Introduction

The genus Streptomyces was proposed by Waksman and Henrici1 to encompass an aerobic, spore-forming group of actinomycetes. The Streptomyces is widely distributed in nature and encompasses more than 630 species.2 Many members of this genus are a source of a broad range of novel bioactive compounds. According to statistics, actinomycete sources account for about 53% of all microbial bioactive secondary metabolites with 7600 of these compounds (75%) being produced by Streptomyces.3 However, it is becoming increasingly difficult to find new leading compounds from common actinomycetes and leads to the costly rediscovery of known compounds.4 On the other hand, the demand of anticancer and anti-infectious drug discovery is growing. Hence, the investigation for new actinomycete resource is an important element in drug discovery.

Nowadays, marine actinomycetes have been widely explored for potential source of novel compounds as the environmental conditions of the sea are entirely different from the terrestrial conditions.5,6 Although new actinomycetes and antibiotics have been discovered from sponges, algae, mangrove plants and other marine animals and plants,7,8 the full suite of actinomycete occupying some special ecological niches have not been fully investigated. Therefore our work was focused on the isolation of actinomycetes from a wide variety of marine animals of the Bohai Sea, such as blue mussel, jellyfish, shrimp, fish, crabs, starfish, sea urchins etc., and on screening for their ability to produce antimicrobials. During our continuous screening for new antibiotics, a putatively novel Streptomyces strain 11A07T from a fish in the Bohai Sea had the genetic potential to produce secondary metabolites related to both polyketides and nonribosomal peptides, as well as showed potent antimicrobial activities.9 In this study, we report the classification and identification of this new isolate based on polyphasic taxonomy. Further, the name Streptomyces bohaiensis sp. nov. 11A07T is proposed as a novel Streptomyces species.

Materials and methods

Strain and maintenance condition

The young Scomberomorus niphonius (long, slender, laterally flattened, pelagic fish with longitudinal dark spots on the sides and ~15 cm in fork length) was collected from the Bohai Sea, located in eastern Liaoning Peninsula in northern China (39°20′ N, 122°17′ E). The specimen was rinsed five times with sterile artificial seawater to remove any transient and loosely attached microorganisms from the surface of the S. niphonius. 1 cm3 sections of specimen tissue were excised with a sterile scalpel and ground with a sterilized mortar and pestle. Then tissue homogenates were inoculated on oatmeal agar international streptomyces project (ISP 3) plates using a standard dilution plating method, containing nalidixic acid (25 mg l−1) and cycloheximide (50 mg l−1) to inhibit growth of bacteria and fungi. After incubation at 28 °C for 3 weeks, an isolate designated 11A07T was picked. The strain was maintained on plates of modified Gauze’s synthetic medium No.1 (soluble starch 20.0 g; KNO3 1.0 g; MgSO4·7 H2O 0.5 g; K2HPO4 0.5 g; FeSO4·7 H2O 10.0 mg; agar 15.0 g; artificial sea water 1.0 l; adjust pH 7.0) at 4 °C and as suspensions of spores or mycelia fragments in glycerol (20%, v/v) at −80 °C.

Phenotypic characterization

Strain 11A07T was cultured for 14 days at 28 °C on tryptic soy agar (TSA) using the coverslip technique.10 Morphological properties were observed by a light microscope (BH-2; Olympus, Tokyo, Japan) and a scanning electron microscope (Quanta200; FEI, Hillsboro, OR, USA).11 Cultural properties were determined using standard ISP media,12 Czapek’s agar, nutrient agar (Difco, Sparks, MD, USA) and potato dextrose agar (Difco) after incubation at 28 °C for 14 days. Colors of the aerial, substrate mycelia and diffusible pigments were determined using color chips from the ISCC-NBS color charts. Growth at various NaCl concentrations (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 10.0, 11.0, 12.0, 15.0%, w/v) and different temperatures (4, 10, 28, 37, 45, 50 °C) was examined by growing the strain on TSA medium as the basal medium. Growth at different pH values (4.0–11.0, at intervals of 1.0 pH unit) was examined on modified TSA medium using the buffer system described by Xu et al.13 Oxidase activity was determined by the oxidation of tetramethyl-p-phenylenediamine. Catalase activity was determined by 3% H2O2, and gas production was identified as a positive reaction. Hydrolysis of starch, gelatin, Tweens 20, 40, 60 and 80, milk coagulation and peptonization were carried out according to methods described by Tindall et al.14 Nitrate reduction and H2S production were determined using conventional procedures.14,15 Carbon-source utilization tests were performed according to the methods described by Shirling and Gottlieb12 and Athalye et al.16 using modified basal medium17 supplemented with 0.05% yeast extract.

