Antonie van Leeuwenhoek DOI 10.1007/s10482-014-0180-x
ORIGINAL PAPER
Streptomyces calidiresistens sp. nov., isolated from a hot spring sediment Yan-Yan Duan • Hong Ming • Lei Dong Yi-Rui Yin • Yi Zhang • En-Min Zhou • Lan Liu • Guo-Xing Nie • Wen-Jun Li
•
Received: 16 March 2014 / Accepted: 18 April 2014 Ó Springer International Publishing Switzerland 2014
Yan-Yan Duan and Hong Ming have contributed equally to this work.
between strain YIM 78087T and its closest neighbours, these analyses indicated that this new isolate represents a different genomic species in the genus Streptomyces. The predominant menaquinones of strain YIM 78087T were identified as MK-9 (H4) and MK-9 (H6). The major fatty acids were identified as anteiso-C15:0 (28.4 %), anteiso-C17:0 (23.0 %) and iso-C16:0 (15.1 %). The whole-cell hydrolysates found to contain glucose, mannose and ribose. The DNA G?C content was determined to be 73.0 mol%. Based on the comparative analysis of phenotypic and genotypic characteristics, it is proposed that strain YIM 78087T represents a novel species of the genus Streptomyces, for which the name Streptomyces calidiresistens sp. nov., is proposed. The type strain is YIM 78087T (=BCRC 16955T=DSM 42108T=JCM 19629T).
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0180-x) contains supplementary material, which is available to authorized users.
Keywords Streptomyces calidiresistens sp. nov. Hot spring Tengchong
Y.-Y. Duan Y. Zhang G.-X. Nie (&) College of Fisheries, Henan Normal University, Xinxiang 453007, People’s Republic of China e-mail: [email protected]
H. Ming College of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, People’s Republic of China
Y.-Y. Duan H. Ming L. Dong Y.-R. Yin E.-M. Zhou L. Liu W.-J. Li (&) Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China e-mail: [email protected]
W.-J. Li State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China
Abstract A Streptomyces-like actinomycete strain, designated as YIM 78087T, was isolated from a sediment sample collected from Hehua hot spring in Tengchong, Yunnan province, south-west China. The taxonomic position of strain YIM 78087T was investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain YIM 78087T belongs to the genus Streptomyces and is closely related to Streptomyces fimbriatus DSM 40942T, Streptomyces marinus DSM 41968T and Streptomyces qinglanensis DSM 42035T (97.18, 97.05 and 97.1 % similarity, respectively). Combined with the low values of DNA–DNA hybridization
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Introduction In Bergey’s Manual of Systematic Bacteriology, the genus Streptomyces within the family Streptomycetaceae was originally described by Waksman and Henrici (1943), and since then the description of the genus has been emended repeatedly (Williams et al. 1983, 1989; Witt and Stackebrandt 1990). So far, the genus Streptomyces encompasses more than 600 species with validly published names (http://www.bacterio.net/ index.html), and so contains the largest number of species of any genus in the domain Bacteria although it is evident that the taxon is underspeciated (Antony-Babu and Goodfellow 2008; Okoro et al. 2009; Antony-Babu et al. 2010). The members of the genus Streptomyces are rich sources of novel pharmaceutically important compounds and notably antibiotics (Watve et al. 2001; Goodfellow and Fiedler 2010; Rateb et al. 2011a, b; Kim et al. 2012a). Nowadays, it is important to isolate new therapeutic compounds from novel species in order to avoid the costly rediscovery of known compounds from Streptomyces species which have already been identified before (Busti et al. 2006; Lam 2007; Antony-Babu and Goodfellow 2008; Goodfellow and Fiedler 2010). Therefore, we are focusing on the isolation of actinomycetes from hot springs. The high temperature of hot springs leads the isolates to adapt through unique physiological mechanisms, which may lead to the evolution of specific genes which will lead to novel chemicals. During our study on thermophilic actinobacterial resources from hot springs, the strain YIM 78087T was isolated from a sediment sample collected from the Hehua hot spring in Yunnan province, southwest China. Strain YIM 78087T has been characterized by phenotypic, chemotaxonomic and phylogenetic analysis. Based on the phylogenetic results, the strain YIM 78087T was affiliated to the genus Streptomyces. The experimental data we obtained also indicated that isolate YIM 78087T represents a novel species of the genus Streptomyces, named here Streptomyces calidiresistens sp. nov.
Materials and methods Strain and culture conditions For the isolation of thermophilic actinobacteria, 2 g of sediment sample collected from Hehua hot spring was
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taken into a flask with 18 ml sterile hot spring water and several glass beads. The mixture were shaken at 45 °C with 200 rpm for 1 h. One millilitre of the sample mixture was diluted 10 and 100 fold, and then 200 ll of the 10-2 dilution suspension were spread on T5 medium (Ming et al. 2014) agar plates adjusted to pH 8.0, supplemented with nalidixic acid (25 mg l-1) and nystatin (50 mg l-1). The plates were incubated at 45 °C for 14 days. Strain YIM 78087T was selected, purified and routinely cultured on T5 medium at 45 °C and stored as a glycerol suspension (20 %, w/v) at -80 °C. Three closely related type strains i.e. Streptomyces fimbriatus DSM 40942T (Millaed and Burr 1926), Streptomyces marinus DSM 41968T (Khan et al. 2010) and Streptomyces qinglanensis DSM 42035T (Hu et al. 2012) were obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures) and used as reference strains. They were routinely grown on ISP 2 medium at 28 °C for 3 days except S. marinus DSM 41968T where the medium was supplemented with 50 % seawater. Biomass for chemical and molecular tests was obtained by cultivation in shaken flasks (about 200 rpm) using ISP 2 medium, T5 broth medium or Tryptic Soy Broth (TSB, containing 15 g tryptone, 5 g soya peptone and 5 g NaCl per litre) for 3 days. Strain YIM 78087T was grown at 45 °C in medium adjusted to pH 8.0, where as the reference strains were grown at 28 °C in medium adjusted to pH 7.0. Cells were harvested and washed three times with distilled water and then freeze-dried. Phenotypic characteristics Morphological and cultural characteristics were tested on ISP 2, oatmeal agar (ISP 3), inorganic salts-starch agar (ISP 4), glycerol-asparagine agar (ISP 5) (Shirling and Gottlieb 1966), potato-dextrose agar (PDA), Czapek’s and nutrient agar (Waksman 1967). The colours of the colonies were determined by using the ISCC–NBS colour charts (Kelly 1964). The morphological characteristics of strain YIM 78087T were determined by light microscopy (Philips XL30) and scanning electron microscopy (ESEM-TMP) after the cultures were grown on T5 medium at 45 °C for 7 days. The spore-chain morphology, spore size and surface ornamentation of isolate were observed. Growth was tested at temperatures of 4, 15, 28, 30, 37, 42, 45, 50, 55 and 60 °C on T5 medium plates.
