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Taxonomic re-evaluation of whorl-forming Streptomyces (formerly Streptoverticillium) species by using phenotypes, DNA–DNA hybridization and sequences of gyrB, and proposal of Streptomyces luteireticuli (ex Katoh and Arai 1957) corrig., sp. nov., nom. rev.

Kazunori Hatano^1,, Tadashi Nishii¹ and Hiroaki Kasai²

¹ Institute for Fermentation, Osaka, 17-85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
² Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan

Correspondence
Kazunori Hatano
hatano-kazunori{at}nite.go.jp

	ABSTRACT

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The taxonomic status of 64 strains of whorl-forming Streptomyces (formerly Streptoverticillium) species was re-evaluated and strains were reclassified on the basis of their phenotypes, DNA–DNA hybridization data and partial sequences of gyrB, the structural gene of the B subunit of DNA gyrase. These strains, which consisted of 46 species and eight subspecies with validly published names and 13 species whose names have not been validly published [including 10 strains examined by the International Streptomyces Project (ISP)], were divided into two groups, namely typical and atypical whorl-forming Streptomyces species, based on their phenotypes and gyrB gene sequences. The typical whorl-forming species (59 strains) were divided into six major clusters of three or more species, seven minor clusters of two species and five single-member clusters, based on the threshold value of 97 % gyrB sequence similarity. Major clusters were typified by Streptomyces abikoensis, Streptomyces cinnamoneus, Streptomyces distallicus, Streptomyces griseocarneus, Streptomyces hiroshimensis and Streptomyces netropsis. Phenotypically, members of each cluster resembled each other closely except for the S. distallicus cluster, which was divided phenotypically into the S. distallicus and Streptomyces stramineus subclusters, and the S. netropsis cluster, which was divided into the S. netropsis and Streptomyces eurocidicus subclusters. Strains in each minor cluster closely resembled each other phenotypically. DNA–DNA relatedness between the representative species and others in each major cluster and/or subcluster, and between strains in the minor clusters, was >70 %, indicating that the major clusters and/or subclusters and the minor clusters each comprise a single species. It was concluded that 59 strains of typical whorl-forming Streptomyces species consisted of the following 18 species, including subjective synonym(s): S. abikoensis, Streptomyces ardus, Streptomyces blastmyceticus, S. cinnamoneus, S. eurocidicus, S. griseocarneus, S. hiroshimensis, Streptomyces lilacinus, ‘Streptomyces luteoreticuli’, Streptomyces luteosporeus, Streptomyces mashuensis, Streptomyces mobaraensis, Streptomyces morookaense, S. netropsis, Streptomyces orinoci, S. stramineus, Streptomyces thioluteus and Streptomyces viridiflavus.

The GenBank/EMBL/DDBJ accession numbers for the gyrB gene sequences are shown in Fig. 1.

Present address: Department of Biotechnology, National Institute of Technology and Evaluation (NITE), 2-5-8, Kazusakamatari, Kisarazu, Chiba 292-0818, Japan.

	INTRODUCTION

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The genus Streptoverticillium was proposed by Baldacci (1958) for a distinctive group of whorl-forming Streptomyces species, in which 40 species have been classified into 12 groups based on aerial and substrate mycelium colour (Baldacci & Locci, 1974). Furthermore, Locci & Schofield (1989) classified 64 Streptoverticillium species in 10 major clusters and 14 single-member species by numerical taxonomy based on phenotypes. Witt & Stackebrandt (1990), however, unified the genera Streptoverticillium and Streptomyces, based on phylogenetic similarities of partial 16S rRNA gene sequences. At present, 46 species and eight subspecies with validly published names comprise the whorl-forming Streptomyces species.

