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Gram-Positive Bacteria

Classification of Streptomyces griseus (Krainsky 1914) Waksman and Henrici 1948 and related species and the transfer of ‘Microstreptospora cinerea’ to the genus Streptomyces as Streptomyces yanii sp. nov.

Zhiheng Liu, Yanlin Shi, Yamei Zhang, Zhihong Zhou, Zhitang Lu, Wei Li, Ying Huang, Carlos Rodríguez, Michael Goodfellow

  • 1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
  • 2School of Biology, King George VIth Building, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
  • Correspondence
    Michael Goodfellow
    m.goodfellow{at}ncl.ac.uk

    • International Journal of Systematic and Evolutionary Microbiology 2005; 55(4):1605–1610 · https://doi.org/10.1099/ijs.0.63654-0

    View at publisher PubMed

    Abstract

    A soil actinomycete, strain 80-133T, with the non-validly published name ‘Microstreptospora cinerea’, was the subject of a polyphasic study designed to clarify its taxonomic status. Comparative 16S rRNA gene sequence studies indicated that the organism belonged to the genus Streptomyces, a result in line with previous chemotaxonomic and morphological data. The strain belonged to the Streptomyces griseus clade, but could be distinguished from representatives of species assigned to this taxon by using DNA–DNA relatedness and phenotypic data. In light of these findings, it is proposed that the organism should be recognized as a novel species of the genus Streptomyces. The name proposed for this taxon is Streptomyces yanii sp. nov., with isolate 80-133T (=AS 4.1146T=JCM 3331T) as the type strain. It was also shown that representative strains of Streptomyces argenteolus, Streptomyces caviscabies, S. griseus and Streptomyces setonii belong to the same genomic species and have key phenotypic properties in common. It is proposed that S. caviscabies and S. setonii should be considered as later heterotypic synonyms of S. griseus and that S. argenteolus AS 4.1693T should also be assigned to this taxon.

    • Published online ahead of print on 1 March 2005 as DOI 10.1099/ijs.0.63654-0.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Streptomyces yanii AS 4.1146T is AB015854.

    It is clear from 16S rRNA gene sequence data that the type strains of Streptomyces caviscabies Goyer et al. 1996, Streptomyces griseus (Krainsky 1914) Waksman and Henrici 1948 and Streptomyces setonii (Millard and Burr 1926) Waksman 1953 form a distinct clade in the Streptomyces tree together with representative strains of Streptomyces sanglieri Manfio et al. 2003. Streptomyces argenteolus AS 4.1693T also belongs to the S. griseus clade. This organism is considered to be the type strain of S. argenteolus, a taxon that is incorrectly attributed to Tresner et al. (1961) in the Approved Lists of Bacterial Names (Skerman et al., 1980) and in the wider microbiological literature (Shirling & Gottlieb, 1968; Williams et al., 1989; Manfio et al., 2003). This organism was first described in the patent literature (Perlman et al., 1955; Fried et al., 1958). S. griseus and its near relatives show close phylogenetic relationships with several species of Streptomyces with validly published names, notably Streptomyces laceyi Manfio et al. 2003, Streptomyces peucetius Grein et al. 1963, Streptomyces purpureus (Matsumae and Hata 1968) Goodfellow et al. 1986b and Streptomyces venezuelae Ehrlich et al. 1948.

    The taxonomy of some members of the S. griseus clade has been the source of considerable confusion, as exemplified by the proposal that S. griseus NRRL-ISP 5236T and S. setonii NRRL-ISP 5395T should be recognized as heterotypic synonyms of Streptomyces anulatus (Williams et al., 1989) and by the assignment of S. argenteolus NRRL-ISP 5226T to the Streptomyces antibioticus and Streptomyces chromofuscus species-groups in the respective numerical phenetic surveys of Kämpfer et al. (1991) and Williams et al. (1983). It is now apparent that additional comparative studies are needed to resolve the finer taxonomic relationships between members of the S. griseus clade, especially since representative strains of S. argenteolus, S. caviscabies, S. griseus and S. setonii share key morphological features (Shirling & Gottlieb, 1968, 1969; Goyer et al., 1996) and have identical or nearly identical 16S rRNA gene sequences (Manfio et al., 2003). It is also evident from the present study that the strain previously described as ‘Microstreptospora cinerea’ belongs to the S. griseus 16S rRNA gene clade.