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The pH range for growth (4.0–10.0, at intervals of 1.0 pH units) and NaCl tolerance (0, 1, 3, 5, 7, 8, 9, 10, 15, 20 and 25 % w/v) were tested at 45 °C for 7 days by culturing the strains in T5 broth. Milk peptonization and coagulation, gelatin liquefaction, nitrate reduction, H2S production, degradation of Tweens, starch and cellulose, activities of urease, oxidase and catalase were tested according to the procedures described by Williams et al. (1989) and Gordon et al. (1974). The single carbon source utilization was tested with Pridham and Gottlieb’s basal mineral salts medium and the acid production were performed with carbohydrates. Nitrogen source utilization was observed in a basal liquid medium (Nie et al. 2012). API ZYM strips were used to determine some enzyme activities according to the instructions of the manufacturer (bioMe´rieux). Other physiological and biochemical characteristics were assessed by using the media and methods described by Gordon et al. (1974). Chemotaxonomy Chemotaxonomic characteristics were determined following standard procedures. The isomer of diaminopimelic acid of the cell wall and sugars analyses of whole-cell hydrolysates were performed as described by Hasegawa et al. (1983), Staneck and Roberts (1974) and Tang et al. (2009). Polar lipids were extracted and separated by two-dimensional thin-layer chromatography (TLC) and identified using the described procedures (Minnikin et al. 1979; Collins and Jones 1980). Menaquinones were extracted from lyophilized cells as described by Collins et al. (1977) and Minnikin et al. (1984) and then purified and analyzed by HPLC (Kroppenstedt 1982; Hu et al. 2001). For analysis of cellular fatty acids, strain YIM 78087T was cultured at 45 °C, and the reference strains S. fimbriatus DSM 40942T, S. marinus DSM 41968T and S. qinglanensis DSM 42035T were cultured at 28 °C with TSB medium for 4 days. The cellular fatty acids were extracted, methylated and analyzed by using the protocol of the Sherlock Microbial Identification System (MIDI) (Sherlock Version 6. 1; MIDI database: TSBA6) (Sasser 1990). The G?C content of the genomic DNA was determined by HPLC (Mesbah et al. 1989) with Escherichia coli JM-109 as the reference strain.
Molecular analysis Genomic DNA extraction, PCR amplification of the 16S rRNA gene sequence was performed as described by Li et al. (2007). The amplicon was purified using a Sangon PCR purification kit (China). The full-length 16S rRNA gene sequence of strain YIM 78087T was compared against other species via BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al. 1990) and type strains via Eztaxon database (http://eztaxon-e.ezbiocloud.net/) (Kim et al. 2012b). Multiple alignments were performed using the CLUSTAL_X software package (Thompson et al. 1997). The Kimura two-parameter model (Kimura 1980, 1983, 1985) was used to calculate evolutionary distance. Phylogenetic trees were constructed by the neighbour-joining (Saitou and Nei 1987), maximumlikelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) tree-making algorithms drawn from the MEGA version 5.0 package (Tamura et al. 2011). Bootstrap analysis with 1,000 resamplings was used to evaluate the topology of each tree (Felsenstein 1985). DNA–DNA relatedness was studied by applying the fluorometric micro-well method (Ezaki et al. 1989; Christensen et al. 2000; He et al. 2005) at the optimal hybridization temperature (47 °C). The experiments were set with eight replications between strain YIM 78087T and its closest phylogenetic neighbours S. fimbriatus DSM 40942T, S. marinus DSM 41968T and S. qinglanensis DSM 42035T.
Results and discussion Strain YIM 78087T was determined to exhibit typical morphological properties of the members of the genus Streptomyces. Observation of a 2 weeks old culture of strain YIM 78087T revealed that both aerial and vegetative hyphae were abundant and the spores are rhabditiform in shape with smooth surfaces (Supplementary Figure S1). YIM 78087T was observed to grow well on ISP 4, Czapek’s agar and nutrient agar, and to grow weakly on ISP 5, PDA, ISP 2 and ISP 3 media. Aerial mycelium was found to be produced on all tested media except PDA medium, and to have cream white or white colours on all media except PDA. The substrate mycelium was observed to have
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yellow-brown or olive-brown colours when grown on all media, and diffusible pigment was produced on ISP 4, ISP 5, Czapek’s agar and nutrient agar (Supplementary Table S1). Growth was observed at 28–50 °C (optimum 37–45 °C), pH 4.0–10.0 (optimum pH 8.0–9.0) and 0–9 % (w/v) NaCl (optimum 1–3 %). Activity of catalase was found to be positive, but urease and oxidase were negative. Milk coagulation and peptonization, hydrolysis of starch, cellulose and gelatin were found to be positive but melanin formation was negative. Tweens 20, 40 and 60 were found to be degraded but Tween 80 was not. Nitrate was found to be reduced but H2S was not produced. Differences in various characteristics demonstrated that strain YIM 78087T is phenotypically distinct from its closest phylogenetic relatives and are shown in Table 1. The detailed physiological characteristics of strain YIM 78087T are given in the species description. The diagnostic cell wall di-amino acid of the strain YIM 78087T was determined to be LL-diaminopimelic acid (LL-DAP). The whole organism hydrolysates were found to be rich in glucose, mannose and ribose, and galactose and rhamnose were also detected as minor components. The polar lipids of strain YIM 78087T were determined to consist of diphosphatidylglycerol, phosphatidylethanolamine, four phospholipids, two aminolipids and an unknown lipid (Supplementary Figure S2). The major respiratory menaquinones of strain YIM 78087T were identified as MK-9 (H4) (84 %) and MK-9 (H6) (16 %). The fatty acids profile ([5 %) was determined to be anteiso-C15:0 (28.4 %), anteiso-C17:0 (23.0 %), isoC16:0(15.1 %), iso-C15:0 (10.0 %), iso-C16:1 H(7.4 %) and iso-C17:0 (5.0 %). Detailed fatty acid profiles of strain YIM 78087T and the reference type strains of the most closely related species are given in Table 2. The DNA G?C content of strain YIM 78087T was determined to be 73.0 mol%, which is in accordance with the values for the genus Streptomyces (from 67 to 78 mol%). These characteristics are consistent with the members of the genus Streptomyces. To determine the phylogenetic position of strain YIM 78087T, the full-length 16S rRNA gene sequence (1588 nt; GenBank accession number KJ361473) of strain YIM 78087T was determined. Comparison of the sequence with the corresponding 16S rRNA gene sequences retrieved from GenBank/EMBL/DDBJ
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Table 1 Differential characteristics between strain YIM 78087T and its related members of the genus Streptomyces Characteristic
1
2
3
4
Utilization of D-Sucrose
?