Classification of whorl-forming Streptomyces species by using DNA–DNA hybridization has been examined by several researchers. Toyama et al. (1974) examined DNA–DNA relatedness of 34 Streptoverticillium strains and found that Streptoverticillium netropsis ISP 5259^T and Streptoverticillium flavopersicum ISP 5053^T, and Streptoverticillium ehimense ISP 5253^T, Streptoverticillium luteoverticillatum ISP 5038^T and ‘Streptomyces reticuli var. latumcidicus’ At-79 are closely related by DNA–DNA homology, indicating that they are synonyms. Recently, Labeda (1996) also examined DNA–DNA relatedness of 32 species and three subspecies of whorl-forming Streptomyces and revealed that Streptomyces biverticillatus ISP 5272^T, Streptomyces fervens NRRL 2755^T, Streptomyces roseoverticillatus NRRL B-1993^T and ‘S. rubrochlorinus’ NRRL B-12558 are subjective synonyms of Streptomyces baldaccii NRRL B-3500^T; Streptomyces kentuckensis NRRL B-1831^T and Streptomyces flavopersicus NRRL 2820^T are subjective synonyms of S. netropsis NRRL 2268^T; and Streptomyces distallicus NRRL 2886^T is a marginal subjective synonym of S. netropsis NRRL 2268^T.

Determination of exact taxonomic status of Streptomyces species by using traditional methods such as phenotypes and DNA–DNA relatedness is laborious and time-consuming; we have therefore searched for a simple and precise method for classification and identification of Streptomyces species. Kataoka et al. (1997) analysed 89 strains of the genus Streptomyces that belonged to eight major clusters of category I in Bergey's Manual of Systematic Bacteriology (Williams et al., 1989) by using phylogenetic analysis of a 120 bp 16S rDNA fragment that contains a highly variable region between positions 158 and 277 in the numbering system of Streptomyces ambofaciens (Pernodet et al., 1989); the authors concluded that these 120 bp nucleotide sequences are useful for rapid identification of Streptomyces species. However, more comparative data, including DNA–DNA hybridization and phenetic data, are needed to evaluate whether this method is a useful tool for discrimination at species level in Streptomyces. Furthermore, phylogenetic analysis of partial sequences of the gyrB gene, which encodes the B subunit of DNA gyrase, has been used for classification of several types of bacteria and has been shown to be a useful tool for discrimination at species level (Yamamoto & Harayama 1995, 1996, 1998; Yamamoto et al., 1999; Kasai et al., 2000). Therefore, we undertook phylogenetic analysis based on partial gyrB gene sequences for reclassification of the whorl-forming Streptomyces species, which are suspected to include many synonyms because of their similar morphology and narrow range of sugars utilized for growth.

This paper deals with reclassification of the whorl-forming Streptomyces species by using their phenotypes, DNA–DNA relatedness and phylogenetic analysis of gyrB gene sequences, and also assesses whether phylogenetic analysis of gyrB is a useful tool for classification and identification of whorl-forming Streptomyces species. This paper also deals with the proposal of Streptomyces luteireticuli corrig., sp. nov., nom. rev.

	METHODS

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Organisms used and culture conditions.
Sixty-four strains of whorl-forming Streptomyces species were used in this study. Strains were cultured in 5 ml YG medium (1 % glucose, 1 % yeast extract, pH 7·0) in a test tube at 28 °C for 2 days on a reciprocal shaker.

Characterization of phenotypes.
Cultured mycelia were harvested, washed three times with sterile distilled water and suspended in 5 ml sterile water. Washed mycelia were inoculated onto International Streptomyces Project (ISP) media: yeast extract/malt extract agar (ISP medium 2), oatmeal agar (ISP medium 3), inorganic salts/starch agar (ISP medium 4), glycerol/asparagine agar (ISP medium 5), yeast extract/iron agar (ISP medium 6), tyrosine agar (ISP medium 7) and Pridham–Gottlieb carbon utilization agar medium (ISP medium 9). After incubation at 28 °C for 14 days, morphological, cultural and physiological characteristics of strains were observed and described according to the method of the ISP (Shirling & Gottlieb, 1966).