Chemotaxonomic characterization

The biomass used for analyses of cellular fatty acids was obtained from cultures grown in tryptic soy broth for 7 days in flasks shaken at 180 r.p.m. at 28 °C. The cells were harvested by centrifugation at 4500 r.p.m. for 10 min and washed twice with sterile distilled water. Cellular fatty acids were extracted, methylated and analyzed using the Sherlock Microbial Identification System according to the method of Sasser18 as the manufacturer’s instructions. Fatty acids were then analyzed by GC (Agilent Technologies 7890A GC System, Wilmington, DE, USA) using the Microbial Identification software package (Sherlock Version 6.1; Sherlock Microbial Identification System database TSBA6). The diaminopimelic acid isomer was identified by TLC.19 The whole-cell sugars were separated by HPLC after precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone.20 Menaquinones were extracted21 and determined by HPLC.22 Polar lipids were extracted and examined according to published procedures.23,24

Molecular characterization

Genomic DNA isolation, PCR amplification of 16S rRNA gene, cloning and sequencing were performed with conventional methods.25 Sequence analysis of 16S rRNA gene was performed using Basic Local Alignment Search Tool for Nucleotides (BLASTN). The 16S rRNA gene sequence of strain 11A07T was compared with sequences from EzBioCloud using basic local alignment search tool (http://eztaxon-e.ezbiocloud.net/).26 Multilocus sequence analysis was based on five house-keeping genes atpD (ATP synthase F1, beta subunit), gyrB (DNA gyrase B subunit), recA (recombinase A), rpoB (RNA polymerase beta subunit) and trpB (tryptophan synthetase, beta subunit) using the primers and protocols described previously.27 Sequence data for the five house-keeping loci for each strain were deposited in GenBank with the accession numbers shown in Table 1. All these gene sequences were aligned with corresponding sequences (retrieved from the GenBank/EMBL/DDBJ database) using CLUSTAL X1.83.28 The 16S rRNA gene and five house-keeping gene sequence-concatenated phylogenetic trees were constructed by the Neighbor-Joining,29 Maximum Parsimony30 and Maximum-Likelihood31 tree-making algorithms by using the software packages MEGA version 5.0.32 Concatenated sequences of all five protein-coding loci were joined head-to-tail in-frame. Evolutionary distance matrices were generated according to the Kimura’s two parameter model.33 The topologies of the resultant trees were evaluated by bootstrap analysis with 1000 replicates.34 The G+C content of genomic DNA prepared by the method of Marmur35 was determined using the HPLC method as described by Mesbah et al.36

Table 1 House-keeping gene sequences of the new isolate and those related Streptomyces strains used in the present study

Results and Discussion

Strain 11A07T was Gram staining positive and aerobic growth. The strain produced extensively branched substrate mycelia and aerial hyphae, but not fragmented. Scanning electron microscopy revealed straight or rectiflexibile chains of smooth surfaced spores were borne on aerial hyphae. Spore chains were generally straight and very long containing >50 spores (Figure 1). These characteristics supported strain 11A07T belonging to the genus Streptomyces.37 Strain 11A07T showed abundant growth on ISP 2, ISP 3, ISP 5, TSA, Czapek solution agar and Nutrient agar, but moderate growth on ISP 4 and poor growth on potato dextrose agar. White aerial mycelium was observed on these media, but variety of substrate mycelium color was displayed on different media. Brown diffusible pigments were detected on ISP 2 and Czapek solution agar (Table 2). The growth temperature for strain 11A07T was 15–40 °C (optimum 28 °C). The strain 11A07T grew at pH 6–10 (optimum 8–9) and 0–11% (w/v) NaCl (optimum 1). It could utilize glucose, l-rhamnose, D-galactose, inositol, raffinose, D-mannitol, salicin, sorbitol, sucrose and sodium citrate as sole carbon source for growth, but not l-arabinose, d-fructose and d-xylose. Other physiological characteristics are given in Table 3 and in the species description.