?
?
W
L-Arabinose
-
?
?
?
D-Xylose
-
?
?
?
Sorbitol
W
?
?
W
D-Mannitol
W
?
?
?
D-Xylitol
?
?
w
?
Lactose
-
?
?
?
Inositol
-
W
?
?
D-Sorbitol
W
?
?
?
Dulcitol
?
?
?
W
Sodium pyruvate
?
?
?
w
Raffinose
?
?
?
-
Trehalose
?
?
W
?
Sodium soccinate L-Arginine
W ?
? ?
W W
? ?
Tween 80
-
?
?
?
CM-cellulose
?
?
-
?-
Starch
?
-
-
-
Esterase lipase (C8)
?
-
?
-
Lipase (C14)
?
-
-
-
Leucine arylamidase
?
-
?
?
Valine arylamidase
?
-
-
-
Cystine arylamidase
?
?
?
-
Acidic phosphatase
-
?
?
-
Naphthol-AS-BI-phosphohydrolase
?
?
-
?
b-Galactosidase
-
-
?
-
a-Glucosidase b-Glucosidase
?
? -
? ?
-
N-Acetyl-b-glucosaminidase
-
?
?
?
a-Mannosidase
-
-
?
28
Degradation of
Activity of
Growth Optimum temperature (°C)
45
28
28
Optimum pH
8.0
7.0
7.0
7.0
8 % (w/v) NaCl
W
-
-
?
Data obtained during this study; analyses were carried out under identical growth conditions except the temperature 1, YIM 78087T; 2, S. fimbriatus DSM 40942T; 3, S. marinus DSM 41968T; 4, S. qinglanensis DSM 42035T; ? (positive), utilized; - (negative), not utilized; w (weakly positive), utilized
Antonie van Leeuwenhoek Table 2 Fatty acid profiles (%) of the new isolate YIM 780087T and its closely related type strains in the genus Streptomyces Fatty acid
1
2
3
4
C16:0
2.0
4.7
3.9
3.1
Iso-C14:0
1.5
2.4
3.8
2.8
Iso-C15:0
10.0
5.2
21.6
8.8
Iso-C16:0
15.1
27.1
12.6
18.4
Iso-C17:0
5.1
3.1
4.8
6.8
Iso-C16:1 H Anteiso-C15:0
7.4 28.4
6.0 18.1
0.7 34.3
1.8 31.9
Anteiso-C17:0
23.0
15.3
Anteiso-C17:1 w9c
2.6
4.4
15.3
21.8
-
0.9
Summed feature 3a
2.7
5.0
1.0
0.7
Summed feature 9a
1.6
2.5
-
-
Data were obtained in this study under identical conditions except temperature Strains 1, YIM 78087T; 2, S. fimbriatus DSM 40942T; 3, S. marinus DSM 41968T; 4, S. qinglanensis DSM 42035T; -, negative or absent a
Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature three contains one or more of the fatty acids C16:1 x7c and C16:1 x6c. Summed feature nine contains one or more of the fatty acids iso-C17:1 x9c and C16:0 10-methyl
clearly demonstrated that strain YIM 78087T is a member of the genus Streptomyces. Strain YIM 78087T was found to be closely related to S. fimbriatus DSM 40942T (97.18 % similarity), S. marinus DSM 41968T (97.05 % similarity) and S. qinglanensis DSM 42035T (97.1 % similarity). From the neighbourjoining phylogenetic tree (Fig. 1), strain YIM 78087T formed a branch with S. marinus DSM 41968T (Khan et al. 2010) and S. qinglanensis DSM 42035T with 87 % bootstrap value, but was distantly related to S. fimbriatus DSM 40942T. This phylogenetic relationship was also supported in the trees generated with maximum-parsimony phylogenetic tree (Supplementary Figure S3) and maximum-likelihood phylogenetic tree (Supplementary Figure S4) with high bootstrap values (81 and 83 %, respectively). These data suggest that strain YIM 78087T is closely related to Streptomyces clade 125 (containing S. marinus) as delineated by Labeda et al. (2012). DNA–DNA relatedness values between strain YIM 78087T and the type strains S. fimbriatus DSM 40942T, S. marinus DSM 41968T and S. qinglanensis DSM 42035T were 24.2 ± 1.8, 27.7 ± 1.4 and
42.6 ± 1.5 %, respectively, which are notably lower than the threshold value (70 %) for the recognition of genomic species (Stackebrandt and Goebel 1994). Thus we suggest that strain YIM 78087T should be considered as a representative of a different genomic species of the genus Streptomyces. The combination of phylogenetic analysis, morphological, cultural, physiological and biochemical characteristics support the proposal that strain YIM 78087T is a member of the genus Streptomyces. Meanwhile, the differences in biochemical characteristics, 16S rRNA gene sequences similarities, DNA– DNA relatedness and fatty acid composition can be used to distinguish strain YIM 78087T from its closest relatives, S. fimbriatus DSM 40942T, S. marinus DSM 41968T and S. qinglanensis DSM 42035T. Therefore, based on these results, strain YIM 78087T represents a novel species of genus Streptomyces, for which the name S. calidiresistens sp. nov. is proposed. Description of Streptomyces calidiresistens sp. nov. Streptomyces calidiresistens (ca.li.di.ri.re.sis’tens; L. adj. calidus, hot; L. part adj. resistens, resisting; N. L. part. adj. calidiresistens, hot resisting). Cells are Gram-positive and aerobic, form extensively branched substrate and aerial mycelia. Substrate mycelia are typically yellow-brown or olive-brown colours and aerial mycelia are typically cream white or white colours. Diffusible pigments (brown-gray or yellow-brown colours) are produced on some media. Growth occurs at 28–50 °C, pH 4.0–10.0 and in the presence of 0–9 % (w/v) NaCl. Can utilize cellobiose, dulcitol, D-fructose, D-galactose, glycerol, D-glucose, maltose, D-mannitol, D-mannose, raffinose, sodium acetate, sodium malate, sodium pyruvate, sodium succinate, D-sorbitol, D-sucrose, trehalose, trisodium citrate and D-xylitol as sole carbon and energy sources, but not L-arabinose, lactose, inositol and D-xylose. Acid is produced from L-arabinose, cellobiose, dulcitol, D-fructose, D-glucose, inositol, lactose, maltose, Dmannitol, D-mannose, sodium malate, sodium pyruvate, D-sorbitol, D-sucrose, trehalose, trisodium citrate, D-xylitol and D-xylose. Utilises L-alanine, L-arginine, L-asparagine, cystine, glycine, L-glutamic acid, hypoxanthine, L-lysine, L-ornithine, L-phenylalanine, Lserine, L-threonine, L-tyrosine and L-valine as sole nitrogen sources. Milk coagulation and peptonization,
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S. ambofaciens ATCC 23877 (M27245) T S. marokkonensis Ap1 (AJ965470) T 76 S. atrovirens NRRL B-16357 (DQ026672) T S. albogriseolus NRRL B-1305 (AJ494865) T S. caelestis NRRL 2418 (X80824) T S. fimbriatus NBRC 15411 (AB184659) T S. fumanus NBRC 13042 (AB184273) T S. variegatus LMG 20315 (AJ781371) T S. mashuensis DSM 40221 (X79323) T S. tacrolimicus ATCC 55098 (FN429653) T S. koyangensis VK-A60 (AY079156) T S. chlorus BK125 (FR692094) T S. hebeiensis YIM 001 (AY277529) T 100* S. sannanensis NRRL B-24303 (AY999924) T S. albus subsp. pathocidicus NBRC 13812 (AB184501) T S. calidiresistens YIM 78087 (KJ361473) T S. qinglanensis 172205 (HQ660227) T S. marinus DSM 41968 (AB473556) T S. haliclonae DSM 41970 (AB473554) T S. xishensis YIM M 10378 (HQ585118) 68
