streptomyces wuyuanensis sp. nov., an actinomycete from soil
TRANSCRIPT
Streptomyces wuyuanensis sp. nov., an actinomy-cete from soil
Xuefang Zhang,1 Jianli Zhang,1 Jimei Zheng,1 Di Xin,1 Yuhua Xin2
and Huancheng Pang3
Correspondence
Jianli Zhang
1School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China
2China General Microbiological Culture Collection Center, Institute of Microbiology,Chinese Academy of Sciences, Beijing 100101, PR China
3Institute of Agri-resources and Regional Planning, CAAS, Beijing 100081, PR China
A novel actinomycete, strain FX61T, was isolated from a saline sample collected from the Inner
Mongolian Autonomous Region in China and subjected to a taxonomic study using a polyphasic
approach. The predominant menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The major fatty
acids were iso-C16 : 0, anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 1 H, C16 :0, iso-C14 : 0 and anteiso-C17 :0. The
phospholipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethano-
lamine, phosphatidylinositol, two phosphatidylinositol mannosides and an unidentified phospholipid.
The G+C content of the genomic DNA was 72 mol%. The 16S rRNA gene sequence of the isolate
had greater than 98 % similarity with those of Streptomyces griseoincarnatus ATCC 23623T (98.2 %),
Streptomyces labedae DSM 41446T (98.2 %), Streptomyces variabilis ATCC 19815T (98.2 %),
Streptomyces erythrogriseus ATCC 27427T (98.2 %), Streptomyces matensis ATCC 23935T
(98.2 %), Streptomyces althioticus ATCC 19724T (98.2 %) and Streptomyces luteosporeus ATCC
33049T (98.0 %), showing that the novel strain should be assigned to the genus Streptomyces. DNA–
DNA hybridizations with the seven above-mentioned members of the genus Streptomyces showed
29.8, 28.5, 27.0, 25.5, 25.0, 23.5 and 22.0 % relatedness, respectively. On the basis of the phenotypic
characteristics and genotypic distinctiveness, strain FX61T should be classified as a novel species of
the genus Streptomyces, for which the name Streptomyces wuyuanensis sp. nov. is proposed. The
type strain is FX61T (5CGMCC 4.7042T5KCTC 29112T).
The genus Streptomyces was initially introduced byWaksman & Henrici (1943) to encompass aerobic, spore-forming actinomycetes. At the time of writing, the genusStreptomyces encompasses nearly 600 species and subspe-cies with validly published names. Members of the genusStreptomyces are Gram-positive and have relatively highDNA G+C contents; produce extensively branchedsubstrate and aerial mycelia; LL-diaminopimelic acid (LL-A2pm) of the cell wall is found and characteristic sugarsare absent in the cell wall (Locci, 1989; Anderson &Wellington, 2001). Many species of the genus Streptomycesare known to produce biologically active secondarymetabolites, including antibiotics, enzymes, enzyme inhi-bitors, anti-tumour agents and anti-fungal compounds(Chun et al., 1997; Kim & Hwang, 2003). In this study,strain FX61T, a Streptomyces-like soil isolate, was char-acterized using a polyphasic approach.
Strain FX61T was isolated from a saline soil sample fromthe Inner Mongolian Autonomous Region in China, after7 days of incubation at 28 uC on glucose-yeast extract-malt extract (ISP 2; Shirling & Gottlieb, 1966) agar.Morphological, physiological and biochemical character-izations of strain FX61T were carried out following thestandard protocol of the International Streptomyces Project(ISP; Shirling & Gottlieb, 1966, 1968a, b, 1969). Colourswere determined by using colour chips from the ISCC-NBScolour charts (standard samples, no. 2106; Kelly, 1964).Plates were incubated for 4 weeks at 28 uC. Formation ofspores and mycelia was determined by light microscopy(BH-2; Olympus) and scanning electron microscopy (JSM-5600LV; JEOL) of strain FX61T grown on ISP 2 medium at28 uC for 4 weeks. Carbon source utilization was deter-mined according to the methods of Shirling & Gottlieb(1966). Growth at different temperatures (4–65 uC), NaClconcentrations (0, 1, 2, 3, 4, 5, 10, 15, 20 and 25 %, w/v)and pH (4.0–10.0 at intervals of 1.0 pH unit) wasdetermined on ISP 2 for 3–4 weeks (Zhao et al., 2009).Acid production from carbohydrates was assessed asdescribed by Gordon et al. (1974). Oxidase activity was
The GenBank/EMBL/DDBJ accession number for the 16S rRNA genesequence of strain FX61T is JX010725.
