain TH-19T was menaquinone MK-6. The cellular fatty acid profiles of strain TH-19T and the type strains of related species of the genus Myroides are shown in Table 2. Major fatty acids (.10 %) of strain TH-19T were iso-C15 : 0 (34.2 %), iso-C17 : 0 3-OH (12.5 %) and summed features 3 comprising C16 : 1v7c and/ or iso-C15 : 0 2-OH (11.4 %). This fatty acid profile was similar to the other species of the genus Myroides. The DNA G+C content of strain TH-19T was 37.2 mol%, which was close to that found for other species of the genus Myroides. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain TH-19T is a member of the family Flavobacteriaceae and forms a coherent cluster with species of the genus Myroides (Fig. 1). Strain TH-19T was related most closely to M. odoratimimus LMG 4029T (98.7 % sequence similarity) and M. profundi D25T
Myroides odoratimimus JCM 7460T (AJ854059) Myroides profundi DSM 19823T (EU204978) Myroides xuanwuensis TH-19T (HF947513) Myroides marinus JCM 16529T (GQ857652)
Myroides phaeus DSM 23313T (GU253339) Myroides pelagicus JCM 21782T (AB176662) Myroides odoratus JCM 7458T (M58777)
96
Flavobacterium terrae KACC 11731T (EF117329)
48
Flavobacterium aquatile ATCC 11947T (M62797)
99
Flavobacterium frigoris LMG 21922T (AJ557887)
67 100 64
Flavobacterium omnivorum JCM 11313T (AF433174) Flavobacterium xinjiangense JCM 11314T (AF433173) Kordia algicida KCTC 8814PT (AY195836) Capnocytophaga granulosa LMG 16022T (U41347)
45
Croceibacter atlanticus HTCC2559T (AY163576) Sphingobacterium spiritivorum DSM 2582 (AJ459411)
Fig. 1. Neighbour-joining tree based on almost complete 16S rRNA gene sequences showing the relationship between strain TH-19T and representative members of the family Flavobacteriaceae. Numbers at the nodes indicate levels of bootstrap support (over 45 %) based on the neighbour-joining analysis of 1000 resampled datasets using Jukes–Cantor distances. Sphingobacterium spiritivorum DSM 2582 was used as an outgroup. Bar, 0.02 substitutions per nucleotide position. 622
International Journal of Systematic and Evolutionary Microbiology 64
Myroides xuanwuensis sp. nov.
Table 1. Differential phenotypic characteristics of strain TH-19T and type strains of related species of the genus Myroides Strains: 1, TH-19T; 2, M. odoratimimus JCM 7460T; 3, M. profundi DSM 19823T; 4, M. marinus JS-08T. Habitats and DNA G+C content values of reference strains taken from Cho et al. (2011) and Yan et al. (2012). +, Positive; 2, negative. Characteristic Isolation source Cell morphology Pigment Range for growth Temperature (uC) NaCl (%) pH Hydrolysis of: Casein Tween 80 Utilization of (Biolog): Dextrin Leucine Glycogen DNA G+C content (mol%)
1
2
3
4
Forest soil Rods Pale yellow
Wound swab Rods Pale yellow
Deep-sea sediment Spindle-shaped rod Pale yellow, diffusible
Seawater Short rods Yellow to orange
10–42 0–6 4–10
10–37 0–6 6–9
8–42 0–8 5–9
10–37 0–5 5–9
+ +
2 2
+ +
2 2
2 2 2 37.2
+ + + 33.8
+ + 2 33
2 2 2 34.2
(98.2 %), but formed an independent phylogenetic line. DNA–DNA hybridization values between strain TH-19T and M. marinus JS-08T, M. odoratimimus JCM 7460T and M. profundi DSM 19823T were 61.0 %, 60.9 % and 37.3 % respectively. It is generally accepted that strains with ,70 % DNA–DNA relatedness or .97 % 16S rRNA gene sequence dissimilarity can be considered as belonging to separate species (Stackebrandt & Goebel, 1994). These results indicated that strain TH-19T represents a novel species of the genus Myroides. Phylogenetic analysis and differences in physiological and biochemical characteristics (Table 1) together with the fatty acid profile (Table 2) clearly distinguish strain TH-19T from closely related species of the genus Myroides. Thus, on the basis of the data presented, we suggest that strain TH19T represents a novel species of the genus Myroides, for which the name Myroides xuanwuensis TH-19T sp. nov. is proposed. Description of Myroides xuanwuensis sp. nov. Myroides xuanwuensis (xuan.wu.en9sis. N.L. masc. adj. xuanwuensis