    The monospecific genus ‘Microstreptospora’ was proposed by Yan et al. (1983, 1987) for an actinomycete, strain 80-133T, which formed single or short chains of spores on the substrate mycelium, chains of spores on aerial hyphae and had whole-organism hydrolysates rich in ll-diaminopimelic acid. The organism was classified as ‘M. cinerea’, but was subsequently considered to be closely related to the genus Elytrosporangium (Itoh et al., 1987), a later synonym of the genus Streptomyces (Goodfellow et al., 1986a). T. Itoh and colleagues were of the view that strain 80-133T should be classified as belonging to the genus Streptomyces as it had chemical and morphological properties that were typical of members of this taxon. The binomial ‘M. cinerea’ is not cited in the Approved Lists of Bacterial Names (Skerman et al., 1980) and hence has no formal standing in nomenclature.

    The present study was designed to establish the taxonomic position of ‘M. cinerea’ strain 80-133T and to clarify the taxonomy of members of the S. griseus clade by using a combination of genotypic and phenotypic data. It is evident from the resultant data that the representatives of S. argenteolus, S. caviscabies and S. setonii should be classified as S. griseus, and that strain 80-133T forms a distinct centre of taxonomic variation in the genus Streptomyces, for which the name Streptomyces yanii sp. nov. is proposed.

    Strain 80-133T and the other strains included in the DNA–DNA relatedness studies, namely S. argenteolus AS 4.1693T, S. caviscabies DSM 41811T, S. griseus AS 4.1419T and AS 4.1321, S. laceyi AS 4.1832T, S. sanglieri AS 4.1831T, S. setonii AS 4.1774T, S. peucetius AS 4.1799T, S. purpureus AS 4.1225T and S. venezuelae AS 4.1307T, were maintained as suspensions of spores and mycelial fragments in glycerol (20 %, v/v) and on Gauze's agar (1 g KNO3, 0·5 g K2HPO4, 0·5 g MgSO4.7H2O, 0·5 g NaCl, 10 mg FeSO4, 20 g starch, 30 g agar, 1 litre distilled water) at room temperature. Biomass for the molecular systematic studies was prepared from 7-day-old cultures grown on Gauze's agar at 30 °C. Genomic DNA was extracted from the biomass and purified following an established procedure (Kim et al., 1998). The undisturbed arrangement of hyphae, notably aerial hyphae, and the spore chain morphology of strains 80-133T and AS 4.1693T were observed on Gauze's agar after 7 days, using the coverslip technique of Kawato & Shinobu (1959). All of the strains were examined by using a range of phenotypic tests according to standard procedures (Williams et al., 1983).

    Purified genomic DNA from strain 80-133T was sequenced according to a standard procedure (Kim et al., 1998). The resultant sequence was aligned manually with corresponding almost-complete sequences of representatives of available Streptomyces species that that been retrieved from GenBank/EMBL/DDBJ using the clustal_x program (version 1.64b; Thompson et al., 1997). The resultant dataset was examined by using the least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms from the phylip suite of programs (Felsenstein, 1993). Evolution distance matrices for the least-squares and neighbour-joining methods were generated according to Jukes & Cantor (1969). Tree topologies were evaluated by a bootstrap analysis based on 1000 resamplings of the neighbour-joining dataset by using the seqboot and consense programs from the phylip package (Felsenstein, 1993). A partial nucleotide sequence (120 bp) from the variable γ-region of the 16S rRNA gene of the tested strain was compared with corresponding nucleotide sequences of nearly 500 strains belonging to the genus Streptomyces retrieved from GenBank (). A phylogenetic tree based on these partial nucleotide sequences was constructed by using the neighbour-joining algorithm (Saitou & Nei, 1987). DNA–DNA relatedness studies were carried out (Table 1⇓) by using the thermal denaturation procedure described by De Ley et al. (1970) and a UV-1206 spectrophotometer (Shimadzu) equipped with a TB-85 thermobath.

    Table 1.

    Percentage DNA–DNA relatedness between representatives of species assigned to the S. griseus clade

    Comparison of the almost-complete sequence of strain 80-133T (1463 nt) with corresponding sequences of the reference Streptomyces strains showed that the strain formed a phyletic line within the S. griseus subclade (Fig. 1⇓). The tested organism was also distinguished from representatives of over 500 members of the genus Streptomyces in a comparative study based on the 120 bp γ-region of the 16S rRNA gene (data not shown). It was evident from these studies that the tested strain is a bone fide member of the genus Streptomyces, a conclusion that was supported by chemotaxonomic data drawn from previous studies (Yan et al., 1983, 1987; Itoh et al., 1987). The morphological data acquired in the present investigation are in line with those from these earlier studies in showing that strain 80-133T forms single or short chains of spores on substrate hyphae and aerial hyphae that differentiate into short chains of spores.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining tree showing the position of strain 80-133T in relation to phylogenetically close organisms in the 16S rRNA Streptomyces gene tree. Asterisks indicate branches that were also recovered using the least-squares, maximum-likelihood and maximum-parsimony tree-making algorithms. f and p indicate branches that were also recovered using the least-squares and maximum-parsimony methods, respectively. Numbers at nodes are percentage bootstrap values based on a neighbour-joining analysis of 1000 resampled datasets; only values above 50 % are given. Bar, 0·1 substitutions per site.