Preparation of DNA.
Total DNA was extracted from 2-day cultured cells by the method of Saito & Miura (1963) with a minor modification: washed cells were suspended in 2 ml 10 mM Tris/HCl buffer (pH 8·0) that contained 1 mg achromopeptidase ml^-1 (Wako Pure Chemical Industries) and 5 mg lysozyme ml^-1 (egg white; Wako Pure Chemical Industries). The suspension was incubated for 2 h at 37 °C, then lysed completely by adding 0·2 ml 20 % (w/v) SDS and heating for 10 min at 65 °C. Total crude DNA was extracted with phenol and precipitated with cold ethanol, rinsed with 70 % ethanol and dried. A solution of dried DNA in 0·2 ml 1x TE (1 mM EDTA in 10 mM Tris/HCl buffer, pH 8·0) was treated with RNase A (Sigma) and proteinase K (Wako Pure Chemical Industries), extracted with phenol/chloroform/isoamyl alcohol (25 : 24 : 1, v/v) and precipitated with ice-cold ethanol. The precipitate was rinsed three times with 70 % ethanol, dried and dissolved in 0·5 ml distilled water. Purity and concentration of the prepared DNA solution were measured with a Beckman model DU-65 spectrophotometer. Solutions with an A₂₆₀/A₂₈₀ ratio above 1·9 were used for PCR and DNA–DNA hybridization.

DNA–DNA hybridization.
DNA–DNA hybridization was performed by the method of Ezaki et al. (1989) at 55 °C in 2x SSC (1x SSC: 0·15 M NaCl plus 0·015 M sodium citrate, pH 7·0) that contained 50 % formamide. The experiment was performed at least three times; DNA–DNA relatedness was expressed as a mean percentage of the homologous DNA binding value. Standard deviation (SD) was <5 %.

PCR amplification of gyrB.
The gyrB gene was amplified by PCR (Saiki et al., 1988) by using TaKaRa Taq LX and one of the following pairs of primers: UP1 (forward: 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3') and UP2r (reverse: 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3') (Yamamoto & Harayama, 1995); or PF-1 (forward: 5'-GAGGTCGTGCTGACCGTGCTGCACGCGGGCGGCAAGTTCGGC-3'), complementary to positions 355–396 in the numbering system of Streptomyces coelicolor A3(2) gyrB sequence (Calcutt, 1994) and PR-2 (reverse: 5'-GTTGATGTGCTGGCCGTCGACGTCGGCGTCCGCCAT-3'), complementary to positions 1624–1659, which were newly designed from conserved regions of the gyrB sequence of S. coelicolor A3(2) (Calcutt, 1994) and other Streptomyces species (H. Kasai, unpublished data). Amplification was performed in a total volume of 50 µl, which contained 10 pmol each primer, 0·1 µg target DNA, 5 µl 10x buffer and 2·5 U Taq polymerase, in a 0·5 ml microtube. DNA was amplified under the following conditions: 95 °C for 3 min for denaturation of target DNA; 30 cycles of denaturation at 95 °C for 0·5 min, primer annealing at 65 °C for 0·5 min and primer extension at 72 °C for 1 min; 72 °C for 4 min for completion of amplification; and cooling at 4 °C.

