Introduction

Approximately 12 000 bioactive compounds produced by Actinobacteria have been identified, of which three-quarters are produced by Streptomyces species, which constitute the majority of Actinobacteria in soil.1, 2 For this reason, a large number of Streptomyces strains have been isolated from soil samples in the search for new bioactive compounds. To date, nearly 600 species of Streptomyces have been identified.3 Among these isolates, numerous bioactive compounds have been characterized. We have also found avermectin4 produced by Streptomyces avermectinius (S. avermitilis) MA-4680T, herbimycin5 produced by Streptomyces hygroscopicus subsp. hygroscopicus AM-3672 and a lot of other compounds. In addition, two Streptomyces strains, OM-6519T and K04–0144T, which produce lactacystin and cyslabdan, respectively, have also been identified;6, 7 however, their taxonomic positions are unclear.

Streptomyces strain OM-6519T was originally isolated from soil and produces lactacystin, a bioactive compound that induces neuritogenesis in neuroblastoma cells.6, 8 Lactacystin also inhibits proteasome activity by interacting with the β subunit of the 20S proteasome.9 The functional mechanism9, 10 biosynthetic pathway11 and total synthesis12 of lactacystin have been determined, and the compound is frequently used as a biochemical reagent.

Strain K04–0144T was also isolated from soil and produces several bioactive compounds, including cyslabdan,7 nosokomycins, which are a group of novel antibiotics that are active against methicillin-resistant Staphylococcus aureus,13 amphotericin B and nocardamine. Cyslabdan is a new compound that consists of a unique labdane-type diterpene and N-acetylcysteine residue, and is of particular interest because it potentiates the activity of β-lactams against methicillin-resistant Staphylococcus aureus.14, 15 Although some plants and fungi produce compounds that contain labdane-type diterpenes, such as pacovatinins16 and botryosphaerins,17 strain K04–0144T is the first member of the genus Streptomyces to reportedly produce labdane-type diterpene.

In this paper, we report the taxonomic characterization of strains OM-6519T and K04–0144T.

Materials and methods

Strains OM-6519T and K04–0144T were previously isolated from soil samples collected near lake at Inba, Chiba, Japan and under a tree on Ishigaki Island, Okinawa, Japan, respectively.6, 7 To observe cultural and morphological characteristics, the strains were cultured for 2 weeks at 27 °C on yeast extract-malt agar (ISP 2; Difco, Detroit, MI, USA), oatmeal agar (ISP 3; Nihon Pharmaceutical Co., Ltd., Tokyo, Japan), inorganic salts-starch agar (ISP 4; Difco), glycerol-asparagine agar (ISP 5; Nihon Pharmaceutical Co., Ltd.), peptone-yeast extract-iron agar (ISP 6; Nihon Pharmaceutical Co., Ltd.) and tyrosine agar (ISP 7; Nihon Pharmaceutical Co., Ltd.).18 The color of aerial and vegetative mycelia and soluble pigments were determined according to the Color Harmony Manual.19 Morphological characteristics were observed by light microscopy and scanning electron microscopy (JEOL-JSM 5600; JEOL Ltd., Tokyo, Japan). The temperature range, pH range and NaCl tolerance for growth were determined using ISP 2. ISP 4 was used to detect starch hydrolysis, and potassium nitrate broth (ISP 8) (beef extract (Difco) 0.3%, peptone (Difco) 0.5% and KNO3 0.1%) was used to examine nitrate reduction.20 Glucose–peptone–gelatin medium (glucose 2.0%, peptone 0.5%, gelatin (Difco) 20%, pH 7.0) was used to detect gelatin hydrolysis at 20 °C, and 10% skim milk (Difco) was used to assess the coagulation and peptonization of milk at 37 °C.20 Utilization of carbohydrates as the sole carbon source was tested using a basal medium (ISP 9; Difco), according to the method of Pridham and Gottlieb.21 Nitrogen source utilization was examined using the basal medium recommended by Williams et al.22 Biomass for molecular systematics and chemotaxonomic studies was obtained after cultivation on a rotary shaker in yeast extract-glucose broth (yeast extract (Difco) 1.0% and glucose 1.0%, pH 7.0) for 3 days at 27 °C. Isomers of diaminopimelic acid were determined by TLC using whole-cell hydrolysis.23 Whole-cell sugar composition was analyzed according to the method of Becker et al.23 Cell phospholipids were extracted and identified by the method of Minnikin et al.24 Isoprenoid quinones were extracted from cells as described by Collins et al.25 and were analyzed by LC/MS (JMS-T 100LP; JEOL Ltd.) using a CAPCELL PAK C18 UG120 column (Shiseido Co. Ltd., Tokyo, Japan) with methanol/2-propanol (7:3). Methyl esters of cellular fatty acids were prepared by direct transmethylation with methanolic hydrochloride, and were analyzed on a GLC system (HP 6890; Hewlett Packard, Palo Alto, CA, USA). Identification and quantification of the fatty acid methyl esters, as well as numerical analysis of fatty acid profiles, were performed according to the instructions of the Microbial Identification System (MIDI, Newark, DE, USA) using the ACTIN 6 database.26 Genomic DNA from both isolates was prepared by sonication of cell suspensions, and 16S rRNA gene sequences were analyzed as described previously.27 The phylogenetically closest neighbors of strains OM-6519T and K04–0144T were identified by BLAST search using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net).3 Evolutionary distances were estimated using SeaView version 4.2.28, 29 Multiple alignments with selected sequences were performed using the ClustalW2 program. A phylogenetic tree was constructed based on the neighbor-joining method,30 maximum-likelihood method31 and maximum-parsimony method.32 Data were re-sampled with 1000 bootstrap replications.33 The values of sequence similarities with the closest strains were determined using the EzTaxon server.3 For determination of the G+C content and performing DNA–DNA hybridizations, genomic DNA was prepared in accordance with the procedure of Saito and Miura.34 DNA G+C content was determined by HPLC, according to the method of Tamaoka and Komagata,35 and DNA–DNA hybridization was performed using the photobiotin-labeling method of Ezaki et al.36