Figure 1
figure 1

Scanning electron micrographs of spore chains of strain 11A07T after incubation at 28 °C for 14 days on TSA medium. (a) Bar 10 μm; (b) Bar 4 μm.

Table 2 Growth and cultural characteristics of strain 11A07T on different growth media after incubation for 2 weeks at 28 °C
Table 3 Differential phenotypic and chemotaxonomic characteristics of strain 11A07AT, and its closest phylogenetic neighbors Streptomyces panacagri Gsoil 519T and Streptomyces albus subsp. albus KCTC 1082T(=NRRL B-2365T), Streptomyces sodiiphilus YIM 80305T

LL-diaminopimelic acid was detected in the peptidoglycan of strain 11A07T. The cell wall sugars were mannose, ribose, glucose, galactose and xylose. The predominant menaquinones were found with MK-9 (H2) 12.7%, MK-9 (H4) 60.3% and MK-9 (H6) 25.0%. The fatty-acid profile contained iso-C16:0 (37.5%), C18:0 10-methyl, tuberculostearic acid (20.0%) and anteiso-C17:0 (11.8%) as the major compositions (Supplementary Table S1). The polar lipids identified were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unknown phospholipid (Figure 2). These characteristics were consistent with those of genus Streptomyces.

Figure 2
figure 2

The polar lipid profiles of stains 11A07T stained with molybdophosphoric acid after separation by two-dimensional TLC. DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannoside; PL, unknown phospholipid. A full color version of this figure is available at The Journal of Antibiotics journal online.

The DNA G+C content of the genomic DNA of this strain was 71.4 mol%. An almost complete 16S rRNA gene sequence (1520 bp) was obtained for isolate 11A07T. The 16S rRNA gene sequence of strain 11A07T was submitted in the GenBank Database under the accession number KF682221. Sequence analysis showed that 16S rRNA gene sequence of strain 11A07T to be most similar to those of Streptomyces rimosus subsp. paromomycinus DSM 41429T, Streptomyces chrestomyceticus DSM 40545T, and Streptomyces panacagri Gsoil 519T, with sequence identities of 97.41%, 97.40% and 97.30%, respectively. Further phylogenetic analyses revealed that strain 11A07T formed a distinct branch separate from other representatives of the genus Streptomyces and most closely related with S. panacagri Gsoil 519T (similarity 97.30%), Streptomyces albus subsp. albus NRRL B-2365T (similarity 97.10%) and Streptomyces sodiiphilus YIM 80305T (similarity 96.83%; Figure 3). Three phylogenetic tree showed that strain 11A07T forms a cluster with three of the above species, with the exception of S. sodiiphilus YIM 80305T by maximum-likelihood algorithm (Supplementary Figures S1 and S2). However, this relationship is not supported by significant bootstrap values nor by the stability. Stackebrandt and Ebers38 demonstrated that strains showing <98.5% 16S rRNA gene sequence similarity presented DNA reassociation values always <70%. And further, Kim et al.39 suggested that 98.65% 16S rRNA gene sequence similarity can be used as the threshold for differentiating two species. In view of the lower 16S rRNA gene sequence similarity values of strains S. panacagri Gsoil 519T, S. sodiiphilus YIM 80305T, S. albus subsp. albus NRRL B-2365T and other close relatives (similarities 97.41%), and the distinct phylogenetic positions of these strains, DNA–DNA hybridizations were not investigated in this study.

Figure 3
figure 3

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain 11A07T in the genus Strepomyces. Filled circles indicate branches that were also recovered using the maximum parsimony and maximum-likelihood trees. Bootstrap values (expressed as percentages of 1000 replications) of above 50% are shown at the branch points. Bar, 0.002 sequence divergence.