referring to Xuanwu district, Nanjing, Jiangsu Province, PR China, where the organism was first isolated). Cells are Gram-reaction-negative, aerobic, straight and single rods, 0.4–0.5 mm in diameter and 1.2–2.0 mm in length. Cells lack flagella (Fig. S1). Neither cellular gliding movement nor swarming growth is observed. Colonies grown on Marine agar 2216 (Difco) are circular, convex and pale yellow. The temperature range for growth is 10– 42 uC, but no growth occurs at 4 or 45 uC. The pH range for growth is pH 4–10. NaCl is not required for growth, up to 6 % NaCl (w/v) is tolerated. Catalase reaction http://ijs.sgmjournals.org
is positive, whereas oxidase reaction is weakly positive. Voges–Proskauer test is positive. Citrate is utilized. Indole is not produced. Nitrate and nitrite are not reduced, and hydrogen sulphide is not produced. DNA and Tweens 40 and 80 are hydrolysed, but casein, cellulose and starch are not. According to API ZYM and API 20E strips, alkaline and acid phosphatase, esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase, lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, urease and gelatinase activities are present, but lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuroidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, b-galatosidase and tryptophan deaminase activities are absent. Acid is not produced from glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin or arabinose. Using the Biolog GN2 microplates, the following 26 compounds exhibit positive reactions: Tween 40, Tween 80, methyl pyruvate, monomethyl succinate, acetic acid, a-ketoglutaric acid, aketovaleric acid, DL-lactic acid, succinic acid, bromosuccinic acid, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic, Lglutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, L-ornithine, L-proline, L-serine, L-threonine, urocanic acid, inosine, uridine and thymidine. The predominant menaquinone is MK-6. The major fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH and two summed features consisting of C16 : 1v7c and/or iso-C15 : 0 2-OH and/or C16 : 1v6c, and iso-C17 : 1v9c and/or 10-methyl C16 : 0. The type strain, TH-19T (5CCTCC AB 2013145T5JCM 19200T), was isolated from forest soil in Xuanwu, Nanjing, Jiangsu province, PR China. The G+C content of DNA of the type strain is 37.2 mol%. 623
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Table 2. Fatty acid contents of strain MH1-19 and the type strains of recognized species of the genus Myroides Strains: 1, TH-19T; 2, M. odoratimimus JCM 7460T; 3, M. profundi DSM 19823T; 4, M. marinus JS-08T. –, Less than 1.0 % of the total or not detected. Data for reference strains from Cho et al. (2011). All strains were grown on Marine agar 2216 (Difco) at 30 uC for 48h. Fatty acid Straight-chain C16 : 0 Branched saturated iso-C13 : 0 iso-C14 : 0 iso-C15 : 0 anteiso-C15 : 0 iso-C16 : 0 iso-C17 : 0 anteiso-C17 : 0 Unsaturated acid C13 : 1 at 12-13 C17 : 1v6c C18 : 1v6c Branched unsaturated C15 : 0 2-OH C15 : 0 3-OH iso-C15 : 0 3-OH C16 : 0 3-OH iso-C16 : 0 3-OH C17 : 0 3-OH iso-C17 : 0 3-OH iso-C17 : 1v9c Summed features* 3 9
1
2
3
4
Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977).
Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230. De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative
measurement of DNA hybridization from renaturation rate. Eur J Biochem 12, 133–142. Ka¨mpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of
fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005. Kim, S. B., Lonsdale, J., Seong, C. N. & Goodfellow, M. (2003).
2.9
1.4
1.3
1.3
3.0 1.2 34.2 – 4.7 – –
2.0 1.5 44.7 3.8 2.7 – –
1.0 1.7 27.6 – 4.3 2.2 3.8
– 2.6 24.2 1.1 3.4 – –
1.3 1.5 –
– – –
– – –
– – 1.8
– – 4.2 6.3 1.2 – 12.5 –
– – 3.6 6.5 2.2 – 16.9 2.8
1.5 – 4.0 5.2 2.2 – 14.5 3.3
2.8 2.3 4.7 6.9 3.0 1.3 18.1 2.4
11.4 8.9
9.2 –
15.4 –
16.8 –
*Summed feature 3 contains C16 : 1v7c and/or iso-C15 : 0 2-OH and/or C16 : 1v6c. Summed feature 9 contains iso-C17 : 1v9c and/or 10-methyl C16 : 0.