Results and Discussion

Taxonomic study of strain OM-6519T

Strain OM-6519T grew well on all of the tested media and formed brown colonies. Substrate mycelia fragmented under submerged growth conditions, but did not fragment when cultured on agar media. Gray aerial mycelia were abundantly produced on ISP 2 and 3. A yellow soluble pigment was also produced (Supplementary Table S1). The mature spore chains were straight and had more than 20 spores per chain. The spores were cylindrical with a rugose surface and size of 1.1–1.3 × 0.6–0.7 μm (Figure 1). The strain grew at 15–37 °C, pH 5–10 and 0–4% (w/v) NaCl. Optimum growth was observed at 18–30 °C and pH 6–10. Melanoid pigment was produced on ISP 7. Coagulation and peptonization of milk, nitrate reduction and hydrolysis of starch were positive, but gelatin liquefaction and cellulose hydrolysis were negative. The strain utilized d-glucose, d-fructose and sucrose as sole carbon sources, but did not utilize l-arabinose, d-xylose, raffinose, melibiose, d-mannitol, l-rhamnose or myo-inositol. l-Aspartic acid, l-threonine, glycine, l-phenylalanine, l-arginine and l-ornithine were utilized as sole nitrogen sources, but the strain did not utilize l-methionine or d-valine. Strain OM-6519T contained LL-diaminopimelic acid as the diamino acid in whole-cell hydrolysates and the whole-cell sugar pattern consisted of glucose, ribose and rhamnose. Phosphatidylethanolamine was detected, but phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and phosphatidylinositol were not found. The major menaquinones were MK-9(H6) (71%) and MK-9(H8) (29%), and the predominant fatty acids were C16:0 (29.6%), anteiso-C15:0 (17.7%) and iso-C15:0 (13.3%) (Supplementary Table S2). The G+C content of the genomic DNA was 73 mol%. These phenotypic and chemotaxonomic characteristics indicated that strain OM-6519T belonged to the genus Streptomyces.

Figure 1
figure 1

Scanning electron micrograph of strain OM-6519T grown on ISP 4 for 2 weeks at 27 °C.

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The nearly complete 16S rRNA gene sequence (1482 bp) of strain OM-6519T was determined and deposited under the DDBJ accession number AB915215. Phylogenetic analysis with the type strains of 20 species showing the highest similarities to strain OM-6519T indicated that the strain clustered with Streptomyces xanthocidicus NBRC 13469T, S. chrysomallus subsp. fumigatus NBRC 15394T and S. aburaviensis NRRL B-2218T (Figure 2). The similarity values of the 16S rRNA gene sequences between the three strains and strain OM-6519T were 99.7, 99.6 and 99.5%, respectively. Strain OM-6519T was differed from the related strains with respect to spore surface morphology, as its closest phylogenetic neighbors had spores with a smooth surface, as opposed to rugose. Moreover, these strains also differed in several physiological characteristics, as only strain OM-6519T utilized L-phenylalanine and L-ornithine as nitrogen sources, and did not utilize D-xylose as a sole source of carbon (Table 1). The levels of DNA–DNA relatedness between strain OM-6519T and S. xanthocidicus NBRC 13469T, S. chrysomallus subsp. fumigatus NBRC 15394T and S. aburaviensis NRRL B-2218T were 57±7%, 41±10% and 44±6%, respectively. These values were clearly below the 70% threshold value for assigning strains to the same species.37