55*
65* 75 50* 87
93*
87* 93* 87* 83*
0.005
Fig. 1 Neighbour-joining phylogenetic tree showing the phylogenetic relationship of strain YIM 78087T and other closely related Streptomyces species based on 16S rRNA gene sequences. Asterisks indicate branches that were also recovered
using the maximum-parsimony and maximum-likelihood methods. Bootstrap values (expressed as percentages of 1,000 replications) of above 50 % are shown at branch points. Bar 0.005 substitutions per nucleotide position
hydrolysis of starch, cellulose and gelatin are positive, but melanin formation is negative. Tweens 20, 40 and 60 are degraded, but Tween 80 is not. Nitrate is reduced but H2S is not produced. In API ZYM tests, activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, naphthol-AS-BI-phosphohydrolase, b-glucosidase are present. Activities of trypsin, a-chymotrypsin, acidic phosphatase, a-galactosidase, b-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-glucosidase, a-mannosidase and b-fucosidase are absent. The whole-cell hydrolysates contain glucose, mannose, ribose, rhamnose and galactose. MK-9 (H4) and MK-9 (H6) are the predominant menaquinones. The major fatty acids ([10 %) are anteiso-C15:0, anteisoC17:0, iso-C16:0, and iso-C15:0. The polar lipids profile contains diphosphatidylglycerol, phosphatidylethanolamine, four phospholipids, two aminolipids and an unknown lipid. The DNA G?C content of the type strain is 73.0 mol%. The type strain YIM 78087T (=BCRC 16955T=DSM 42108T=JCM 19629T) was isolated from a sediment sample collected from Hehua hot spring in Tengchong, Yunnan province, south-west
China. The 16S rRNA gene sequence of strain YIM 78087T has been deposited in GenBank under the accession number KJ361473.
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Acknowledgments The authors are grateful to Prof. Dr. HansPeter Klenk (DSMZ, Germany) for his kind providing of reference type strains. This research was supported by Key Project of International Cooperation of Ministry of Science & Technology (MOST) (No. 2013DFA31980), National Science Foundation Grant (OISE-0968421), Natural Science Foundation of China (Nos. 31070007 and 31372545), Key Project of Yunnan Provincial Natural Science Foundation (2013FA004), Scientific Research Fund of Xinxiang Medical University (2013QN126) and Research Project of Education Department of Henan Province of China (2011A180025).
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EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120 Kimura M (1983) The neutral theory of molecular evolution. Cambridge University Press, Cambridge Kimura M (1985) The neutral theory of molecular evolution. Cambridge University Press, Cambridge Kroppenstedt RM (1982) Separation of bacterial menaquinones by HPLC using reverse phase (RP18) and a silver loaded ion exchanger as stationary phases. J Liq Chromatogr 5:2359–2367 Labeda DP, Goodfellow M, Brown R, Ward AC, Lanoot B, Vanncanneyt M, Swings J, Kim SB, Liu Z, Chun J, Tamura T, Oguchi A, Kikuchi T, Kikuchi H, Nishii T, Tsuji K, Yamaguchi Y, Tase A, Takahashi M, Sakane T, Suzuki KI, Hatano K (2012) Phylogenetic study of the species within the family Streptomycetaceae. Antonie Van Leeuwenhoek 101:73–104 Lam KS (2007) New aspects of natural products in drug discovery. Trends Microbiol 15:279–289 Li WJ, Xu P, Schumann P, Zhang YQ, Pukall R, Xu LH, Stackebrandt E, Jiang CL (2007) Georgenia ruanii sp. nov., a novel actinobacterium isolated from forest soil in Yunnan (China) and emended description of the genus Georgenia. Int J Syst Evol Microbiol 57:1424–1428 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 Millaed W, Burr S (1926) A study of twenty four strains of actinomyces and their relation to types of common scab of potato. Ann Appl Biol 13:580–644 Ming H, Yin YR, Li S, Nie GX, Yu TT, Zhou EM, Liu L, Dong L, Li WJ (2014) Thermus caliditerrae sp. nov., a novel thermophilic species isolated from a geothermal area. Int J Syst Evol Microbiol 64:650–656 Minnikin DE, Collins MD, Goodfellow M (1979) Fatty acid and polar lipid composition in the classification of Cellulomonas, Oerskovia and related taxa. J Appl Bacteriol 47:87–95 Minnikin DE, O’Donnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, Parlett JH (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2:233–241 Nie GX, Ming H, Li S, Zhou EM, Cheng J, Tang X, Feng HG, Tang SK, Li WJ (2012) Amycolatopsis dongchuanensis sp. nov., a novel actinobacterium isolated from dry-hot valley in Yunnan, south-west China. Int J Syst Evol Microbiol 62:2650–2656 Okoro CK, Brown R, Jones AL, Andrews BA, Asenjo JA, Goodfellow M, Bull AT (2009) Diversity of culturable actinomycetes in hyper-arid soils of the Atacama Desert, Chile. Antonie Van Leeuwenhoek 95:121–133 Rateb ME, Houssen WE, Arnold M, Abdelrahman MH, Deng H, Harrison WT, William TA, Okoro CK, Asenjo JA, Andrews BA, Ferguson G, Bull AT, Goodfellow M, Ebel R, Jaspars M (2011a) Diverse metabolic profiles of a Streptomyces strain isolated from a hyper-arid environment. J Nat Prod 74:1965–1971
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Antonie van Leeuwenhoek Rateb ME, Houssen WE, Arnold M, Abdelrahman M-H, Deng H, Harrison WTA, Okoro CK, Asenjo JA, Andrews BA, Ferguson G, Bull AT, Goodfellow M, Ebel R, Jaspars M (2011b) Chaxamycins A-D, bioactive ansamycins from a hyper-arid desert Streptomyces sp. J Nat Prod 74:1491–1499 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic tree. Mol Biol Evol 4:406–425 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. USFCC Newsl 20:16 Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340 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 Staneck JL, Roberts GD (1974) Simplified approached to identification of aerobic actinomycetes by thin-layer chromatography. Appl Mirobiol 28:2731–2739 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using aaximum likelhood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Tang SK, Wang Y, Chen Y, Lou K, Cao LL, Xu LH, Li WJ (2009) Zhihengliuella alba sp. nov., and emended description of the genus Zhihengliuella. Int J Syst Evol Microbiol 59:2025–2032
