Introduction

The genus Streptomyces was originally proposed by Waksman and Henrici1 and later unified with the genus Streptoverticillium.2 The genus Kitasatospora was also unified to the genus Streptomyces3 but was separated anew.4 The genus Streptomyces is known as the most diverse genus of actinomycetes with more than 600 recognized species.5 The genus is characterized by aerobic, Gram-stain-positive, formation of highly branched mycelia and spores, high DNA G + C content, presence of LL-diaminopimelic acid and absence of characteristic sugars in their cell wall. Numerous novel bioactive compounds have been isolated from marine-derived actinomycetes, especially, members of the genus Streptomyces, which have been recognized as an important source for drug discovery.6, 7, 4 Most of marine-derived actinomycetes do not require seawater or salt supplementation for growth, and thus, it is assumed that they are transported from land into sea. However, the existence of some marine-adapted actinomycetes was indicated by phylogenetic analyses of marine actinomycete isolates and their secondary metabolism is suggested to be different from those of terrestrial counterparts.8 Hyaluronidase is a target enzyme of anti-inflammatory and anti-allergic drug deveropment.9 For example anti-allergic and anti-inflammatory agents such as disodium cromoglycate and glycyrrhizin show hyaluronidase inhibitory activity.10, 11 During the course of screening for hyaluronidase inhibitors from microbial metabolites, a novel actinomycete strain was isolated from a marine invertebrate. The objective of the present study is to clarify the taxonomic position of the isolate using a polyphasic approach.

Materials and methods

Bacterial strains and isolation

A novel actinomycete strain, designated MB-PO13T, was isolated from a tunicate (Molgula manhattensis) collected in Tokyo Bay, Japan, as a producer of hyaluronidase inhibitor.12 After the collection, the tunicate was immediately washed three times with sterilized natural seawater, cut into small pieces, and then crushed using a homogenizer (model POLYTRON PT2100; Kinematica, Luzern, Switzerland). Five hundred microliter of suspension of the tunicate was spread onto inorganic salts−starch agar (ISP 4) supplemented with nalidixic acid (20 mg l–1) and cycloheximide (50 mg l–1). After incubation at 27 °C for 14 days, a single colony was repeatedly transferred onto the same agar medium to obtain the pure isolate of strain MB-PO13T. The strain was maintained on a yeast extract−malt extract medium (ISP 2). Streptomyces graminisoli NBRC 108883T, Streptomyces shenzhenensis DSM 42034T, Streptomyces gramineus NBRC 107863T and Streptomyces rhizophilus NBRC 108885T were used as reference strains.

Morphological, physiological and biochemical tests

Colony appearance was examined after incubation at 28 °C for 14 days. Cell morphology was observed under a light microscope (model BX-51; Olympus, Tokyo, Japan) and a scanning electron microscope (model JSM-6060; JEOL, Tokyo, Japan). Growth under anaerobic conditions was examined by incubating the strain in an anaerobic chamber with an O2-absorbing and CO2-generating agent (Anaero-Pack; Mitubishi Gas Chemical, Tokyo, Japan). Gram staining was performed as described by Hucker and Conn.13 Catalase activity was determined by production of oxygen bubbles after the addition of a drop of 3% (v/v) aqueous hydrogen peroxide solution. The cultural properties of strain MB-PO13T were observed according to the ISP guideline14 on ISP 2, oatmeal agar (ISP 3), ISP 4, glycerol asparagine agar (ISP 5), peptone-yeast extract-iron agar (ISP 6), tyrosine agar (ISP 7), glucose−asparagine agar, nutrient agar, sucrose–nitrate agar, skim milk agar and yeast extract-starch agar after 14 days of incubation at 28 °C. Colors of aerial and substrate mycelia were determined using The Japanese Industrial Standard Common Color Names (JIS Z 8102: 2001). The temperature range and optimum temperature for growth were determined by incubating the strain at 5, 10, 15, 20, 28, 37, 42 and 50 °C on ISP 2 agar plates for 14 days. The pH range and the optimum pH for initial growth were determined by incubating at 28 °C in ISP 2 broth, of which pH was adjusted to 3−12 by 1 m HCl or 1 m Na2CO3. Tolerance to NaCl was tested by incubation on ISP 2 agar plates containing 2, 3, 5, 7, 9 and 12% (w/v) NaCl at 28 °C. Enzyme activity test was performed using API ZYM kit (bioMérieux, Tokyo, Japan) according to the supplier’s instruction. Carbohydrate utilization was determined on Pridham−Gottlieb carbon utilization agar (ISP 9) supplemented with sterilized carbon sources.14