Two supplementary figures are available with the online version of thispaper.
International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2945–2950 DOI 10.1099/ijs.0.047050-0
047050 G 2013 IUMS Printed in Great Britain 2945
determined from the oxidation of tetramethyl-p-phenyle-nediamine. Catalase activity was determined with 3 %H2O2 according to standard methods. Other phenotypiccharacteristics were tested using standard procedures(Goodfellow, 1971; Williams et al., 1983). Antimicrobialactivities of strain FX61T were tested using the double-layeragar method (Wan et al., 2006). Reference strains,purchased from the China General MicrobiologicalCulture Collection Center (CGMCC), were Escherichiacoli CGMCC 1.797, Staphylococcus aureus CGMCC 1.89,Bacillus subtilis CGMCC 1.1849 and Aspergillus nigerCGMCC 3.2915. Susceptibility to antibiotics was testedby placing antibiotic-impregnated discs on ISP 2 plates thatwere seeded with suspensions of the test strain. Theantibiotics (mg per disc unless otherwise stated) used were:chloramphenicol (30), gentamicin (10), kanamycin (30),nalidixic acid (30), novobiocin (5), penicillin G (20international units), rifampicin (5), streptomycin (50),tetracycline (30), tobramycin (10) and sulfamethoxazole/trimethoprim (23.75/1.25).
Biomass for chemotaxonomic studies was obtained afterincubation at 28 uC for 7 days in shake flasks of ISP 2 brothmedium. Fatty acid analyses were performed with cellsgrown on Tryptone Soy Broth Agar (TSBA) medium for1 day. Amino acids and peptides in cell-wall hydrolysateswere analysed by the methods described by Hasegawa et al.(1983). Whole-cell sugars were analysed by the methods ofLechevalier & Lechevalier (1970). Isoprenoid quinones wereextracted according to the method of Collins et al. (1987)and analysed using reversed-phase HPLC and a YMC ODS-A (25064.6 mm) column. Phospholipids were examinedand identified using the method of Minnikin et al. (1979).The fatty acids were extracted and fatty acid methyl esterswere prepared according to the standard protocol of theMIDI/Hewlett Packard Microbial Identification System(Sasser, 1990; Kampfer & Kroppenstedt, 1996); the databaseused was TSBA40. The DNA G+C content was determinedby thermal denaturation method (Marmur & Doty, 1962).
Cell biomass for DNA extraction was obtained fromcultivation in ISP 2 at 28 uC. Extraction of genomic DNA,PCR amplification and sequencing of the 16S rRNA genewere performed as described by Li et al. (2007). TheEzTaxon-e server (http://eztaxon-e.ezbiocloud.net/) wasused for determining the closest known relatives of thestrain FX61T and calculating their sequence similarities.The resultant 16S rRNA gene sequence was compared withthose available from the GenBank database by using theBLAST program provided by the NCBI (Altschul et al.,1997). Multiple alignments with closely related species wereperformed using CLUSTAL X 1.8 software (Thompson et al.,1997). Evolutionary distances were calculated by usingdistance options according to Kimura’s two-parametermodel (Kimura, 1980). Phylogenetic trees were recon-structed with the neighbour-joining (NJ; Saitou & Nei,1987) and maximum-parsimony (MP; Kluge & Farris,1969) methods using the MEGA5 software program(Tamura et al., 2011). DNA–DNA hybridization of strain
FX61T and its closest neighbours (Streptomyces griseoin-carnatus ATCC 23623T, Streptomyces labedae DSM 41446T,Streptomyces variabilis ATCC 19815T, Streptomyces erythro-griseus ATCC 27427T, Streptomyces matensis ATCC 23935T,Streptomyces althioticus ATCC 19724T and Strepto-myces luteosporeus ATCC 33049T) were carried out usinga thermal denaturation procedure (De Ley et al., 1970;Huß et al., 1983) with a UV-1206 spectrophotometer(Shimadzu) fitted with a TB-85 thermobath and standardsoftware (Jahnke, 1992). Hybridization was performed withfive replications for each sample. The highest and lowestvalues obtained in each sample were excluded and themeans of the remaining three values were quoted as DNA–DNA relatedness values.