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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 Waksman SA (1967) The actinomycetes: a summary of current knowledge. Ronald Press, New York Waksman SA, Henrici AT (1943) Family I. Streptomycetaceae. Nov. In: Boone DR, Castenholz RW (eds) Bergey’s manual of systematic bacteriology, vol 5, 2nd edn. Springer, New York, pp 1446–1767 Watve MG, Tikoo R, Jog MM, Bhole BD (2001) How many antibiotics are produced by the genus Streptomyces? Arch Microbiol 176:386–390 Williams ST, Goodfellow M, Alderson G, Wellington EMH, Sneath PHA, Sackin MJ (1983) Numerical classification of Streptomyces and related genera. J Gen Microbiol 129:1743–1813 Williams ST, Goodfellow M, Alderson G (1989) Genus Streptomyces Waksman and Henrici 1943, 339AL. In: Williams ST, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol 4. Williams and Willkins, Baltimore, pp 2453–2492 Witt D, Stackebrandt E (1990) Unification of the genera Streptoverticillium and Streptomyces, and amendation of Streptomyces Waksman and Henrici, 1943, 339AL. Syst Appl Microbiol 13:361–371
ORIGINAL PAPER
Streptomyces calidiresistens sp. nov., isolated from a hot spring sediment Yan-Yan Duan • Hong Ming • Lei Dong Yi-Rui Yin • Yi Zhang • En-Min Zhou • Lan Liu • Guo-Xing Nie • Wen-Jun Li
•
Received: 16 March 2014 / Accepted: 18 April 2014 Ó Springer International Publishing Switzerland 2014
Yan-Yan Duan and Hong Ming have contributed equally to this work.
between strain YIM 78087T and its closest neighbours, these analyses indicated that this new isolate represents a different genomic species in the genus Streptomyces. The predominant menaquinones of strain YIM 78087T were identified as MK-9 (H4) and MK-9 (H6). The major fatty acids were identified as anteiso-C15:0 (28.4 %), anteiso-C17:0 (23.0 %) and iso-C16:0 (15.1 %). The whole-cell hydrolysates found to contain glucose, mannose and ribose. The DNA G?C content was determined to be 73.0 mol%. Based on the comparative analysis of phenotypic and genotypic characteristics, it is proposed that strain YIM 78087T represents a novel species of the genus Streptomyces, for which the name Streptomyces calidiresistens sp. nov., is proposed. The type strain is YIM 78087T (=BCRC 16955T=DSM 42108T=JCM 19629T).
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0180-x) contains supplementary material, which is available to authorized users.
Keywords Streptomyces calidiresistens sp. nov. Hot spring Tengchong
Y.-Y. Duan Y. Zhang G.-X. Nie (&) College of Fisheries, Henan Normal University, Xinxiang 453007, People’s Republic of China e-mail: [email protected]
H. Ming College of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, People’s Republic of China
Y.-Y. Duan H. Ming L. Dong Y.-R. Yin E.-M. Zhou L. Liu W.-J. Li (&) Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China e-mail: [email protected]
W.-J. Li State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China
Abstract A Streptomyces-like actinomycete strain, designated as YIM 78087T, was isolated from a sediment sample collected from Hehua hot spring in Tengchong, Yunnan province, south-west China. The taxonomic position of strain YIM 78087T was investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain YIM 78087T belongs to the genus Streptomyces and is closely related to Streptomyces fimbriatus DSM 40942T, Streptomyces marinus DSM 41968T and Streptomyces qinglanensis DSM 42035T (97.18, 97.05 and 97.1 % similarity, respectively). Combined with the low values of DNA–DNA hybridization
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Introduction In Bergey’s Manual of Systematic Bacteriology, the genus Streptomyces within the family Streptomycetaceae was originally described by Waksman and Henrici (1943), and since then the description of the genus has been emended repeatedly (Williams et al. 1983, 1989; Witt and Stackebrandt 1990). So far, the genus Streptomyces encompasses more than 600 species with validly published names (http://www.bacterio.net/ index.html), and so contains the largest number of species of any genus in the domain Bacteria although it is evident that the taxon is underspeciated (Antony-Babu and Goodfellow 2008; Okoro et al. 2009; Antony-Babu et al. 2010). The members of the genus Streptomyces are rich sources of novel pharmaceutically important compounds and notably antibiotics (Watve et al. 2001; Goodfellow and Fiedler 2010; Rateb et al. 2011a, b; Kim et al. 2012a). Nowadays, it is important to isolate new therapeutic compounds from novel species in order to avoid the costly rediscovery of known compounds from Streptomyces species which have already been identified before (Busti et al. 2006; Lam 2007; Antony-Babu and Goodfellow 2008; Goodfellow and Fiedler 2010). Therefore, we are focusing on the isolation of actinomycetes from hot springs. The high temperature of hot springs leads the isolates to adapt through unique physiological mechanisms, which may lead to the evolution of specific genes which will lead to novel chemicals. During our study on thermophilic actinobacterial resources from hot springs, the strain YIM 78087T was isolated from a sediment sample collected from the Hehua hot spring in Yunnan province, southwest China. Strain YIM 78087T has been characterized by phenotypic, chemotaxonomic and phylogenetic analysis. Based on the phylogenetic results, the strain YIM 78087T was affiliated to the genus Streptomyces. The experimental data we obtained also indicated that isolate YIM 78087T represents a novel species of the genus Streptomyces, named here Streptomyces calidiresistens sp. nov.