16S rRNA gene sequence determination and phylogenetic analysis

Genomic DNA of strain MB-PO13T was extracted by modified bead-beating phenol−chloroform method.15 PCR amplification was carried out with a Dice mini TP 100 (Takara Bio, Tokyo, Japan). Primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492r (5′-TACGGYTACCTTGTTACGACTT-3′) were used for PCR amplification of 16S rRNA gene.16 The PCR product was purified with a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA), and sequenced with a BigDye cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 310 Genetic analyzer (Applied Biosystems). Phylogenetic neighbors were identified and pairwise 16S rRNA gene sequence similarities were calculated using EzTaxon-e server.17 The CLUSTAL-W program18 was used to align an almost complete 16S rRNA gene sequence (1429 nucleotides) of strain MB-PO13T with the corresponding sequences from related Streptomyces species. Phylogenetic trees were reconstructed by the neighbor-joining,19 maximum-likelihood20 and maximum-parsimony21 algorithms by using the MEGA 6.0 program.22 The resultant tree topologies were evaluated by bootstrap analysis23 based on 1000 replicates.

G + C content of DNA and DNA–DNA hybridization

Genomic DNAs for G + C content analysis and DNA−DNA hybridization were obtained using the method of Saito and Miura.24 The G + C content was determined according to the method described by Hamada et al.25 The microplate hybridization method developed by Ezaki et al.26 was used to analyze the DNA−DNA relatedness. DNA−DNA hybridizations were carried out five times; after the highest and lowest values for each sample were excluded, the means of the remaining three values were obtained.

Chemotaxonomic tests

Biomass for chemotaxonomic studies was prepared by incubating the strains in flasks on a shaker (100 r.p.m.) at 28 °C for 72 h. Diaminopimelic acid (DAP) in whole-cell hydrolysate was analyzed by modified method of Hasegawa et al.27 Whole-cell sugar and menaquinone compositions were analyzed according to the methods described previously.25 The polar lipids were analyzed by TLC as described by Hamada et al.,28 using chloroform−methanol-water (65:25:4, v/v/v) in the first direction and chloroform−acetic acid−methanol-water (80:18:12:5, v/v/v/v) in the second. Preparation and analysis of cellular fatty acid methyl esters were performed using the protocol of MIDI Sherlock Microbial Identification System29 and gas chromatography (model 6890N; Agilent Technologies, Santa Clara, CA, USA) with the Sherlock MIDI software (version 6.2) and the ACTINO database (version 6.2; MIDI Inc., Newark, DE, USA).

Nucleotide sequence accession number

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain MB-PO13T is AB184533.

Results and discussion

Strain MB-PO13T formed circular and cream color colonies and exhibited well developed, branched substrate and aerial mycelia. Fragmentation of mycelia was not observed. The aerial mycelia were branched monopodially and 0.4–0.6 μm in diameter. Spore chain was spiral type and contained about 6–10 helixes and 30–50 spores per chain. The spores were cylindrical, 0.5–0.8 × 1.0–1.5 μm in size and had a smooth surface (Figure 1). Sporulation was not observed on nutrient-rich media examined in this study. A number of spore chains were observed on three-fold diluted ISP 4 and ten-fold diluted ISP 2 agar plates. Sporangia were not observed. The substrate mycelia appeared in cream color after a few days, which later turned reddish when grown on all ISP media except for ISP 6, glucose−asparagine and yeast extract-starch agars (Table 1). In contrast to the reference strains, strain MB-PO13T produced soluble reddish pigments on an ISP 2 agar plate (Supplementary Figure S1). The major component of the pigments was identified as an aromatic polyketide, hyaluromycin9 (Supplementary Figure S2). Yellowish-white aerial mycelia were formed on ISP 4 medium, which became light gray at sporulation. Sporulation was not observed on other media. The strain grew at 10–37 °C (optimum temperature 28 °C) and at pH 4.0–9.0 (optimum pH 7.0). The strain exhibited growth with 0–2% (w/v) NaCl (optimum 0% NaCl). The strain was Gram-stain-positive, catalase-positive and did not grow under anaerobic conditions. The results of other physiological and biochemical analyses have been summarized in the species description.

Figure 1
figure 1

Scanning electron micrograph of strain MB-PO13T grown on ten-fold diluted ISP 2 medium for 14 days at 28 °C. Bar, 10 μm.