Sequencing of gyrB.
PCR products were run on a 1·5 % (w/v) agarose gel to remove primers; bands of amplified DNA (approx. 1·3 kb) were cut out with a scalpel and purified by using a QIAquick Gel Extraction kit (Qiagen) according to the manufacturer's instructions. Purified PCR products were subjected to cycle sequencing by using a BigDye Terminator Cycle Sequencing kit by Amplitaq FS (PE Applied Biosystems) and a Gene Amp PCR System 9700 (PerkinElmer) according to the manufacturer's protocol with the following seven primers: F-1 (5'-GAGGTCGTGCTGACCGTGCTGCA-3', positions 355–378), F-352 (5'-TACCACTACGAGGGCGGCATC-3', positions 779–799), F-701 (5'-AGCCGCAGTTCGAGGGCCAGAC-3', positions 1128–1149), R-1 (5'-GTTGATGTGCTGGCCGTCGACGT-3', positions 1637–1659), R-996 (5'-CTCGACGATGAAGATCTCGCAC-3', positions 1393–1414), R-728 (5'-GTCTTGGTCTGGCCCTCGAACTG-3', positions 1133–1155) and R-4 (5'-CGCTCCTTGTCCTCGGCCTC-3', positions 866–885). Conditions for thermal cycling were 25 cycles of denaturation at 96 °C for 10 s, primer annealing at 50 °C for 5 s and primer extension at 60 °C for 4 min. Products were purified as recommended by PE Applied Biosystems and were analysed with a model ABI PRISM 310 Genetic Analyser (PE Applied Biosystems) according to the manufacturer's protocol.

Phylogenetic analysis.
GyrB amino acid sequences, translated from gyrB gene sequences, were aligned by using CLUSTAL W software, version 1.7 (Thompson et al., 1994) and corrected manually. Sequences of the gyrB gene were aligned according to the alignments of GyrB sequences. Phylogenetic analyses were performed by using the neighbour-joining (NJ) (Saitou & Nei, 1987), maximum-parsimony (MP) (Swofford, 2000) and maximum-likelihood (ML) (Adachi & Hasegawa, 1992) methods. Evolutionary distances and similarity values based on Kimura's two-parameter model (Kimura, 1980) were calculated by using CLUSTAL W software, version 1.7. A phylogenetic tree was generated from the NJ method by using NJplot (Perrière & Gouy, 1996). Stability of the tree was assessed by bootstrap analysis with the resampling method of Felsenstein (1993) with 1000 replications, by using CLUSTAL W version 1.7. Nucleotide sequence data reported in this paper are available in DDBJ, EMBL and GenBank under the accession numbers given in Fig. 1.

View larger version (63K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree of whorl-forming Streptomyces species inferred by the NJ method (Saitou & Nei, 1987), using Kimura's evolutionary distance (Kimura, 1980) and based on a comparison of 1197 nt of gyrB gene sequences. The tree is depicted by NJplot (Perrière & Gouy, 1996). Bootstrap values >50 %, expressed as percentages of 1000 replications (Felsenstein, 1993), are given at nodes. Accession numbers of gyrB sequences are given in parentheses. Bar, 0·01 Knuc.

	RESULTS AND DISCUSSION

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Phylogenetic structure based on gyrB sequences and DNA–DNA relatedness
DNA fragments of about 1·3 kb that corresponded to approximately 64 % of the whole gyrB gene sequence of S. coelicolor A3(2) were amplified by PCR with the primers described in Methods. For phylogenetic analysis, sequences of 1197 nt [positions 397–1623, according to the numbering system of S. coelicolor A3(2) (Calcutt, 1994)] of the fragments were used. Sequence similarity values among the typical whorl-forming strains were 90·1–100 %. Phylogenetic tree topologies inferred from NJ, MP and ML were very similar to one another, except that ‘Streptomyces luteoreticuli’ NBRC 13422 and S. thioluteus NBRC 13341^T formed a cluster by MP and ML, but not by NJ. Fig. 1 shows the phylogenetic tree inferred from NJ. The 64 strains formed two independent clusters: typical whorl-forming Streptomyces species (59 strains) and atypical whorl-forming ones (five strains), which is the same as the classification of the strains by phenotype. Typical whorl-forming strains formed 18 clusters that consisted of six major clusters, seven minor clusters and five single-member clusters, based on the threshold value of 97 % gyrB sequence similarity. These clusters were roughly in accord with phenotypic groups (Table 1). The six major clusters were typified by S. abikoensis, S. cinnamoneus, S. distallicus, S. griseocarneus, S. hiroshimensis and S. netropsis.