Figure 2
figure 2

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences from strain OM-6519T and the type strains of related species in the genus Streptomyces. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Branches marked with a solid circle were also recovered in the maximum-likelihood tree; asterisks show branches recovered in the maximum-parsimony tree. Bar, 0.005 nucleotide substitutions per site.

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Table 1 Morphological and cultural features and physiologically differential characteristics of strain OM-6519T and type strains of related species
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Based on the above results, we propose that strain OM-6519T represents a novel species in the genus Streptomyces, for which the name Streptomyces lactacystinicus sp. nov. is proposed.

Taxonomic study of strain K04–0144T

Strain K04–0144T grew well on ISP 2, 4, 6 and 7, and formed brown colonies. Substrate mycelia did not show fragmentation. Gray aerial mycelia were abundantly produced on ISP 2, 3, 4 and 7. A yellow soluble pigment was produced (Supplementary Table S3). The mature spore chains were spiral and had more than 20 spores per chain. Spores were cylindrical with a smooth surface and size of 1.0–1.3 × 0.7–0.8 μm (Figure 3). Strain K04–0144T grew at 12–42 °C, pH 4–10 and 0–7% (w/v) NaCl. Optimum growth was observed at 16–36 °C and pH 5–10. The strain did not produce melanoid pigment. Coagulation and peptonization of milk, nitrate reduction, gelatin liquefaction and starch hydrolysis were positive, but cellulose hydrolysis was negative. The strain utilized d-glucose, d-xylose, raffinose, melibiose, d-mannitol, d-fructose and l-rhamnose as sole carbon sources, but did not utilize l-arabinose, myo-inositol or sucrose. l-Aspartic acid, l-threonine, glycine, l-phenylalanine, l-arginine, l-methionine and l-ornithine were utilized as sole nitrogen sources, but d-valine was not utilized. Strain K04–0144T contained LL-diaminopimelic acid as a diamino acid in whole-cell hydrolysates, the whole-cell sugar pattern consisted of glucose and mannose. Phosphatidylethanolamine and diphosphatidylglycerol were detected, but phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol were not found. The major menaquinones were MK-9 (H8) (62%) and MK-9 (H6) (35%) and the predominant fatty acids were iso-C16:0 (22.5%), anteiso-C15:0 (22.5%) and iso-C15:0 (13.7%) (Supplementary Table S2). The G+C content of the genomic DNA was 73 mol%. These phenotypic and chemotaxonomic characteristics indicated that strain K04–0144T belonged to the genus Streptomyces.

Figure 3
figure 3

Scanning electron micrograph of strain K04–0144T grown on ISP 2 for 2 weeks at 27 °C.

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The nearly complete 16S rRNA gene sequence (1484 bp) of strain K04–0144T was determined and was deposited under the DDBJ accession number AB915216. Phylogenetic analysis based on the sequences of the type strains of the 20 species showing the highest similarities to strain K04–0144T showed that strain K04–0144T clustered with eight strains (Figure 4). Streptomyces corchorusii NBRC 13032T showed the highest similarity with strain K04–0144T (99.4%), followed by S. olivaceoviridis NBRC 15394T (99.4%) and S. canarius NRRL B-2218T (99.3%). However, strain K04–0144T differed physiologically from these three species with respect to nitrate reduction, gelatin liquefaction, utilization of L-arabinose, myo-inositol and sucrose as sole carbon sources, and utilization of l-threonine and l-methionine as sole nitrogen sources (Table 2). The levels of DNA–DNA relatedness between strain K04–0144T and S. corchorusii NBRC 13032T, S. olivaceoviridis NBRC 15394T and S. canarius NRRL B-2218T were 32±15%, 35±13% and 38±18%, respectively. These values are clearly below the 70% threshold value for assigning strains to the same species.37

Figure 4
figure 4

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences from strain K04–0144T and the type strains of related species in the genus Streptomyces. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Branches marked with a solid circle were also recovered in the maximum-likelihood tree asterisks show branches recovered in the maximum-parsimony tree. Bar, 0.01 nucleotide substitutions per site.