Morphological observation of a 28-day-old culture ofstrain FX61T grown on ISP 2 (yeast extract-malt extractagar) revealed that strain FX61T had the typical character-istics of members of the genus Streptomyces. Aerialmycelium and substrate mycelium were well-developedwithout fragmentation. Cultural characteristics on variousagar media are given in Table 1. Long spore chains werespiral and spores were smooth and non-motile (Fig. 1).Strain FX61T grew well between pH 4.0 and pH 12.0, withoptimum growth at pH 6.0–8.0. It grew at 15–40 uC, withoptimum growth at 28 uC. Strain FX61T grew in thepresence of 0–9 % (w/v) NaCl. Mycelia of strain FX61T
were well-developed on some media tested, including ISP2, ISP 3 (oatmeal agar), nutrient agar, Czapek’s agar,potato-dextrose agar (glycerol-asparagine agar) and ISP 4(inorganic salts-starch agar), but growth was poor on ISP5. The antimicrobial activities of strain FX61T wereinvestigated; it displayed excellent activity against B.subtilis CGMCC 1.1849, but showed no activities againstE. coli CGMCC 1.797, Staphylococcus aureus CGMCC 1.89and Aspergillus niger CGMCC 3.2915.
The cell wall of strain FX61T contained LL-diaminopimelicacid as the major diamino acid. Whole-cell hydrolysatescontained glucose, galactose, ribose and mannose.The menaquinones were MK-9(H6) (59.6 %), MK-9(H8)(27.0 %) and MK-9(H4) (8.6 %) and the phospho-lipids were diphosphatidylglycerol, phosphatidylglycerol,
Table 1. Cultural characteristics of strain FX61T on variousmedia after 4 weeks at 28 6C
Agar medium Colour of mycelium
Aerial mycelium Substrate mycelium
Czapek’s Grey White
Potato-dextrose Grey Yellow
Nutrient Absent Yellow
ISP 2 Grey Yellow
ISP 3 Grey Yellowish-white
ISP 4 Oyster white Yellow
ISP 5 Oyster white Yellow
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phosphatidylethanolamine, phosphatidylinositol, two phos-phatidylinositol mannosides and an unidentified phospho-lipid (Fig S1 available in IJSEM Online). The major fattyacids were iso-C16 : 0 (31.03 %), anteiso-C15 : 0 (10.55 %), iso-C15 : 0 (8.79 %), iso-C16 : 1 H (8.30 %), C16 : 0 (6.33 %), iso-C14 : 0 (5.81 %) and anteiso-C17 : 0 (5.20 %). The DNA G+Ccontent of strain FX61T was 72 mol%. All morphologicaland chemical features of strain FX61T were consistent withits assignment to the genus Streptomyces.
An almost complete 16S rRNA gene sequence (1403 nt) ofstrain FX61T was obtained and compared with theGenBank/EMBL/DDBJ databases using a BLAST search.The sequence of the novel strain showed one remarkabledifference to the other closely related type strains, betweenE. coli positions 1133–1139 (corresponding to positions1050–1057 in the sequence of the novel strain).Comparative 16S rRNA gene sequence analyses showedthat strain FX61T was affiliated phylogenetically with
Fig. 1. Scanning electron micrograph showing spiral spore chainsand smooth spores of strain FX61T after growth on ISP 2 mediumat 28 6C for 4 weeks. Bar, 10 mm.