Streptacidiphilus gen. nov., acidophilic actinomycetes with wall chemotype I and emendation of the family Streptomycetaceae (Waksman and Henrici (1943)AL) emend. Rainey et al. 1997. Antonie van Leeuwenhoek 83, 107–116. Maull, K., Hickey, M. & Lee, J. (2012). The study and identification of
bacterial spoilage species isolated from catfish during refrigerated storage. J Food Process Technol 10, S11-003. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Park, M. S., Jung, S. R., Lee, M. S., Kim, K. O., Do, J. O., Lee, K. H., Kim, S. B. & Bae, K. S. (2005). Isolation and characterization of
bacteria associated with two sand dune plant species, Calystegia soldanella and Elymus mollis. J Microbiol 43, 219–227. Park, M. S., Jung, S. R., Lee, K. H., Lee, M. S., Do, J. O., Kim, S. B. & Bae, K. S. (2006). Chryseobacterium soldanellicola sp. nov. and
Chryseobacterium taeanense sp. nov., isolated from roots of sand-dune plants. Int J Syst Evol Microbiol 56, 433–438. Spiteller, D., Dettner, K. & Boland, W. (2000). Gut bacteria may be
involved in interactions between plants, herbivores and their predators: microbial biosynthesis of N-acylglutamine surfactants as elicitors of plant volatiles. Biol Chem 381, 755–762. Stackebrandt, E. & Goebel, B. (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. Vancanneyt, M., Segers, P., Torck, U., Hoste, B., Bernardet, J. F., Vandamme, P. & Kersters, K. (1996). Reclassification of Flavobacterium
odoratum (Stutzer 1929) strains to a new genus, Myroides, as Myroides odoratus comb. nov. and Myroides odoratimimus sp. nov. Int J Syst Bacteriol 46, 926–932. Xie, C. H. & Yokota, A. (2003). Phylogenetic analyses of Lampropedia
Acknowledgements This work was supported by National Natural Science Foundation of China (project nos 41071173, 41201521). Professor Yuzhong Zhang (Shandong University, China) kindly provided M. odoratimimus JCM 7460T and M. profundi D25T, and Professor XiaoHua Zhang (Ocean University of China) provided M. marinus JS-08T.
References Bogan, B. W., Sullivan, W. R., Kayser, K. J., Derr, K. D., Aldrich, H. C. & Paterek, J. R. (2003). Alkanindiges illinoisensis gen. nov., sp. nov., an
obligately hydrocarbonoclastic, aerobic squalane-degrading bacterium isolated from oilfield soils. Int J Syst Evol Microbiol 53, 1389–1395.
hyalina based on the 16S rRNA gene sequence. J Gen Appl Microbiol 49, 345–349. Yan, S., Zhao, N. & Zhang, X. H. (2012). Myroides phaeus sp. nov.,
isolated from human saliva, and emended descriptions of the genus Myroides and the species Myroides profundi Zhang et al. 2009 and Myroides marinus Cho et al. 2011. Int J Syst Evol Microbiol 62, 770– 775. Yoon, J., Maneerat, S., Kawai, F. & Yokota, A. (2006). Myroides
pelagicus sp. nov., isolated from seawater in Thailand. Int J Syst Evol Microbiol 56, 1917–1920. Zhang, X. Y., Zhang, Y. J., Chen, X. L., Qin, Q. L., Zhao, D. L., Li, T. G., Dang, H. Y. & Zhang, Y. Z. (2008). Myroides profundi sp. nov., isolated
from deep-sea sediment of the southern Okinawa Trough. FEMS Microbiol Lett 287, 108–112. Zhao, F., Qiu, G., Huang, Z., He, L. Y. & Sheng, X. F. (2013).
marinus sp. nov., a member of the family Flavobacteriaceae, isolated from seawater. Int J Syst Evol Microbiol 61, 938–941.
Characterization of Rhizobium sp. Q32 isolated from weathered rocks and its role in silicate mineral weathering. Geomicrobiol J 30, 616–622.