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Table 1 Growth and cultural characteristics of strain MB-PO13T on various media
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Phylogenetic analysis, based on 16S rRNA gene sequences, showed that strain MB-PO13T represented a member of the genus Streptomyces. In the neighbor-joining tree, strain MB-PO13T and S. graminisoli formed a monophyletic cluster with a bootstrap resampling value of 89% (Figure 2). This cluster was supported in the trees generated by the maximum-likelihood and maximum-parsimony algorithms (Supplementary Figures S3 and S4). The highest similarity value was observed with S. graminisoli JR-12T (99.72%), followed by S. shenzhenensis 172115T (99.23%), S. jiujiangensis JXJ 0074T (98.87%), S. rhizophilus JR-41T (98.58%) and S. gramineus JR-43T (98.53%). The DNA−DNA relatedness values between strain MB-PO13T and the type strains of S. graminisoli and S. shenzhenensis were 40.6% (reciprocal reaction: 56.9%) and 22.4% (37.9%), respectively. These values are sufficiently below the 70% cutoff point of DNA−DNA relatedness that is a standard criterion for the assignment of bacterial strains to the same genomic species.30 The G + C content of the genomic DNA of strain MB-PO13T was 73.6 mol%.

Figure 2
figure 2

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of strain MB-PO13T and its taxonomic neighbors. Kitasatospora setae KM-6054T (AB022868) was used as the outgroup. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. Filled circles indicate that the corresponding nodes are also recovered in the trees generated with the maximum-likelihood and maximum-parsimony algorithms (Supplementary Figures S3). Bar, 0.01 substitutions per nucleotide position.

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The whole-cell hydrolysate of strain MB-PO13T contained LL-DAP, glucose and mannose. The menaquinones were identified as MK-9(H8), MK-9(H6), MK-9(H4) and MK-9(H10) (57:37:5:1). The principal polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol (Supplementary Figure S5). Six minor unidentified polar lipids were also detected. The major cellular fatty acids (>10%) were anteiso-C15:0 (24.9%), iso-C16:0 (23.4%), iso-C14:0 (15.0%) and C16:0 (10.7%; Supplementary Table S1). These chemotaxonomic features were in good accordance with those of the genus Streptomyces.

The results of the phylogenetic analyses based on 16S rRNA gene sequences suggested that strain MB-PO13T belonged to the genus Streptomyces, and its chemotaxonomic characteristics were also in good agreement with those of the genus Streptomyces. Meanwhile, DNA−DNA relatedness between strain MB-PO13T and the type strains of the closest species was low, and physiological and biochemical tests also distinguished strain MB-PO13T from these species (Table 2). On the basis of these findings, strain MB-PO13T was proposed to be classified as a representative of a novel species of the genus Streptomyces, with the name Streptomyces hyaluromycini sp. nov.

Table 2 Differential characteristics of strain MB-PO13T and its closest phylogenetic neighbors
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Description of Streptomyces hyaluromycini sp. nov.

Streptomyces hyaluromycini (hya.lu.ro.my.ci’ni. N. L. neut. n. hyaluromycinum hyaluromycin; N. L. gen. neut. n. hyaluromycini of the hyaluronidase inhibitor hyaluromycin).

Cells are aerobic and Gram-stain-positive. The aerial mycelia are branched and yellowish white in color, which became light gray at sporulation and the substrate mycelia are yellow to red. Smooth surface spores (0.5–0.8 × 1.0–1.5 μm) in spiral chains are formed when cultured on nutritionally poor media. Soluble red pigments are produced on ISP 2, ISP 3, ISP 4, ISP 7, glucose−asparagine, nutrient and yeast−starch media. Melanin pigments are produced on ISP 6 medium. Growth occurs at 10–37 °C (optimum 28 °C), at pH 4.0–9.0 (optimum pH 7.0) and in the presence of <2% NaCl (w/v). The API ZYM assays are positive for esterase (C4), leucine arylamidase, acid phosphatase, α-galactosidase, β-galactosidase and α-glucosidase but negative for valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, β-glucosidase, α-mannosidase and α-fucosidase. Weakly positive for alkaline phosphatase, esterase lipase (C8), naphtol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase activities. Utilizes l-arabinose, d-fructose, d-glucose, inositol, d-mannitol, rhamnose and d-xylose as sole carbon source for energy and growth, but not raffinose and sucrose (all at 1%, w/v). Hydrolyzes casein and starch, but not gelatin and cellulose. Peptidoglycan contains LL-DAP, glucose and mannose. The predominant menaquinones are MK-9(H8) and MK-9(H6). The major cellular fatty acids (>10% of total) are anteiso-C15:0, iso-C16:0, iso-C14:0 and C16:0. The principal polar lipids are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol.

The type strain, MB-PO13T (=NBRC 110483T =DSM 100105T), was isolated from a tunicate (Molgula manhattensis) collected at Tokyo Bay, Minato-ku, Tokyo, Japan. The DNA G + C content of the type strain is 73.6 mol%.