S. variabilis ATCC 19815T (DQ442551) S. erythrogriseus ATCC 27427T (AJ781328)
S. griseoincarnatus ATCC 23623T (AJ781321)
S. griseorubens ATCC 19767T (AB184139)
S. griseoflavus ATCC 25456T (AJ781322)
S. althioticus ATCC 19724T (AY999808)
S. matensis ATCC 23935T (AB184221)
S. griseoloalbus ATCC 23624T (AB184275) S. paradoxus ATCC 15813T (AB184628)
S. malachitofuscus ATCC 25471T (AB184282)
S. tendae ATCC 19812T (D63873) S. thinghirensis DSM 41919T (FM202482)
S. marokkonensis DSM 41918T (AJ965470) S. eurythermus ATCC 14975T (D63870)
S. phaeoluteichromatogenes DSM 41898T (AJ391814) S. misionensis ATCC 14991T (EF178678)
S. indiaensis ATCC 33330T (AB184553)
S. massasporeus ATCC 19785T (AB184152)
S. griseoaurantiacus ATCC 19840T (AB184676)
Streptomyces wuyuanensis FX61T (JX010725) S. luteosporeus ATCC 33049T (DQ442525)
S. catenulae ATCC 12476T (AJ621613) S. griseocarneus ATCC 12628T (X99943)
S. lilacinus ATCC 23930T (AB184819)
S. mobaraensis ATCC 29032T (DQ442528)
S. werraensis ATCC 14424T (DQ442558)
Actinomadura madurae ATCC 19425T (X97889)60
5577
90
10099
7999
87
77
98 78
71
75
0.01
S. labedae DSM 41446T (AB184704)
Fig. 2. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships between strainFX61T and closely related species. Filled circles indicate that the corresponding branches were also recovered in the maximum-parsimony tree. Numbers at the branch nodes are bootstrap values, expressed as a percentage of 1000 replicates; only values.50 % are shown. Bar, 0.01 substitutions per nucleotide position.
Streptomyces wuyuanensis sp. nov.
http://ijs.sgmjournals.org 2947
members of the genus Streptomyces. Strain FX61T sharedsimilarity above 98 % to Streptomyces griseoincarnatusATCC 23623T (98.2 %), Streptomyces labedae DSM 41446T
(98.2 %), Streptomyces variabilis ATCC 19815T (98.2 %),Streptomyces erythrogriseus ATCC 27427T (98.2 %), Strepto-myces matensis ATCC 23935T (98.2 %), Streptomycesalthioticus ATCC 19724T (98.2 %) and Streptomycesluteosporeus ATCC 33049T (98.0 %). The region of the 16SrRNA gene used for the reconstruction of the phylogenetictrees corresponded to positions 84–1485 of the E. colisequence. It was apparent from the neighbour-joining tree(Fig. 2) that strain FX61T formed a tight cluster with speciesof the genus Streptomyces. The maximum- parsimony tree(Fig. S2) also supported the clustering of strain FX61T andrecognized species of the genus Streptomyces. Strain FX61T
exhibited DNA–DNA relatedness values of less than 30 %to Streptomyces griseoincarnatus ATCC 23623T (29.8 %),Streptomyces labedae DSM 41446T (28.5 %), S. variabilis ATCC19815T (27.0 %), S. erythrogriseus ATCC 27427T (25.5 %),Streptomyces matensis ATCC 23935T (25.0 %), Streptomycesalthioticus ATCC 19724T (23.5 %) and Streptomycesluteosporeus ATCC 33049T (22.0 %), which are well belowthe 70 % cut-off point for recognition of genomic species(Stackebrandt & Goebel, 1994), suggesting that strain FX61T
should be considered to represent a different genomic speciesof the genus Streptomyces.
In summary, phylogenetic results and chemotaxonomicanalyses indicated that strain FX61T belonged to the genus
Streptomyces. A phenotypic comparison between strainFX61T and closely related species of the genus Streptomyceswas carried out and differential characteristics of the abovestrains were identified (Table 2). Low DNA–DNA related-ness values were detected between the novel isolate andclosely related species of the genus Streptomyces. It isapparent that strain FX61T is not affiliated to anyrecognized species of the genus Streptomyces. Therefore,on the basis of the data presented above, strain FX61T
represents a novel species of the genus Streptomyces, forwhich the name Streptomyces wuyuanensis sp. nov. isproposed.
Description of Streptomyces wuyuanensissp. nov.
Streptomyces wuyuanensis (wu.yu.an.en9sis. N.L. masc. adj.wuyuanensis of or pertaining to Wuyuan, Inner MongolianAutonomous Region, China, the site from which the typestrain was isolated).
