hyphae grew well on various media after 4 weeks. ... Media and procedures used for determination .... evaluated by bootstrap resampling (Felsenstein, 1985).
International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1695–1699
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The Key Laboratory for Microbial Resources of Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan 650091, China College of Biological Sciences, Hebei University, Baoding 071002, China
DOI : 10.1099/ijs.0.02077-0
Streptomyces beijiangensis sp. nov., a psychrotolerant actinomycete isolated from soil in China Wen-Jun Li,1 Li-Ping Zhang,2 Ping Xu,1 Xiao-Long Cui,1 Zhi-Tang Lu,2 Li-Hua Xu1 and Cheng-Lin Jiang1 Author for correspondence : Cheng-Lin Jiang. Tel : j86 871 5034139. Fax : j86 871 5173878. e-mail : lihxu!ynu.edu.cn
A psychrotolerant actinomycete, strain YIM6T, was isolated from a soil sample collected from Beijiang in Xinjiang Province, China. Both vegetative and aerial hyphae grew well on various media after 4 weeks. Long spore chains, borne on the aerial mycelium, were straight to flexuous or occasionally Retinaculiaperti and non-motile. The novel isolate grew well at between 8 and 20 mC ; the lower and upper temperature limits for growth were 0 and 32 mC. The cell wall of strain YIM6T contained LL-diaminopimelic acid (A2pm) and traces of meso-A2pm. Whole-cell hydrolysates contained mainly glucose and small amounts of xylose, galactose and arabinose. The predominant menaquinones were MK9(H6) and MK-9(H8) and phosphatidylethanolamine was the diagnostic phospholipid. The predominant cellular fatty acids were 15 :0 anteiso, 16 :0 iso and 17 :0 cyclo. Phylogenetic analysis indicated that strain YIM6T belongs to the genus Streptomyces. In its morphology, physiological and biochemical characteristics and 16S rDNA sequence, strain YIM6T differed from other members of Streptomyces. Thus, strain YIM6T (l CCTCC 99005T l AS 4.1718T l DSM 41794T) is proposed as the type strain of a novel species, Streptomyces beijiangensis sp. nov.
Keywords : Streptomyces beijiangensis sp. nov.
The genus Streptomyces was proposed by Waksman & Henrici (1943) for aerobic, spore-forming actinomycetes. Streptomycetes are a rich source of bioactive compounds, notably antibiotics, enzymes, enzyme inhibitors and pharmacologically active agents (Be! rdy, 1995 ; Chun et al., 1997 ; Labeda et al., 1997). Compared with mesophilic or thermophilic taxa, psychrophilic and psychrotolerant streptomycetes have been relatively poorly studied. During a taxonomic study of extremophilic actinomycetes, several psychrophilic and psychrotolerant actinomycetes were isolated from soil samples from Beijiang in Xinjiang, the mostwesterly province of China.
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Published online ahead of print on 7 June 2002 as DOI 10.1099/ ijs.0.02077-0. Abbreviations : A2pm, diaminopimelic acid ; ISP, International Streptomyces Project. The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of strain YIM6T is AF385681. 02077 # 2002 IUMS Printed in Great Britain
Strain YIM6T was isolated on glycerol\asparagine agar [International Streptomyces Project (ISP) medium 5 (ISP 5) ; Shirling & Gottlieb, 1966] and incubated at 8 mC for about 4 weeks. The strain was maintained on ISP 2 and ISP 5 agar slants at 4 mC and as glycerol suspensions (20 %, v\v) at k20 mC. Biomass for chemical and molecular systematic studies was obtained by growing in shake flasks (about 150 r.p.m.) of ISP 2 broth supplemented with the vitamin mixture of HV medium (Hayakawa & Nonomura, 1987) at 15 mC for 3 weeks. Cultural characteristics were determined after 4 weeks at 8 mC by methods used in the ISP (Shirling & Gottlieb, 1966). Morphological observations of spores and mycelia were made by light microscopy (Olympus microscope BH2) and scanning electron microscopy (model EPMA8705). Media and procedures used for determination of physiological features and carbon-source utilization were those described by Shirling & Gottlieb (1966) and Locci (1989). Colour determination was done with colour chips from the ISCC-NBS Color Charts Standard Samples no. 2106 (Kelly, 1964). 1695
W.-J. Li and others Table 1. Cultural characteristics of strain YIM6T on various media .................................................................................................................................................................................................................................................................................................................