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Cho, S. H., Chae, S. H., Im, W. T. & Kim, S. B. (2011). Myroides
DOI 10.1099/ijs.0.056739-0
Myroides xuanwuensis sp. nov., a mineral-weathering bacterium isolated from forest soil Zhen-Dong Zhang, Lin-Yan He, Zhi Huang and Xia-Fang Sheng Correspondence Xia-Fang Sheng
Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
[email protected]
A Gram-reaction-negative, aerobic, non-motile, yellow-pigmented, rod-shaped bacterium, designated strain TH-19T, was isolated from a forest soil sample in Jiangsu province, China. On the basis of 16S rRNA gene sequence similarity, strain TH-19T was shown to belong to the genus Myroides, a member of the phylum Bacteroidetes, and was related to Myroides odoratimimus LMG 4029T (98.7 % similarity), Myroides profundi D25T (98.2 %) and Myroides marinus JS-08T (97.5 %). Strain TH-19T contained menaquinone-6 (MK-6) as the predominant menaquinone, and the dominant fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH. The DNA G+C content of strain TH-19T was 37.2 mol%. The DNA–DNA relatedness values of strain TH-19T with Myroides odoratimimus JCM 7460T, Myroides profundi D25T and Myroides marinus JS-08T were below 70 %. Based on phenotypic, genotypic and phylogenetic evidence, it is suggested that strain TH19T represents a novel species of the genus Myroides, for which the name Myroides xuanwuensis sp. nov. is proposed. The type strain is TH-19T (5CCTCC AB 2013145T5JCM 19200T).
The genus Myroides, belonging to the family Flavobacteriaceae, was proposed in 1996 to reclassify strains of the species Flavobacterium odoratum (Vancanneyt et al., 1996). At the time of writing, the genus Myroides comprised six recognized species, namely Myroides odoratimimus, Myroides profundi, Myroides marinus, Myroides phaeus, Myroides pelagicus and Myroides odoratus (Cho et al., 2011; Vancanneyt et al., 1996; Yan et al., 2012; Yoon et al., 2006; Zhang et al., 2008). Most strains of M. odoratus, M. odoratimimus and M. phaeus originate from clinical specimens (Yan et al., 2012), whereas those of the other species come from the marine environment. Species of the genus Myroides also occur in freshwater fish (Maull et al., 2012) and the insect gut (Spiteller et al., 2000). Here, we isolated a strain from a forest soil. Preliminary analysis of the 16S rRNA gene sequence indicated that strain TH-19T belongs to the genus Myroides. The exact taxonomic status of strain TH-19T was determined by using a polyphasic approach. Strain TH-19T was isolated from a forest soil sample collected from Nanjing, Jiangsu Province, China by using the standard dilution plating technique on 1/5-strength Luria–Bertani (LB) agar. Strain TH-19T was able to weather biotite (a silicate mineral) and release Si, Al and Fe from the mineral. Mineral dissolution experiments [based on the method of Zhao et al. (2013)] showed that The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain TH-19T is HF947513. Three supplementary figures and one supplementary table are available with the online version of this paper.