Gram-stain-positive, aerobic and non-motile. Long sporechains are spiral; spores are smooth and non-motile. Aerialmycelium is oyster white to grey, substrate mycelium iswhite to yellow. Soluble pigments are not produced.Additional cultural characteristics on various agar mediaare given in Table 1. The temperature range for growthis 15–40 uC (optimum, 28 uC). Growth occurs at pH 4.0–12.0 (optimum, pH 6.0–8.0) and in the presence of 0–9 % NaCl. Catalase is produced. Negative result for
Table 2. Differentiating physiological characteristics of strain FX61T and its closest phylogenetic neighbours
Taxa: 1, strain FX61T; 2, Streptomyces griseoincarnatus ATCC 23623T; 3, Streptomyces labedae DSM 41446T; 4, Streptomyces variabilis ATCC 19815T;
5, Streptomyces erythrogriseus ATCC 27427T, 6, Streptomyces matensis ATCC 23935T; 7, Streptomyces althioticus ATCC 19724T; 8, Streptomyces
luteosporeus ATCC 33049T. +, Well-utilized/present; M, moderately well-utilized/present. W, weakly utilized; 2, not utilized/absent; ND, not
determined. G, grey colour series; GR, grey–red; OD, olive grey–dark reddish-brown; RO, red–orange; RV, red–violet; WG, oyster white–grey; WP,
white–pinkish; WY, white–yellow; YB, yellow–dark brown; YY, yellow–yellow brown. All data are from this study.
Characteristic 1 2 3 4 5 6 7 8
Morphology and pigmentation
Aerial mass colour WG GR WP G GR G G GR
Substrate mycelium WY YY OD YB YB WG YB YY
Reverse side pigment 2 RO YB 2 YY RV GR 2
Melanin pigments 2 + ND 2 2 2 2 2
Soluble pigments 2 + + 2 + + + 2
Spore surface ornamentation Smooth Warty Warty Warty Warty Warty Warty ND
Carbon source utilization
L-Arabinose + + M + + + + 2
Sucrose + + M 2 2 M 2 2
D-Xylose + + 2 + + + + 2
myo-Inositol + M + + + + + +
Raffinose W W W 2 W 2 2 2
D-Galactose + W ND ND W + + 2
Liquefaction of gelatin + W + W W + + +
Growth at:
15 uC + 2 2 + ND 2 + 2
40 uC + 2 + 2 + + 2 +
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Voges–Proskauer and methyl red tests, oxidase reaction,production of H2S, nitrate reduction and milk coagulationand peptonization. Starch, gelatin, urea and Tween 20 arehydrolysed, but cellulose and Tweens 40 and 80 are not.Utilizes L-arabinose, fructose, galactose, D-glucose, manni-tol, myo-inositol, raffinose (weakly), a-L-rhamnose, sucroseand D-xylose as sole carbon sources, but not ribose.Adenine, L-alanine, L-arginine, L-asparagine, glycine,hypoxanthine, L-leucine L-lysine, L-phenylalanine, L-tryp-tophan, L-tyrosine and L-valine can be used as sole nitrogensources. Acid is produced from fructose, D-glucose, ribose,starch, trehalose and D-xylose, but not from L-arabinose ormannitol. The diagnostic amino acid in the peptidoglycanis LL-diaminopimelic acid, and glucose, galactose, riboseand mannose are present in whole-cell hydrolysates (cellwall type I). Sensitive to (mg per disc unless otherwisestated): chloramphenicol (30), gentamicin (10), kanamycin(30), novobiocin (5), rifampicin (5), streptomycin (50),tetracycline (30) and tobramycin (10), but resistant tonalidixic acid (30), penicillin G (20 international units) andsulfamethoxazole/trimethoprim (23.75/1.25). The phos-pholipid profile contains diphosphatidylglycerol, phospha-tidylglycerol, phosphatidylethanolamine, phosphatidylinositol,two phosphatidylinositol mannosides and an unidentifiedphospholipid. The quinone system is composed of the majorcompounds MK-9(H6) and MK-9(H8) and minor amounts ofMK-9(H4). The major cellular fatty acids are iso-C16 : 0, anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 1 H, C16 : 0, iso-C14 : 0 and anteiso-C17 : 0.
The type strain, FX61T (5CGMCC 4.7042T5KCTC29112T), was isolated from a soil sample of the InnerMongolian Autonomous Region, China. The DNA G+Ccontent of the type strain is 72 mol%.
Acknowledgements
This research was supported by the National Natural Science
Foundation of China (NSFC, grant numbers 30970009 and
31070002) and by the Special Fund for Public Welfare Industrial
(Agriculture) Research of China (grant number 200903001).
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