ISP media (Shirling & Gottlieb, 1966) are described in the text. Diffusible pigment was not observed on any of the media listed. Colours are taken from Kelly (1964). Characteristic
ISP 2
ISP 3
ISP 4
ISP 5
Potato agar
Nutrient agar
Czapek’s agar
Growth Sporulation Colour of : Aerial mycelium Substrate mycelium
Good Good
Good Moderate
Good Good
Good Good
Good Moderate
Poor Poor
None None
Pale white Moderate yellow
White Deep yellow
White Yellow-white
White Moderate yellow
Grey yellow Light orange-yellow
Green yellow Light yellow
None None
Strain YIM6T developed well on most media including inorganic salts\starch agar (ISP 4), oatmeal agar (ISP 3 medium), glycerol\asparagine agar (ISP 5) and yeast extract\malt extract agar (ISP 2) (Table 1). It showed poor growth on nutrient agar. No growth occurred on Czapek’s agar medium. No diffusible pigments were produced on any medium. Morphological features were observed on ISP 4, ISP 5 and ISP 2. Cultures were incubated for 4 weeks at 8 mC. Strain YIM6T had characteristics typical of the genus Streptomyces. Aerial mycelium and substrate mycelium were well developed (Fig. 1). At maturity, the aerial mycelium formed long spore chains that were straight to flexuous or occasionally Retinaculiaperti and non-motile.
(a)
(b)
The physiological features indicated in Table 2 and in the species description were determined on media commonly used for characterization of Streptomyces species (Shirling & Gottlieb, 1966). Cell walls were purified and amino acids of peptidoglycan were analysed by TLC (Lechevalier & Lechevalier, 1980 ; Jiang et al., 2001). Analysis of whole-cell sugar composition followed procedures described by Becker et al. (1964) and Lechevalier & Lechevalier (1980). Phospholipid analysis was carried out as described by Lechevalier et al. (1981). Menaquinones were determined using the procedures of Collins (1985). Cellular fatty acid composition was determined as described by Takeuchi & Hatano (1998). Cell walls of strain YIM6T contained -diaminopimelic acid (A pm) and trace amounts of meso-A pm. Whole-cell # # hydrolysates contained mainly glucose and small quantities of xylose, galactose and arabinose. The predominant menaquinones were MK-9(H ) and MK9(H ) and the diagnostic phospholipid was' phospha) tidylethanolamine. Cellular fatty acids are depicted in Table 3. Genomic DNA was extracted and 16S rDNA amplified as described by Cui et al. (2001). Reference strains were chosen from (Altschul et al., 1997) search results. Multiple alignments and calculations of levels of sequence similarity were carried out using 1696
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Fig. 1. Scanning electron micrographs of the spore chains of Streptomyces beijiangensis sp. nov. YIM6T grown on inorganic salts/starch agar (ISP 4) (a) and on yeast/malt extract agar (ISP 2) for 28 days at 8 mC. Bars, 5 (a) and 1 (b) µm.
1.74 (Thompson et al., 1994). A phylogenetic tree was reconstructed using the neighbour-joining method of Saitou & Nei (1987) from Knuc values (Kimura, 1980, 1983). The topology of the phylogenetic tree was evaluated by bootstrap resampling (Felsenstein, 1985) with 100 replicates. The almost complete 16S rDNA sequence (1499 nt) was compared to those of Streptomyces species deposited in public databases. None of the reference strains shared more than 97n5 % sequence International Journal of Systematic and Evolutionary Microbiology 52
Streptomyces beijiangensis sp. nov. Table 2. Phenotypic properties of strain YIM6T and its phylogenetic neighbours .................................................................................................................................................................................................................................................................................................................
Data for reference species were taken from Shirling & Gottlieb (1968a, b, 1969, 1972) and Williams et al. (1983). , Not determined ; d, doubtful result ; , weak. All taxa listed are positive for melanin production. Characteristic
Colony colour on ISP 2 Spore shape Spore chain morphology Production of diffusible pigment Milk coagulation Milk peptonization Starch hydrolysis Growth in cellulose H S production # Gelatin liquefaction Utilization of : Rhamnose Arabinose Fructose Sucrose Maltose Xylose Mannitol
S. beijiangensis YIM6 T
Streptomyces setonii
Streptomyces ornatus
Streptomyces virginiae
Streptomyces subrutilus
Streptomyces bikiniensis
Pale white Cylindrical Straight to flexuous to Retinaculiaperti k
Grey-yellow Oval or cylindrical Long and straight to flexuous k
Pale yellow-green Oval or rod Straight to flexuous j
Red or grey Rod or cylindrical Straight to flexuous to spiral j
Grey-yellow Cylindrical Long, straight or flexuous j
Grey Oval Straight to flexuous k
k k j k k k
k j j k j
j j k j j
k j j j j
k j j k j j
k j k j k
k k k k k j k
j j j k j j
k k j d j j
j k j k k
k j j j k k
d d j k j k
Table 3. Fatty acids of strain YIM6T
100 99
Fatty acid
Content (%)
13 : 0 anteiso 14 : 0 iso 14 : 0 15 : 0 iso 15 : 0 anteiso 15 : 0 16 : 1 iso 16 : 0 iso Summed feature 4* 16 : 0 iso 17 : 1ω9c 17 : 0 iso 17 : 0 anteiso 17 : 0 cyclo Summed feature 7*
0n36 2n87 1n10 6n78 30n47 1n08 1n59 18n25 2n82 6n59 3n04 3n17 4n63 12n66 1n84
* Summed features of unknown composition.