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the Si, Al and Fe released from the mineral by strain TH19T were increased by 3.2-fold, 4.2-fold and 40-fold, respectively, compared to the uninoculated controls. Cellular morphology and the presence of flagella were examined using light microscopy (CX21; Olympus) and transmission electron microscopy (H-7650; Hitachi) as described by Bogan et al. (2003) and Zhang et al. (2008). Examination of the biochemical properties of strain TH-19T followed procedures previously described by Cho et al. (2011) and Yan et al. (2012). The following phenotypic tests were performed: Gram-reaction testing, cell morphology, catalase and oxidase activities, and hydrolysis of casein, cellulose, DNA, starch, Tweens 40, 60 and 80 and urea. Growth in Marine broth 2216 (Difco) at different temperatures (6, 10, 18, 28, 37, 42 and 45 uC) and at various pH values (pH 4.0–11.0, at intervals of 1.0 pH unit, adjusted with 1M HCl or 1M NaOH solution before autoclaving) was assayed after 5 days of incubation. The buffers used to keep the pH of the marine broth stable (each at a final concentration of 100 mM) were acetate (for pH 4.0–5.0), phosphate (for pH 6.0–8.0) and Tris (for pH 9.0–11.0). The requirement for and tolerance of NaCl (0, 3, 5, 10, 15 and 20 % NaCl) were investigated by using a basal medium containing10 g peptone and 5 g yeast extract per litre. Other biochemical and physiological properties were examined by using API 20E and API ZYM strips (bioMe´rieux) according to the manufacturer’s instructions. The profile of carbon substrate oxidation was obtained using Biolog GN2 microplates according to the manufacturer’s instructions. 621
Z.-D. Zhang and others
Respiratory quinone, fatty acid methyl ester and DNA G+C content analysis were performed. Briefly, cell mass of strain TH-19T was grown on Marine agar 2216 (Difco) after incubation for 2 days at 30 uC and harvested. Analysis of fatty acid methyl esters was carried out as described by Ka¨mpfer & Kroppenstedt (1996). The DNA G+C content was determined by using the HPLC method (Mesbah et al., 1989). Determination of the respiratory quinone was performed via reversed-phase HPLC as described by Collins et al. (1977) and Xie & Yokota (2003). DNA–DNA hybridization between TH-19T and type strains of members of the genus Myroides was carried out as described by De Ley et al. (1970) using a UV/VIS spectrophotometer (UV1201; Rayleigh). The 16S rRNA gene of strain TH-19T was amplified by PCR from genomic DNA and sequenced as described by Park et al. (2005). The 16S rRNA gene sequence of strain TH-19T was aligned with corresponding sequences of the type strains of recognized species of the genus Myroides from GenBank with CLUSTAL X software. Phylogenetic trees were reconstructed by using the neighbour-joining, maximum-likelihood and minimum-evolution methods following procedures described by Kim et al. (2003) and Park et al. (2006). The neighbour-joining tree is shown in Fig. 1. The maximum-likelihood and minimum-evolution
96 100
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trees are available as Figs S2 and S3 (available in IJSEM Online). Strain TH-19T formed yellow, circular, convex colonies with entire margins on 2216 agar at 30 uC within 48h. Growth occurred between 10 and 42 uC, at pH 4–10 and with 0–6 % NaCl. Cells were aerobic, Gram-reaction-negative, nonspore-forming, non-motile rods. Other phenotypic characteristics are given in the species description and in Table 1. The major quinone system of strain TH-19T was menaquinone MK-6. The cellular fatty acid profiles of strain TH-19T and the type strains of related species of the genus Myroides are shown in Table 2. Major fatty acids (.10 %) of strain TH-19T were iso-C15 : 0 (34.2 %), iso-C17 : 0 3-OH (12.5 %) and summed features 3 comprising C16 : 1v7c and/ or iso-C15 : 0 2-OH (11.4 %). This fatty acid profile was similar to the other species of the genus Myroides. The DNA G+C content of strain TH-19T was 37.2 mol%, which was close to that found for other species of the genus Myroides. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain TH-19T is a member of the family Flavobacteriaceae and forms a coherent cluster with species of the genus Myroides (Fig. 1). Strain TH-19T was related most closely to M. odoratimimus LMG 4029T (98.7 % sequence similarity) and M. profundi D25T
Myroides odoratimimus JCM 7460T (AJ854059) Myroides profundi DSM 19823T (EU204978) Myroides xuanwuensis TH-19T (HF947513) Myroides marinus JCM 16529T (GQ857652)
Myroides phaeus DSM 23313T (GU253339) Myroides pelagicus JCM 21782T (AB176662) Myroides odoratus JCM 7458T (M58777)
96
Flavobacterium terrae KACC 11731T (EF117329)
48
Flavobacterium aquatile ATCC 11947T (M62797)
99
Flavobacterium frigoris LMG 21922T (AJ557887)
67 100 64
Flavobacterium omnivorum JCM 11313T (AF433174) Flavobacterium xinjiangense JCM 11314T (AF433173) Kordia algicida KCTC 8814PT (AY195836) Capnocytophaga granulosa LMG 16022T (U41347)
45
Croceibacter atlanticus HTCC2559T (AY163576) Sphingobacterium spiritivorum DSM 2582 (AJ459411)
Fig. 1. Neighbour-joining tree based on almost complete 16S rRNA gene sequences showing the relationship between strain TH-19T and representative members of the family Flavobacteriaceae. Numbers at the nodes indicate levels of bootstrap support (over 45 %) based on the neighbour-joining analysis of 1000 resampled datasets using Jukes–Cantor distances. Sphingobacterium spiritivorum DSM 2582 was used as an outgroup. Bar, 0.02 substitutions per nucleotide position. 622
International Journal of Systematic and Evolutionary Microbiology 64
Myroides xuanwuensis sp. nov.