    It is evident from Fig. 1⇑ that strain 80-133T lies at the periphery of the S. griseus clade together with the type strain of S. sanglieri; the relationship between these strains was underpinned by the results from all of the tree-making algorithms and by a bootstrap value of 76 % in the neighbour-joining analysis. The taxonomic status of the S. griseus clade is supported by the results from two of the tree-making algorithms and by a bootstrap value of 88 % in the neighbour-joining analysis. The type strains of S. peucetius, S. purpureus and S. venezuelae formed a loose phylogenetic group that was closely related to the S. griseus clade; members of these taxa formed a distinct group that was supported by a bootstrap value of 94 % in the neighbour-joining analysis.

    Strain 80-133T shared relatively high 16S rRNA gene similarity values with other members classified in the S. griseus clade, that is, with S. sanglieri AS 4.1831T (99·4 %), S. argenteolus AS 4.1693T (99·2 %), S. griseus AS 4.1419T (99·2 %), S. caviscabies DSM 41811T (99·1 %) and S. setonii AS 4.1774T (99·1 %), values that correspond to 8, 12, 12, 13 and 13 nucleotide differences, respectively. 16S rRNA gene similarities within this range are consistent with the assignment of closely related streptomycetes to distinct species (Sembiring et al., 2000; Manfio et al., 2003). The 16S rRNA gene data are in line with results from previous studies which showed that strain 80-133T had chemical and morphological properties that were typical of members of the genus Streptomyces (Itoh et al., 1987; Yan et al., 1987).

    DNA–DNA relatedness studies are used routinely to resolve finer taxonomic relationships between closely related actinomycetes (Kim et al., 2003; Groth et al., 2004). The minimum level of DNA relatedness between strains recommended to define genomic species is 70 % (Wayne et al., 1987), although comprehensive studies on specific groups of streptomycetes indicate that relatedness values above 80 % correspond to species level relatedness within this taxon (Labeda, 1993, 1998; Labeda & Lyons, 1992). It is evident from Table 1⇑ that strain 80-133T shared relatedness values that were well below these cut-off points with strains of S. argenteolus, S. caviscabies, S. griseus and S. setonii; it was also sharply separated from its nearest phylogenetic neighbour, the type strain of S. sanglieri. Strain 80-133T showed uniformly low DNA–DNA relatedness values with the type strains of S. laceyi (14 %), S. peucetius (14 %), S. purpureus (11 %) and S. venezuelae (16 %). The DNA–DNA pairing data also provide further evidence that strain AS 4.1321, previously designated ‘Streptomyces ornatus’, belongs to the same genomic species as the type strain of S. griseus (Okanishi et al., 1972).

    It is apparent from Table 2⇓ that strain 80-133T can be distinguished from representatives of species classified in the S. griseus clade by using a combination of phenotypic properties, notably morphological features. Since ‘M. cinerea’ 80-133T can be distinguished from all of these organisms by using a combination of genotypic and phenotypic data, it is proposed that it should be classified as a novel species of the genus Streptomyces. The name proposed for this new taxon is Streptomyces yanii sp. nov.

    Table 2.

    Phenotypic properties that differentiate strain 80-133T from representatives of Streptomyces species classified in the S. griseus clade

    Taxa: 1, strain 80-133T; 2, S. argenteolus AS 4.1693T; 3, S. caviscabies DSM 41811T; 4, S. griseus AS 4.1419T; 5, S. sanglieri AS 4.1831T; 6, S. setonii AS 4.1774T. Spore chain arrangement data for the reference strains were taken from: Shirling & Gottlieb (1968), AS 4.1419T; Goyer et al. (1996), DSM 41811T; Shirling & Gottlieb (1969), AS 4.1774T; Manfio et al. (2003), AS 4.1831T. +, Positive/present; −, negative/absent. All of the strains were positive for degradation of adenine, hypoxanthine and xanthine; hydrolysis of aesculin and arbutin; utilization of (+)-d-fructose, (+)-d-galactose, (+)-d-glucose, (+)-d-lactose, (+)-d-mannitol, (+)-d-mannose, methyl α-d-glucoside, (+)-d-raffinose, (+)-d-ribose and (+)-d-xylose (1 %, w/v) and acetate, formate, malonate and propionate (0·1 %, w/v); and growth in the presence of 7 % NaCl. All of the strains were negative for growth at 45 °C, and production of melanin pigments on either peptone yeast extract iron agar or tyrosine agar.