Materials and methods Strain and culture conditions For the isolation of thermophilic actinobacteria, 2 g of sediment sample collected from Hehua hot spring was
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taken into a flask with 18 ml sterile hot spring water and several glass beads. The mixture were shaken at 45 °C with 200 rpm for 1 h. One millilitre of the sample mixture was diluted 10 and 100 fold, and then 200 ll of the 10-2 dilution suspension were spread on T5 medium (Ming et al. 2014) agar plates adjusted to pH 8.0, supplemented with nalidixic acid (25 mg l-1) and nystatin (50 mg l-1). The plates were incubated at 45 °C for 14 days. Strain YIM 78087T was selected, purified and routinely cultured on T5 medium at 45 °C and stored as a glycerol suspension (20 %, w/v) at -80 °C. Three closely related type strains i.e. Streptomyces fimbriatus DSM 40942T (Millaed and Burr 1926), Streptomyces marinus DSM 41968T (Khan et al. 2010) and Streptomyces qinglanensis DSM 42035T (Hu et al. 2012) were obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures) and used as reference strains. They were routinely grown on ISP 2 medium at 28 °C for 3 days except S. marinus DSM 41968T where the medium was supplemented with 50 % seawater. Biomass for chemical and molecular tests was obtained by cultivation in shaken flasks (about 200 rpm) using ISP 2 medium, T5 broth medium or Tryptic Soy Broth (TSB, containing 15 g tryptone, 5 g soya peptone and 5 g NaCl per litre) for 3 days. Strain YIM 78087T was grown at 45 °C in medium adjusted to pH 8.0, where as the reference strains were grown at 28 °C in medium adjusted to pH 7.0. Cells were harvested and washed three times with distilled water and then freeze-dried. Phenotypic characteristics Morphological and cultural characteristics were tested on ISP 2, oatmeal agar (ISP 3), inorganic salts-starch agar (ISP 4), glycerol-asparagine agar (ISP 5) (Shirling and Gottlieb 1966), potato-dextrose agar (PDA), Czapek’s and nutrient agar (Waksman 1967). The colours of the colonies were determined by using the ISCC–NBS colour charts (Kelly 1964). The morphological characteristics of strain YIM 78087T were determined by light microscopy (Philips XL30) and scanning electron microscopy (ESEM-TMP) after the cultures were grown on T5 medium at 45 °C for 7 days. The spore-chain morphology, spore size and surface ornamentation of isolate were observed. Growth was tested at temperatures of 4, 15, 28, 30, 37, 42, 45, 50, 55 and 60 °C on T5 medium plates.
Antonie van Leeuwenhoek
The pH range for growth (4.0–10.0, at intervals of 1.0 pH units) and NaCl tolerance (0, 1, 3, 5, 7, 8, 9, 10, 15, 20 and 25 % w/v) were tested at 45 °C for 7 days by culturing the strains in T5 broth. Milk peptonization and coagulation, gelatin liquefaction, nitrate reduction, H2S production, degradation of Tweens, starch and cellulose, activities of urease, oxidase and catalase were tested according to the procedures described by Williams et al. (1989) and Gordon et al. (1974). The single carbon source utilization was tested with Pridham and Gottlieb’s basal mineral salts medium and the acid production were performed with carbohydrates. Nitrogen source utilization was observed in a basal liquid medium (Nie et al. 2012). API ZYM strips were used to determine some enzyme activities according to the instructions of the manufacturer (bioMe´rieux). Other physiological and biochemical characteristics were assessed by using the media and methods described by Gordon et al. (1974). Chemotaxonomy Chemotaxonomic characteristics were determined following standard procedures. The isomer of diaminopimelic acid of the cell wall and sugars analyses of whole-cell hydrolysates were performed as described by Hasegawa et al. (1983), Staneck and Roberts (1974) and Tang et al. (2009). Polar lipids were extracted and separated by two-dimensional thin-layer chromatography (TLC) and identified using the described procedures (Minnikin et al. 1979; Collins and Jones 1980). Menaquinones were extracted from lyophilized cells as described by Collins et al. (1977) and Minnikin et al. (1984) and then purified and analyzed by HPLC (Kroppenstedt 1982; Hu et al. 2001). For analysis of cellular fatty acids, strain YIM 78087T was cultured at 45 °C, and the reference strains S. fimbriatus DSM 40942T, S. marinus DSM 41968T and S. qinglanensis DSM 42035T were cultured at 28 °C with TSB medium for 4 days. The cellular fatty acids were extracted, methylated and analyzed by using the protocol of the Sherlock Microbial Identification System (MIDI) (Sherlock Version 6. 1; MIDI database: TSBA6) (Sasser 1990). The G?C content of the genomic DNA was determined by HPLC (Mesbah et al. 1989) with Escherichia coli JM-109 as the reference strain.
Molecular analysis Genomic DNA extraction, PCR amplification of the 16S rRNA gene sequence was performed as described by Li et al. (2007). The amplicon was purified using a Sangon PCR purification kit (China). The full-length 16S rRNA gene sequence of strain YIM 78087T was compared against other species via BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al. 1990) and type strains via Eztaxon database (http://eztaxon-e.ezbiocloud.net/) (Kim et al. 2012b). Multiple alignments were performed using the CLUSTAL_X software package (Thompson et al. 1997). The Kimura two-parameter model (Kimura 1980, 1983, 1985) was used to calculate evolutionary distance. Phylogenetic trees were constructed by the neighbour-joining (Saitou and Nei 1987), maximumlikelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) tree-making algorithms drawn from the MEGA version 5.0 package (Tamura et al. 2011). Bootstrap analysis with 1,000 resamplings was used to evaluate the topology of each tree (Felsenstein 1985). DNA–DNA relatedness was studied by applying the fluorometric micro-well method (Ezaki et al. 1989; Christensen et al. 2000; He et al. 2005) at the optimal hybridization temperature (47 °C). The experiments were set with eight replications between strain YIM 78087T and its closest phylogenetic neighbours S. fimbriatus DSM 40942T, S. marinus DSM 41968T and S. qinglanensis DSM 42035T.