The S. abikoensis cluster (>98·1 % gyrB sequence similarity) was divided into two subclusters: the S. abikoensis subcluster contained six strains with >99·0 % gyrB sequence similarity and the Streptomyces olivoverticillatus subcluster contained three strains with >98·0 % gyrB sequence similarity. DNA–DNA relatedness levels between S. abikoensis NBRC 13860^T and others in the cluster were >70 %, except for Streptomyces parvisporogenes NBRC 13907^T (65 %) (Table 2). In the S. abikoensis cluster, Streptomyces ehimensis NBRC 12858^T, ‘Streptomyces takataensis’ NBRC 13470 and ‘Streptoverticillium rubrireticuli’ NBRC 13082, which exhibited the same phenotype (including sugar utilization pattern; phenotype group IV, Table 1), also exhibited high gyrB sequence similarity (99–99·1 %) and high levels of DNA–DNA homology (99–101 %) (data not shown). In phenetic group V, levels of DNA–DNA relatedness among S. luteoverticillatus NBRC 12887^T, Streptomyces olivoreticuli subsp. olivoreticuli NBRC 12896^T, S. parvisporogenes NBRC 13907^T and ‘Streptomyces paucisporogenes’ NBRC 13070 were 65–83 % (data not shown), in accord with the results reported by Toyama et al. (1974).

The S. distallicus cluster (>97·5 % gyrB sequence similarity) was divided into two subclusters according to distinctive phenotypes: the S. distallicus subcluster, which consisted of S. distallicus NBRC 15815^T and Streptomyces syringium NBRC 15900^T, and the S. stramineus subcluster. Sequence similarity in gyrB and DNA–DNA relatedness between S. distallicus NBRC 15815^T and S. syringium NBRC 15900^T were 99·5 and 97 %, respectively, suggesting that they are synonymous. On the other hand, DNA–DNA relatedness between S. distallicus NBRC 15815^T and S. stramineus NBRC 16131^T was 42–48 %, indicating that these subclusters are independent despite their comparatively high gyrB sequence similarity (97·5 %).

The S. griseocarneus cluster (>97·7 % gyrB sequence similarity) was also divided into two subclusters: S. griseocarneus (two strains, 99·5 % similarity) and Streptomyces septatus (two strains, 99·5 % similarity). DNA–DNA relatedness levels between S. griseocarneus NBRC 12776^T and ‘S. tropicalensis’ NBRC 13428, and between S. septatus NBRC 13471^T and Streptomyces alboverticillatus NBRC 13861^T were 96 and 91–96 %, respectively. In addition, DNA–DNA relatedness between S. griseocarneus NBRC 12776^T and S. septatus NBRC 13471^T was 69–71 %, indicating that the S. griseocarneus cluster consists of a single species. On the other hand, DNA–DNA relatedness and gyrB sequence similarity between S. griseocarneus NBRC 12776^T and Streptomyces ardus NBRC 13430^T, and between S. alboverticillatus NBRC 13861^T and S. ardus NBRC 13430^T, which belong to the same group phenetically (Table 1), were <40 % and approximately 94 %, respectively (data not shown).

The S. hiroshimensis cluster (>97 % gyrB sequence similarity) was divided into four subclusters: S. baldaccii (four strains, 98–100 % gyrB similarity), S. hiroshimensis (three strains, 98·7–98·8 % similarity), Streptomyces rectiverticillatus (three strains, 98·2–100 % similarity) and Streptomyces salmonis. Streptomyces aureoversilis NBRC 13021^T and S. rectiverticillatus NBRC 13079^T, and S. baldaccii NBRC 14693^T and Streptomyces spitsbergensis NBRC 15745^T, had identical gyrB sequences and high levels of DNA–DNA relatedness (97–100 and 94 %, respectively). As shown in Table 2, DNA–DNA relatedness between S. hiroshimensis NBRC 12785^T (the representative species) and others was 68–91 %. Furthermore, levels of DNA–DNA relatedness between two representative species, S. baldaccii NBRC 14693^T and S. roseoverticillatus NBRC 12817^T, and others were 78–99 and 67–92 %, respectively (data not shown). These results indicate that the S. hiroshimensis cluster consists of a single species. On the other hand, DNA–DNA relatedness and gyrB sequence similarity among S. baldaccii NBRC 14693^T, S. distallicus NBRC 15815^T and S. syringium NBRC 15900^T, which belong to the same group phenetically, were 46–55 and 90·8–91·0 %, respectively (data not shown), suggesting that S. distallicus NBRC 15815^T and S. syringium NBRC 15900^T did not belong genetically to the S. baldaccii subcluster, as shown in Fig. 1.