similarity with strain YIM6T. The phylogenetic tree obtained by the neighbour-joining algorithm from Knuc values is shown in Fig. 2. Strain YIM6T exhibited morphological characteristics typical of the genus Streptomyces and the phylogenetic data clearly link this strain to Streptomyces. Although few reports on Streptomyces species include detailed analysis of chemotaxonomic properties, the few quantitative chemotaxonomic data available for actinomycetes (Wellington et al., 1992) indicate that the properties determined for strain YIM6T do occur in Streptomyces species and hence lack discriminating power at the species level. http://ijs.sgmjournals.org
Streptomyces caviscabies ATCC 51928T (AF11260) Streptomyces setonii ATCC 25497T (D63872) ‘Streptomyces ornatus’ DSM 40307 (X79326) strain YIM 6T
100
Streptomyces virginiae IFO 12827T (D85123) Streptomyces subrutilus DSM 40445T (X80825) Streptomyces bikiniensis DSM 40581T (X79851)
87
Streptomyces galbus DSM 40480 (X79325) Streptomyces hygroscopicus ATCC 21431 (X79853) Streptomyces seoulensis IMSNU 21266T (Z71365) Streptomyces bottropensis ATCC 25435T (D63868) Streptomyces somaliensis DSM 40760 (AJ007399)
96
Streptomyces albulus DSM 40492T (AB024440) 2% .................................................................................................................................................
Fig. 2. Phylogenetic dendrogram obtained by distance-matrix analysis of 16S rDNA sequences, showing the position of strain YIM6T among its phylogenetic neighbours. Numbers on branch nodes are bootstrap percentages (100 resamplings). Sequence accession numbers are given in parentheses. The sequence of Arthrobacter globiformis was used a root. Bar, 2 % sequence divergence.
Comparison of the phenotypic characteristics of strain YIM6T and its closest phylogenetic neighbours (Fig. 2), however, reveals significant differences (Table 3), including the ability to grow well at low temperatures (8–20 mC) while the other reference type strains grow optimally at 28 mC. As most of the phenotypic properties determined for Streptomyces caviscabies (Goyer et al., 1996) are different from those determined for the other neighbouring strains, this organism has not been 1697
W.-J. Li and others
included in Table 3. Strain YIM6T differs from the type strain of S. caviscabies, ATCC 51928T, in the absence of diffusible pigment and in xylose (positive) and raffinose (negative) utilization in the former strain. We propose the name Streptomyces beijiangensis sp. nov. for this novel organism, with strain YIM6T as the type strain. Description of Streptomyces beijiangensis sp. nov.
Streptomyces beijiangensis (bei.jiang.enhsis. N.L. adj. beijiangensis pertaining to Beijiang, a place in Xinjiang province in western China). Aerial mycelium and substrate mycelium are well developed. Aerial mycelium at maturity forms long chains of spores that are straight to flexuous or occasionally Retinaculiaperti and are non-motile. Good growth on most media. Optimum growth temperature is between 8 and 20 mC. Diffusible pigment is not produced. The colour of colonies is mediumdependent (Table 1). In addition to the physiological reactions listed in Table 2, glucose, galactose and glycerol are utilized and lactose, mannose, inulin, acetate and oxalate are not utilized. Nitrate reductionand urease-positive. Diagnostic amino acid of peptidoglycan is meso-A pm. Whole-cell hydrolysates con# quantities of xylose, galactose tain glucose and small and arabinose. The predominant menaquinones are MK-9(H ) and MK-9(H ) and phosphatidylethanolamine is ' the diagnostic )phospholipid. Predominant cellular fatty acids are 15 : 0 anteiso, 16 : 0 iso and 17 : 0 cyclo. Isolated from soil in low-temperature habitats collected from Beijiang, western China. The type strain is strain YIM6T (l CCTCC 99005T l AS 4.1718T l DSM 41794T).
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Acknowledgements
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We are grateful to Professor Erko Stackebrandt for help in revising this manuscript. This research was supported by the Ministry of Science and Technology, PR China (project no. 2001CCC 00600), the Key Laboratory for Microbial Resources of Ministry of Education, PR China, and Yunnan Provincial Natural Science Foundation.
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