The strain 11A07T was further characterized by partial sequencing of atpD (981 nt), gyrB (891 nt), recA (911 nt), rpoB (994 nt) and trpB (735 nt). The phylogenetic trees based on concatenated sequences (2511 bp) of atpD (position 101–595), gyrB (position 19–423), recA (position 109–612), rpoB (position 270–807) and trpB (position 45–611) trimmed sequences unambiguously demonstrated that the isolate constituted a separate phylogenetic line within other closely related species of the genus Streptomyces (Figure 4, Supplementary Figures S3 and S4). As well as multilocus sequence analysis evolutionary distances were greater than the species-definitive multilocus sequence analysis distance (0.007)40 with all of the phylogenetically near species as shown in Supplementary Table S2. These evidences supported the proposal that this strain represents a new species in the genus Streptomyces.

Figure 4
figure 4

Maximum-likelihood phylogenetic tree based on five gene (atpD-gyrB-recA-rpoB-trpB) concatenated sequences showing the position of strain 11A07T in the genus Strepomyces. Filled circles indicate branches that were also recovered using the maximum parsimony and neighbor-joining trees. Bootstrap values (expressed as percentages of 1000 replications) of above 50% are shown at the branch points. Bar, 0.02 sequence divergence.

Moreover, strain 11A07T could utilize raffinose as sole carbon source and produce brown diffusible pigments on ISP 2 medium, but could not hydrolyze gelatin. All of these properties could distinguish the strain 11A07T from each of the three most closely related species. Other differences in phenotypic characteristics, which were summarized in Table 3, support strain 11A07T as a novel species. In view of the combination of morphological, physiological and phylogenetic data discussed above, it is evident that strain 11A07T forms a novel taxon within the genus Streptomyces. Therefore, it is proposed that this organism should be recognized as a novel species of the genus Streptomyces, for which the name Streptomyces bohaiensis sp. nov. is proposed.

Description of S. bohaiensis sp. nov.

S. bohaiensis [bo.hai.en’sis. N.L. masc. adj. bohaiensis of or pertaining to Bohai Sea, where the type strain was isolated]: Gram staining positive and aerobic actinomycete that forms extensively branched substrate mycelia and aerial hyphae that carry straight or rectiflexibile spore chains containing >50 smooth spores. Grows well on ISP 2, ISP 3, ISP 5, TSA, Czapek solution agar and nutrient agar. The color of the aerial mycelium is white and the substrate mycelium has different colors on different media. Brown diffusible pigments are produced on ISP 2 and Czapek solution agar. Growth occurs in the presence of 11% NaCl (w/v), temperature 15–40 °C and pH value 6–10, optimum at 1% NaCl, 28 °C, pH 8–9, respectively. Glucose, inositol, raffinose, l-rhamnose, d-galactose, d-mannitol, salicin, sorbitol, sucrose and sodium citrate are utilized as sole carbon source, but not l-arabinose, d-fructose and d-xylose. It can utilize l-valine, l-lysine, l-phenylalanine, l-histidine and l-alanine as sole nitrogen, but not l-methionine and glycine. Test for nitrate reductase is positive. Amylase and catalase activities are produced, but oxidase is not. H2S production, gelatin liquefaction, milk coagulation and peptonization are negative. Tweens 40, 60 and 80 are hydrolyzed, but Tween 20 is not. Cell wall contains LL-diaminopimelic acid. Mannose, ribose, glucose, galactose and xylose are whole-cell sugars. The predominant menaquinones are MK-9 (H2), MK-9 (H4) and MK-9 (H6). The fatty-acid profile contains iso-C16:0, C18:0 10-methyl, tuberculostearic acid and anteiso-C17:0 as the major components. The polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unknown phospholipid. The G+C content of the genomic DNA is 71.4 mol%.

The type strain, 11A07T (=JCM 19630T=CCTCC AA 2013020T=KCTC 29263T−), was isolated from a young fish (S. niphonius) collected from the Bohai Sea of China (39°20′ N, 122°17′ E).