The S. netropsis cluster (>97·1 % gyrB sequence similarity) was divided into two subclusters by phenotype: the S. netropsis subcluster (three strains, 98·9–100 % gyrB similarity) and the Streptomyces eurocidicus subcluster (two strains, 98·7 % similarity). S. netropsis NBRC 12893^T and S. flavopersicus NBRC 12769^T exhibited identical gyrB sequences. DNA–DNA relatedness of three strains in the S. netropsis subcluster was 75–93 %, which agrees with the results of Labeda (1996). DNA–DNA relatedness and gyrB sequence similarity between S. eurocidicus NBRC 13491^T and Streptomyces albireticuli NBRC 12737^T were 96–97 and 99·1 %, respectively. On the other hand, DNA–DNA relatedness between S. netropsis NBRC 12893^T and S. eurocidicus NBRC 13491^T was 32–44 %, indicating that these two subclusters are independent of each other.

Table 3 shows DNA–DNA relatedness levels and gyrB sequence similarity in the seven minor clusters: strains in each cluster resembled each other closely in phenotype. Strains in each cluster exhibited species levels of DNA–DNA relatedness with each other (72–100 %) and high gyrB sequence similarity (97–99·8 %), suggesting that each cluster consists of a single species. Streptomyces ladakanum NBRC 13476^T exhibited species levels of identity with Streptomyces mobaraensis NBRC 13819^T, with high DNA–DNA relatedness (97 %) and gyrB sequence similarity (99·1 %). This result is consistent with that of Labeda (1996). DNA–DNA relatedness and gyrB sequence similarity values between ‘S. luteoreticuli’ NBRC 13422 and S. thioluteus NBRC 13341^T, which formed a cluster in MP and ML, were 31 and 95·2 %, respectively, suggesting that these two strains are independent taxa. Streptomyces luteosporeus NBRC 14657^T and S. orinoci NBRC 13466^T are distinct taxa on the basis of gyrB sequence similarity and DNA–DNA relatedness. ‘S. paucisporogenes’ NBRC 13070 is considered to be marginally synonymous with S. abikoensis NBRC 13860^T, as it exhibits 59, 62 and 65 % DNA–DNA relatedness with S. abikoensis NBRC 13860^T, S. ehimensis NBRC 12858^T and S. luteoverticillatus NBRC 12887^T, respectively, which are in accord with the results of Toyama et al. (1974). This strain may be related to S. abikoensis NBRC 13860^T at the subspecies level. S. stramineus NBRC 16131^T is also a distinct taxon in phenotype and DNA–DNA relatedness, although it has 97·5 % gyrB sequence similarity with S. distallicus NBRC 15815^T. S. distallicus NBRC 15815^T was considered to be a subjective synonym of S. netropsis NBRC 12893^T based on DNA–DNA relatedness (73 %), although the gyrB sequence similarity value between S. netropsis NBRC 12893^T and S. distallicus NBRC 15815^T was 95·9 %. This result is in accord with that of Labeda (1996), who indicated that S. distallicus NRRL 2268^T is marginally synonymous with S. netropsis NRRL 2886^T.

Among the atypical whorl-forming Streptomyces species, S. aureoverticillatus NBRC 12742^T and ‘S. nobilis’ NBRC 13386 closely resembled each other in phenotype, except for the ability to form melanin (Table 1). They also had high DNA–DNA relatedness (73–75 %) and gyrB sequence similarity (99·4 %), indicating that they are synonyms.