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Table 2 Morphological and cultural features and physiologically differential characteristics of strain K04–0144T and type strains of related species
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Based on these findings, we propose that strain K04–0144T represents a novel species in the genus Streptomyces, for which the name Streptomyces cyslabdanicus sp. nov. is proposed.

Description of Streptomyces lactacystinicus sp. nov

Streptomyces lactacystinicus (lac.ta.cys.ti'ni.cus. N.L. n. lactacystinum, lactacystin; L. masc. suff. -icus, suffix used with the sense of pertaining to; N.L. masc. adj. lactacystinicus, pertaining to lactacystin, a proteasome inhibitor produced by the organism).

A Gram-positive, aerobic actinomycete that forms straight spore chains. The spores are cylindrical with a rugose surface and size of 1.1–1.3 × 0.6–0.7 μm. Grows well on ISP 2, 3, 4, 5, 6 and 7, and forms brown colonies. Gray aerial mycelia are abundantly produced on ISP 2 and 3. A yellow soluble pigment is produced. Growth occurs at 15–37 °C and pH 5–10. No growth occurs at 5% (w/v) NaCl. Melanoid pigment is not produced. Milk is coagulated and peptonized. Nitrate is reduced to nitrite. Gelatin is not liquefied. Starch is hydrolyzed, but cellulose is not. d-glucose, d-fructose, sucrose, l-aspartic acid, l-threonine, glycine, L-phenylalanine, l-arginine and l-ornithine are utilized as sole carbon and nitrogen sources. l-Arabinose, d-xylose, raffinose, melibiose, d-mannitol, l-rhamnose, myo-inositol, l-methionine and d-valine are not utilized. Whole-cell hydrolysate contains LL-diaminopimelic acid as the diamino acid, in addition to glucose, ribose, and rhamnose. The polar lipids mainly consist of phosphatidylethanolamine. The major menaquinones are MK-9 (H6) and MK-9 (H8). The predominant fatty acids are C16:0, anteiso-C15:0, and iso-C15:0. The G+C content of the genomic DNA of the type strain is 73 mol%. The type strain, OM-6519T (=NBRC 110082T, DSM 43136T), was isolated from soil from Inba, Chiba, Japan, and produces lactacystin, a proteasome inhibitor.

Description of Streptomyces cyslabdanicus sp. nov

Streptomyces cyslabdanicus (cys.lab.da'ni.cus. N.L. n. cyslabdanum, cyslabdan; L. masc. suff. -icus, suffix used with the sense of pertaining to; N.L. masc. adj. cyslabdanicus, pertaining to cyslabdan, an antibiotic produced by the organism).

A Gram-positive, aerobic actinomycete that forms spiral spore chains. The spores are cylindrical with a smooth surface and size of 1.0–1.3 × 0.7–0.8 μm. Mycelia do not fragment. Grows well on ISP 2, 4, 6 and 7, and form brown colonies. Gray aerial mycelia are abundantly produced on ISP 2, 3, 4 and 7. A yellow soluble pigment is produced on agar media. Growth occurs at 12–42 °C, pH 4–10 and 0–7% (w/v) NaCl. Melanoid pigment is not produced. Milk is coagulated and peptonized. Nitrate is reduced to nitrite. Gelatin is liquefied. Starch is hydrolyzed, but cellulose is not. d-glucose, d-xylose, raffinose, melibiose, d-mannitol, d-fructose, d-rhamnose, l-aspartic acid, l-threonine, glycine, l-phenylalanine, l-arginine, l-ornithine and l-methionine are utilized as sole carbon and nitrogen sources. l-Arabinose, myo-inositol, sucrose and d-valine are not utilized. Whole-cell hydrolysate contains LL-diaminopimelic acid as the diamino acid, in addition to glucose and mannose. The main polar lipids are phosphatidylethanolamine and diphosphatidylglycerol. The major menaquinones are MK-9 (H6) and MK-9 (H8). The predominant fatty acids are iso-C16:0, anteiso-C15:0, and iso-C15:0. The G+C content of the genomic DNA of the type strain is 73 mol%. The type strain, K04–0144T (=NBRC 110081T, DSM 42135T), was isolated from soil from Ishigaki Island, Okinawa, Japan, and produces cyslabdan, a bioactive compound that enhances the anti-MRSA activity of imipenem medication.