Table 1. Differential phenotypic characteristics of strain TH-19T and type strains of related species of the genus Myroides Strains: 1, TH-19T; 2, M. odoratimimus JCM 7460T; 3, M. profundi DSM 19823T; 4, M. marinus JS-08T. Habitats and DNA G+C content values of reference strains taken from Cho et al. (2011) and Yan et al. (2012). +, Positive; 2, negative. Characteristic Isolation source Cell morphology Pigment Range for growth Temperature (uC) NaCl (%) pH Hydrolysis of: Casein Tween 80 Utilization of (Biolog): Dextrin Leucine Glycogen DNA G+C content (mol%)
1
2
3
4
Forest soil Rods Pale yellow
Wound swab Rods Pale yellow
Deep-sea sediment Spindle-shaped rod Pale yellow, diffusible
Seawater Short rods Yellow to orange
10–42 0–6 4–10
10–37 0–6 6–9
8–42 0–8 5–9
10–37 0–5 5–9
+ +
2 2
+ +
2 2
2 2 2 37.2
+ + + 33.8
+ + 2 33
2 2 2 34.2
(98.2 %), but formed an independent phylogenetic line. DNA–DNA hybridization values between strain TH-19T and M. marinus JS-08T, M. odoratimimus JCM 7460T and M. profundi DSM 19823T were 61.0 %, 60.9 % and 37.3 % respectively. It is generally accepted that strains with ,70 % DNA–DNA relatedness or .97 % 16S rRNA gene sequence dissimilarity can be considered as belonging to separate species (Stackebrandt & Goebel, 1994). These results indicated that strain TH-19T represents a novel species of the genus Myroides. Phylogenetic analysis and differences in physiological and biochemical characteristics (Table 1) together with the fatty acid profile (Table 2) clearly distinguish strain TH-19T from closely related species of the genus Myroides. Thus, on the basis of the data presented, we suggest that strain TH19T represents a novel species of the genus Myroides, for which the name Myroides xuanwuensis TH-19T sp. nov. is proposed. Description of Myroides xuanwuensis sp. nov. Myroides xuanwuensis (xuan.wu.en9sis. N.L. masc. adj. xuanwuensis referring to Xuanwu district, Nanjing, Jiangsu Province, PR China, where the organism was first isolated). Cells are Gram-reaction-negative, aerobic, straight and single rods, 0.4–0.5 mm in diameter and 1.2–2.0 mm in length. Cells lack flagella (Fig. S1). Neither cellular gliding movement nor swarming growth is observed. Colonies grown on Marine agar 2216 (Difco) are circular, convex and pale yellow. The temperature range for growth is 10– 42 uC, but no growth occurs at 4 or 45 uC. The pH range for growth is pH 4–10. NaCl is not required for growth, up to 6 % NaCl (w/v) is tolerated. Catalase reaction http://ijs.sgmjournals.org
is positive, whereas oxidase reaction is weakly positive. Voges–Proskauer test is positive. Citrate is utilized. Indole is not produced. Nitrate and nitrite are not reduced, and hydrogen sulphide is not produced. DNA and Tweens 40 and 80 are hydrolysed, but casein, cellulose and starch are not. According to API ZYM and API 20E strips, alkaline and acid phosphatase, esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase, lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, urease and gelatinase activities are present, but lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuroidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, b-galatosidase and tryptophan deaminase activities are absent. Acid is not produced from glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin or arabinose. Using the Biolog GN2 microplates, the following 26 compounds exhibit positive reactions: Tween 40, Tween 80, methyl pyruvate, monomethyl succinate, acetic acid, a-ketoglutaric acid, aketovaleric acid, DL-lactic acid, succinic acid, bromosuccinic acid, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic, Lglutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, L-ornithine, L-proline, L-serine, L-threonine, urocanic acid, inosine, uridine and thymidine. The predominant menaquinone is MK-6. The major fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH and two summed features consisting of C16 : 1v7c and/or iso-C15 : 0 2-OH and/or C16 : 1v6c, and iso-C17 : 1v9c and/or 10-methyl C16 : 0. The type strain, TH-19T (5CCTCC AB 2013145T5JCM 19200T), was isolated from forest soil in Xuanwu, Nanjing, Jiangsu province, PR China. The G+C content of DNA of the type strain is 37.2 mol%. 623