Correlation between gyrB sequence similarity and DNA–DNA relatedness
Fig. 2 shows the correlation between gyrB sequence similarity values and levels of DNA–DNA relatedness in whorl-forming Streptomyces species. All strains that exhibited 98·5–100 % gyrB sequence similarity showed almost-identical phenotypes and high DNA–DNA relatedness (70–100 %) without exception, suggesting that these strains are synonyms. This result is in good accord with that of Kasai et al. (2000), who reported that a gyrB genetic distance of about 0·0014, roughly equivalent to 98·5 % gyrB sequence similarity, would correspond to 70 % DNA–DNA relatedness. Furthermore, strains with approximately 97 % or higher gyrB sequence similarity and similar phenotypes in the cluster exhibited DNA–DNA relatedness of >65 %, an acceptable value for proposal of a single species. Three exceptional cases were found: the combinations of S. distallicus NBRC 15815^T and S. stramineus NBRC 16131^T (97·5 % gyrB sequence similarity and 45 % DNA–DNA relatedness), S. netropsis NBRC 12893^T and S. albireticuli NBRC 12737^T (97·4 % sequence similarity and 38 % DNA–DNA relatedness), and S. viridiflavus NBRC 15799^T and S. cinnamoneus subsp. cinnamoneus NBRC 12852^T (97·3 % sequence similarity and 32 % DNA–DNA relatedness); in each combination, the strains are independent species. Strains with 95·5–96·5 % gyrB sequence similarity and similar phenotypes in a cluster exhibited DNA–DNA relatedness of 59–75 %, indicating that they are grouped at the species or subspecies level.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Correlation between DNA–DNA relatedness and gyrB sequence similarity. Horizontal line indicates 70 % DNA–DNA relatedness; vertical line indicates 96·5 % gyrB sequence similarity.

We conclude that phylogenetic analysis of gyrB sequences is the most convenient procedure for classification and/or identification of whorl-forming Streptomyces species at the species level. We believe that this method is a useful tool for discrimination at the species level in Streptomyces. We are currently constructing a database of gyrB sequences for identification and classification of whorl-forming, and other, Streptomyces species.

Taxonomic status of whorl-forming Streptomyces species
On the basis of similarity of phenotypes, gyrB gene sequences and DNA–DNA relatedness, the 59 formerly described whorl-forming Streptomyces species actually consist of the following 18 species with subjective synonym(s):

Streptomyces abikoensis NBRC 13860^T.
Subjective synonyms: S. ehimensis NBRC 12858^T, S. luteoverticillatus NBRC 12887^T, S. olivoreticuli NBRC 12896^T, S. parvisporogenes NBRC 13907^T, ‘S. paucisporogenes’ NBRC 13070, S. olivoreticuli subsp. cellulophilus NBRC 15929^T, ‘S. takataensis’ NBRC 13470, ‘Streptoverticillium rubrireticuli’ NBRC 13082 and ‘Streptoverticillium rubroverticillatum’ NBRC 15818.

Streptomyces ardus NBRC 13430^T.
Subjective synonym: ‘Streptomyces caespitosus’ NBRC 13490.

Streptomyces blastmyceticus NBRC 12747^T.
Subjective synonym: ‘Streptomyces mediocidicus’ NBRC 13202.

Streptomyces cinnamoneus NBRC 12852^T.
Subjective synonyms: S. hachijoensis NBRC 12782^T, S. griseoverticillatus NBRC 13420^T, S. sapporonensis NBRC 13823^T.

Streptomyces eurocidicus NBRC 13491^T.
Subjective synonym: S. albireticuli NBRC 12737^T.

Streptomyces griseocarneus NBRC 12776^T.
Subjective synonyms: S. alboverticillatus NBRC 13861^T, S. septatus NBRC 13471^T and ‘S. tropicalensis’ NBRC 13428.