    The results of the present study provide further evidence that the genus Streptomyces as currently constituted is overspeciated (Hatano et al., 2003; Lanoot et al., 2002, 2004, 2005). It is apparent from Table 1⇑ that the type strains of S. argenteolus, S. caviscabies Goyer et al. 1996, S. griseus (Krainsky 1914) Waksman and Henrici 1948 and S. setonii (Millard and Burr 1926) Waksman 1953 share DNA–DNA relatedness values between 84 and 100 %, results which provide further evidence for the recognition of the 80 % cut-off for species level relatedness within the genus Streptomyces (Labeda, 1993, 1998; Labeda & Lyons, 1992). Members of these taxa also have key phenotypic properties in common, notably spore chain morphology (Table 2⇑). It is therefore proposed that S. argenteolus, S. caviscabies and S. setonii are heterotypic synonyms of S. griseus, the earliest described of these species with validly published names. An emended description of S. griseus is given below.

    Description of Streptomyces yanii sp. nov.

    Streptomyces yanii (ya′ni.i. N.L. gen. n. yanii of Yan, named in honour of Dr Xunchu Yan, a Chinese microbiologist).

    The description is based on data taken from this and earlier studies (Yan et al., 1983, 1987; Itoh et al., 1987). Aerobic, Gram-positive, non-acid–alcohol-fast actinomycete that forms an extensively branched substrate mycelium and aerial hyphae. The substrate mycelium carries abundant spherical, smooth-surfaced spores (about 2·5 μm in diameter) singly or in short chains of 2 to 4 spores on short sporophores. Short chains of ellipsoidal smooth-surfaced spores (0·8×2·0 μm in diameter) are formed on aerial hyphae. The latter are usually formed only on inorganic media; an abundant grey aerial spore mass is apparent on oatmeal and Krass's No. 1 agars. Forms an abundant grey to black substrate mycelium on Czapek's, Krass's and oatmeal agars. Gelatin and starch are degraded, but not cellulose or tyrosine. Nitrate is reduced. H2S is not formed. Milk is coagulated but not peptonized. Additional phenotypic properties are shown in Table 2⇑. Good growth occurs at 18–35 °C and pH 6–9. Whole-organism hydrolysates contain major proportions of ll-diaminopimelic acid, glycine, galactose and mannose, and a trace of ribose. The muramic acid of the peptidoglycan is N-acetylated. The predominant isoprenologues are tetra- and hexa-hydrogenated menaquinones with nine isoprene units, and the major phospholipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The G+C content of the DNA is 69·4 mol%. The sole strain was isolated from a mud sample collected from a sewage ditch in Zhanjiang City, Guangdong Province, China.

    The type strain is 80-133T (=AS 4.1146T=JCM 3331T).

    Emended description of Streptomyces griseus (Krainsky 1914) Waksman and Henrici 1948, 948AL

    Streptomyces griseus (gri′se.us. M.L. masc. adj. griseus grey).

    The description is based on data taken from this and earlier studies (Shirling & Gottlieb, 1968, 1969). Spore chains are rectiflexibiles; the spore surface is smooth. Aerial spore mass colour places the organism in the grey colour series; the reverse side of colonies is grey–yellow. Melanin pigment is not formed nor are soluble pigments produced. Additional phenotypic properties are shown in Table 2⇑. The G+C content of the DNA is 71–72 mol%. The type strain is ATCC 23921T (=AS 4.1419T=DSM 40236T=NRRL-ISP 5236T=JCM 4047T=NCIMB 13023T). Heterotypic synonyms: Streptomyces argenteolus, Streptomyces caviscabies Goyer et al. 1996 and Streptomyces setonii (Millard and Burr 1926) Waksman 1953.

    Acknowledgments

    This work was supported through the Chinese Academy of Sciences–Royal Society Exchange Scheme (grant number Q 814) and by the National Natural Science Foundation of China (grant number 30370002). C. R. gratefully acknowledges receipt of a scholarship from the Ecuadorian FUNDACYT (Foundation for Science and Technology) and an Overseas Development Award. The authors are indebted to Dr M. Takeuchi (NBRC, Japan) for providing some of the type strains.

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