Z.-D. Zhang and others
Table 2. Fatty acid contents of strain MH1-19 and the type strains of recognized species of the genus Myroides Strains: 1, TH-19T; 2, M. odoratimimus JCM 7460T; 3, M. profundi DSM 19823T; 4, M. marinus JS-08T. –, Less than 1.0 % of the total or not detected. Data for reference strains from Cho et al. (2011). All strains were grown on Marine agar 2216 (Difco) at 30 uC for 48h. Fatty acid Straight-chain C16 : 0 Branched saturated iso-C13 : 0 iso-C14 : 0 iso-C15 : 0 anteiso-C15 : 0 iso-C16 : 0 iso-C17 : 0 anteiso-C17 : 0 Unsaturated acid C13 : 1 at 12-13 C17 : 1v6c C18 : 1v6c Branched unsaturated C15 : 0 2-OH C15 : 0 3-OH iso-C15 : 0 3-OH C16 : 0 3-OH iso-C16 : 0 3-OH C17 : 0 3-OH iso-C17 : 0 3-OH iso-C17 : 1v9c Summed features* 3 9
1
2
3
4
Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977).
Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230. De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative
measurement of DNA hybridization from renaturation rate. Eur J Biochem 12, 133–142. Ka¨mpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of
fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005. Kim, S. B., Lonsdale, J., Seong, C. N. & Goodfellow, M. (2003).
2.9
1.4
1.3
1.3
3.0 1.2 34.2 – 4.7 – –
2.0 1.5 44.7 3.8 2.7 – –
1.0 1.7 27.6 – 4.3 2.2 3.8
– 2.6 24.2 1.1 3.4 – –
1.3 1.5 –
– – –
– – –
– – 1.8
– – 4.2 6.3 1.2 – 12.5 –
– – 3.6 6.5 2.2 – 16.9 2.8
1.5 – 4.0 5.2 2.2 – 14.5 3.3
2.8 2.3 4.7 6.9 3.0 1.3 18.1 2.4
11.4 8.9
9.2 –
15.4 –
16.8 –
*Summed feature 3 contains C16 : 1v7c and/or iso-C15 : 0 2-OH and/or C16 : 1v6c. Summed feature 9 contains iso-C17 : 1v9c and/or 10-methyl C16 : 0.
Streptacidiphilus gen. nov., acidophilic actinomycetes with wall chemotype I and emendation of the family Streptomycetaceae (Waksman and Henrici (1943)AL) emend. Rainey et al. 1997. Antonie van Leeuwenhoek 83, 107–116. Maull, K., Hickey, M. & Lee, J. (2012). The study and identification of
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measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Park, M. S., Jung, S. R., Lee, M. S., Kim, K. O., Do, J. O., Lee, K. H., Kim, S. B. & Bae, K. S. (2005). Isolation and characterization of
bacteria associated with two sand dune plant species, Calystegia soldanella and Elymus mollis. J Microbiol 43, 219–227. Park, M. S., Jung, S. R., Lee, K. H., Lee, M. S., Do, J. O., Kim, S. B. & Bae, K. S. (2006). Chryseobacterium soldanellicola sp. nov. and
Chryseobacterium taeanense sp. nov., isolated from roots of sand-dune plants. Int J Syst Evol Microbiol 56, 433–438. Spiteller, D., Dettner, K. & Boland, W. (2000). Gut bacteria may be
involved in interactions between plants, herbivores and their predators: microbial biosynthesis of N-acylglutamine surfactants as elicitors of plant volatiles. Biol Chem 381, 755–762. Stackebrandt, E. & Goebel, B. (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. Vancanneyt, M., Segers, P., Torck, U., Hoste, B., Bernardet, J. F., Vandamme, P. & Kersters, K. (1996). Reclassification of Flavobacterium
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Acknowledgements This work was supported by National Natural Science Foundation of China (project nos 41071173, 41201521). Professor Yuzhong Zhang (Shandong University, China) kindly provided M. odoratimimus JCM 7460T and M. profundi D25T, and Professor XiaoHua Zhang (Ocean University of China) provided M. marinus JS-08T.
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