Streptomyces hiroshimensis NBRC 12785^T.
Subjective synonyms: S. aureoversilis NBRC 13021^T, S. baldaccii NBRC 14693^T, S. biverticillatus NBRC 12845^T, S. fervens subsp. fervens NBRC 13343^T, S. fervens subsp. melrosporus NBRC 15920^T, S. rectiverticillatus NBRC 13079^T, S. roseoverticillatus NBRC 12817^T, S. salmonis NBRC 15865^T, S. spitsbergensis NBRC 15745^T and ‘Streptoverticillium rubrochlorinum’ NBRC 14694.

Streptomyces lilacinus NBRC 12884^T.
Subjective synonym: Streptomyces kashimirensis NBRC 13906^T.

‘Streptomyces luteoreticuli’ NBRC 13422.

Streptomyces luteosporeus NBRC 14657^T.

Streptomyces mashuensis NBRC 12888^T.
Subjective synonym: Streptomyces kishiwadensis NBRC 13052^T.

Streptomyces mobaraensis NBRC 13819^T.
Subjective synonym: S. ladakanum NBRC 13476^T.

Streptomyces morookaense NBRC 13416^T.
Subjective synonym: ‘Streptomyces aspergilloides’ NBRC 13461.

Streptomyces netropsis NBRC 12893^T.
Subjective synonyms: S. distallicus NBRC 15815^T, S. flavopersicus NBRC 12769^T, S. kentuckensis NBRC 12880^T and S. syringium NBRC 15900^T.

Streptomyces orinoci NBRC 13466^T.

Streptomyces stramineus NBRC 16131^T.

Streptomyces thioluteus NBRC 13341^T.

Streptomyces vididiflavus NBRC 15799^T.
Subjective synonym: S. olivoverticillatus NBRC 15273^T.

Description of Streptomyces luteireticuli corrig., sp. nov., nom. rev.
Streptomyces luteireticuli (lu.te.i.re.ti'cu.li. L. adj. luteus yellow; L. n. reticulum net; N.L. gen. n. luteireticuli of a yellow net).

The description of Streptomyces luteireticuli is the same as that given by Shirling & Gottlieb (1972) for ‘S. luteoreticuli’, with some additions from this study. Spore-chain morphology is umbellate monoverticilate. Mature spore-chains are generally short (three to ten or more spores per chain) when grown on oatmeal agar (ISP medium 3) and sometimes on inorganic salts/starch agar (ISP medium 4). Sporulating aerial mycelium is usually thin or absent on yeast/malt agar (ISP medium 2) or glycerol/asparagine agar (ISP medium 5). Spore surface is smooth. Aerial mass colour is in the yellow or grey colour-series on ISP medium 3. Nearest matching colours are pale yellow-green, pale yellow, yellowish-grey and light olive-brown. Reverse-side colour of the colony is not distinctive (olive-brown to dark brown on ISP medium 2 and greyish-yellow or yellowish-brown to olive-brown on ISP media 3, 4 and 5). Melanin pigments are not formed in peptone/yeast/iron agar (ISP medium 6), tyrosine agar (ISP medium 7) or tryptone/yeast broth (ISP medium 1). Yellowish and greenish-yellow pigment is found in ISP media 2, 3, 4 and 5. D-Glucose and i-inositol are utilized for growth. Utilization of L-arabinose, D-xylose, D-mannitol, D-fructose, rhamnose, sucrose and raffinose is doubtful or negative. Utilization of D-fructose is weakly positive in this study.

The type strain is NBRC 13422^T (=ATCC 27446^T=CBS 723.72^T=DSM 40509^T=ISP 5509=JCM 4788^T=RIA 1383^T).

	ACKNOWLEDGEMENTS

 
We are grateful to Tomohiko Tamura and Yasuyoshi Nakagawa for their helpful discussions on phylogenetic analysis of gyrB. We are also grateful to Takuji Kudo of JCM and David Labeda of ARS Culture Collection for kindly providing strains.

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