Results and discussion Strain YIM 78087T was determined to exhibit typical morphological properties of the members of the genus Streptomyces. Observation of a 2 weeks old culture of strain YIM 78087T revealed that both aerial and vegetative hyphae were abundant and the spores are rhabditiform in shape with smooth surfaces (Supplementary Figure S1). YIM 78087T was observed to grow well on ISP 4, Czapek’s agar and nutrient agar, and to grow weakly on ISP 5, PDA, ISP 2 and ISP 3 media. Aerial mycelium was found to be produced on all tested media except PDA medium, and to have cream white or white colours on all media except PDA. The substrate mycelium was observed to have
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yellow-brown or olive-brown colours when grown on all media, and diffusible pigment was produced on ISP 4, ISP 5, Czapek’s agar and nutrient agar (Supplementary Table S1). Growth was observed at 28–50 °C (optimum 37–45 °C), pH 4.0–10.0 (optimum pH 8.0–9.0) and 0–9 % (w/v) NaCl (optimum 1–3 %). Activity of catalase was found to be positive, but urease and oxidase were negative. Milk coagulation and peptonization, hydrolysis of starch, cellulose and gelatin were found to be positive but melanin formation was negative. Tweens 20, 40 and 60 were found to be degraded but Tween 80 was not. Nitrate was found to be reduced but H2S was not produced. Differences in various characteristics demonstrated that strain YIM 78087T is phenotypically distinct from its closest phylogenetic relatives and are shown in Table 1. The detailed physiological characteristics of strain YIM 78087T are given in the species description. The diagnostic cell wall di-amino acid of the strain YIM 78087T was determined to be LL-diaminopimelic acid (LL-DAP). The whole organism hydrolysates were found to be rich in glucose, mannose and ribose, and galactose and rhamnose were also detected as minor components. The polar lipids of strain YIM 78087T were determined to consist of diphosphatidylglycerol, phosphatidylethanolamine, four phospholipids, two aminolipids and an unknown lipid (Supplementary Figure S2). The major respiratory menaquinones of strain YIM 78087T were identified as MK-9 (H4) (84 %) and MK-9 (H6) (16 %). The fatty acids profile ([5 %) was determined to be anteiso-C15:0 (28.4 %), anteiso-C17:0 (23.0 %), isoC16:0(15.1 %), iso-C15:0 (10.0 %), iso-C16:1 H(7.4 %) and iso-C17:0 (5.0 %). Detailed fatty acid profiles of strain YIM 78087T and the reference type strains of the most closely related species are given in Table 2. The DNA G?C content of strain YIM 78087T was determined to be 73.0 mol%, which is in accordance with the values for the genus Streptomyces (from 67 to 78 mol%). These characteristics are consistent with the members of the genus Streptomyces. To determine the phylogenetic position of strain YIM 78087T, the full-length 16S rRNA gene sequence (1588 nt; GenBank accession number KJ361473) of strain YIM 78087T was determined. Comparison of the sequence with the corresponding 16S rRNA gene sequences retrieved from GenBank/EMBL/DDBJ
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Table 1 Differential characteristics between strain YIM 78087T and its related members of the genus Streptomyces Characteristic
1
2
3
4
Utilization of D-Sucrose
?
?
?
W
L-Arabinose
-
?
?
?
D-Xylose
-
?
?
?
Sorbitol
W
?
?
W
D-Mannitol
W
?
?
?
D-Xylitol
?
?
w
?
Lactose
-
?
?
?
Inositol
-
W
?
?
D-Sorbitol
W
?
?
?
Dulcitol
?
?
?
W
Sodium pyruvate
?
?
?
w
Raffinose
?
?
?
-
Trehalose
?
?
W
?
Sodium soccinate L-Arginine
W ?
? ?
W W
? ?
Tween 80
-
?
?
?
CM-cellulose
?
?
-
?-
Starch
?
-
-
-
Esterase lipase (C8)
?
-
?
-
Lipase (C14)
?
-
-
-
Leucine arylamidase
?
-
?
?
Valine arylamidase
?
-
-
-
Cystine arylamidase
?
?
?
-
Acidic phosphatase
-
?
?
-
Naphthol-AS-BI-phosphohydrolase
?
?
-
?
b-Galactosidase
-
-
?
-
a-Glucosidase b-Glucosidase
?
? -
? ?
-
N-Acetyl-b-glucosaminidase
-
?
?
?
a-Mannosidase
-
-
?
28
Degradation of
Activity of
Growth Optimum temperature (°C)
45
28
28
Optimum pH
8.0
7.0
7.0
7.0
8 % (w/v) NaCl
W
-
-
?
Data obtained during this study; analyses were carried out under identical growth conditions except the temperature 1, YIM 78087T; 2, S. fimbriatus DSM 40942T; 3, S. marinus DSM 41968T; 4, S. qinglanensis DSM 42035T; ? (positive), utilized; - (negative), not utilized; w (weakly positive), utilized
Antonie van Leeuwenhoek Table 2 Fatty acid profiles (%) of the new isolate YIM 780087T and its closely related type strains in the genus Streptomyces Fatty acid
1
2
3
4
C16:0
2.0
4.7
3.9
3.1
Iso-C14:0
1.5
2.4
3.8
2.8
Iso-C15:0
10.0
5.2
21.6
8.8
Iso-C16:0
15.1
27.1
12.6
18.4
Iso-C17:0
5.1
3.1
4.8
6.8
Iso-C16:1 H Anteiso-C15:0
7.4 28.4
6.0 18.1
0.7 34.3
1.8 31.9
Anteiso-C17:0
23.0
15.3
Anteiso-C17:1 w9c
2.6
4.4
15.3
21.8
-
0.9
Summed feature 3a
2.7
5.0
1.0
0.7
Summed feature 9a
1.6
2.5
-
-
Data were obtained in this study under identical conditions except temperature Strains 1, YIM 78087T; 2, S. fimbriatus DSM 40942T; 3, S. marinus DSM 41968T; 4, S. qinglanensis DSM 42035T; -, negative or absent a
Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature three contains one or more of the fatty acids C16:1 x7c and C16:1 x6c. Summed feature nine contains one or more of the fatty acids iso-C17:1 x9c and C16:0 10-methyl
clearly demonstrated that strain YIM 78087T is a member of the genus Streptomyces. Strain YIM 78087T was found to be closely related to S. fimbriatus DSM 40942T (97.18 % similarity), S. marinus DSM 41968T (97.05 % similarity) and S. qinglanensis DSM 42035T (97.1 % similarity). From the neighbourjoining phylogenetic tree (Fig. 1), strain YIM 78087T formed a branch with S. marinus DSM 41968T (Khan et al. 2010) and S. qinglanensis DSM 42035T with 87 % bootstrap value, but was distantly related to S. fimbriatus DSM 40942T. This phylogenetic relationship was also supported in the trees generated with maximum-parsimony phylogenetic tree (Supplementary Figure S3) and maximum-likelihood phylogenetic tree (Supplementary Figure S4) with high bootstrap values (81 and 83 %, respectively). These data suggest that strain YIM 78087T is closely related to Streptomyces clade 125 (containing S. marinus) as delineated by Labeda et al. (2012). DNA–DNA relatedness values between strain YIM 78087T and the type strains S. fimbriatus DSM 40942T, S. marinus DSM 41968T and S. qinglanensis DSM 42035T were 24.2 ± 1.8, 27.7 ± 1.4 and
42.6 ± 1.5 %, respectively, which are notably lower than the threshold value (70 %) for the recognition of genomic species (Stackebrandt and Goebel 1994). Thus we suggest that strain YIM 78087T should be considered as a representative of a different genomic species of the genus Streptomyces. The combination of phylogenetic analysis, morphological, cultural, physiological and biochemical characteristics support the proposal that strain YIM 78087T is a member of the genus Streptomyces. Meanwhile, the differences in biochemical characteristics, 16S rRNA gene sequences similarities, DNA– DNA relatedness and fatty acid composition can be used to distinguish strain YIM 78087T from its closest relatives, S. fimbriatus DSM 40942T, S. marinus DSM 41968T and S. qinglanensis DSM 42035T. Therefore, based on these results, strain YIM 78087T represents a novel species of genus Streptomyces, for which the name S. calidiresistens sp. nov. is proposed. Description of Streptomyces calidiresistens sp. nov. Streptomyces calidiresistens (ca.li.di.ri.re.sis’tens; L. adj. calidus, hot; L. part adj. resistens, resisting; N. L. part. adj. calidiresistens, hot resisting). Cells are Gram-positive and aerobic, form extensively branched substrate and aerial mycelia. Substrate mycelia are typically yellow-brown or olive-brown colours and aerial mycelia are typically cream white or white colours. Diffusible pigments (brown-gray or yellow-brown colours) are produced on some media. Growth occurs at 28–50 °C, pH 4.0–10.0 and in the presence of 0–9 % (w/v) NaCl. Can utilize cellobiose, dulcitol, D-fructose, D-galactose, glycerol, D-glucose, maltose, D-mannitol, D-mannose, raffinose, sodium acetate, sodium malate, sodium pyruvate, sodium succinate, D-sorbitol, D-sucrose, trehalose, trisodium citrate and D-xylitol as sole carbon and energy sources, but not L-arabinose, lactose, inositol and D-xylose. Acid is produced from L-arabinose, cellobiose, dulcitol, D-fructose, D-glucose, inositol, lactose, maltose, Dmannitol, D-mannose, sodium malate, sodium pyruvate, D-sorbitol, D-sucrose, trehalose, trisodium citrate, D-xylitol and D-xylose. Utilises L-alanine, L-arginine, L-asparagine, cystine, glycine, L-glutamic acid, hypoxanthine, L-lysine, L-ornithine, L-phenylalanine, Lserine, L-threonine, L-tyrosine and L-valine as sole nitrogen sources. Milk coagulation and peptonization,
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Antonie van Leeuwenhoek T
S. ambofaciens ATCC 23877 (M27245) T S. marokkonensis Ap1 (AJ965470) T 76 S. atrovirens NRRL B-16357 (DQ026672) T S. albogriseolus NRRL B-1305 (AJ494865) T S. caelestis NRRL 2418 (X80824) T S. fimbriatus NBRC 15411 (AB184659) T S. fumanus NBRC 13042 (AB184273) T S. variegatus LMG 20315 (AJ781371) T S. mashuensis DSM 40221 (X79323) T S. tacrolimicus ATCC 55098 (FN429653) T S. koyangensis VK-A60 (AY079156) T S. chlorus BK125 (FR692094) T S. hebeiensis YIM 001 (AY277529) T 100* S. sannanensis NRRL B-24303 (AY999924) T S. albus subsp. pathocidicus NBRC 13812 (AB184501) T S. calidiresistens YIM 78087 (KJ361473) T S. qinglanensis 172205 (HQ660227) T S. marinus DSM 41968 (AB473556) T S. haliclonae DSM 41970 (AB473554) T S. xishensis YIM M 10378 (HQ585118) 68
55*
65* 75 50* 87
93*
87* 93* 87* 83*
0.005
Fig. 1 Neighbour-joining phylogenetic tree showing the phylogenetic relationship of strain YIM 78087T and other closely related Streptomyces species based on 16S rRNA gene sequences. Asterisks indicate branches that were also recovered
using the maximum-parsimony and maximum-likelihood methods. Bootstrap values (expressed as percentages of 1,000 replications) of above 50 % are shown at branch points. Bar 0.005 substitutions per nucleotide position
hydrolysis of starch, cellulose and gelatin are positive, but melanin formation is negative. Tweens 20, 40 and 60 are degraded, but Tween 80 is not. Nitrate is reduced but H2S is not produced. In API ZYM tests, activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, naphthol-AS-BI-phosphohydrolase, b-glucosidase are present. Activities of trypsin, a-chymotrypsin, acidic phosphatase, a-galactosidase, b-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-glucosidase, a-mannosidase and b-fucosidase are absent. The whole-cell hydrolysates contain glucose, mannose, ribose, rhamnose and galactose. MK-9 (H4) and MK-9 (H6) are the predominant menaquinones. The major fatty acids ([10 %) are anteiso-C15:0, anteisoC17:0, iso-C16:0, and iso-C15:0. The polar lipids profile contains diphosphatidylglycerol, phosphatidylethanolamine, four phospholipids, two aminolipids and an unknown lipid. The DNA G?C content of the type strain is 73.0 mol%. The type strain YIM 78087T (=BCRC 16955T=DSM 42108T=JCM 19629T) was isolated from a sediment sample collected from Hehua hot spring in Tengchong, Yunnan province, south-west
China. The 16S rRNA gene sequence of strain YIM 78087T has been deposited in GenBank under the accession number KJ361473.
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Acknowledgments The authors are grateful to Prof. Dr. HansPeter Klenk (DSMZ, Germany) for his kind providing of reference type strains. This research was supported by Key Project of International Cooperation of Ministry of Science & Technology (MOST) (No. 2013DFA31980), National Science Foundation Grant (OISE-0968421), Natural Science Foundation of China (Nos. 31070007 and 31372545), Key Project of Yunnan Provincial Natural Science Foundation (2013FA004), Scientific Research Fund of Xinxiang Medical University (2013QN126) and Research Project of Education Department of Henan Province of China (2011A180025).
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