International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2770–2775 DOI 10.1099/ijs.0.047456-0

Streptomyces halophytocola sp. nov., an endophytic actinomycete isolated from the surface-sterilized stems of a coastal halophyte Tamarix chinensis Lour.

Sheng Qin,1 Guang-Kai Bian,1 Tomohiko Tamura,2 Yue-Ji Zhang,1 Wen-Di Zhang,1 Cheng-Liang Cao1 and Ji-Hong Jiang1

Correspondence 1The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, School of Life Sheng Qin Science, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, PR China [email protected] 2NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, 2-5-8 Ji-Hong Jiang Kazusakamatari, Kisarazu, Chiba 292-0818, Japan [email protected]

A novel actinomycete, designated KLBMP 1284T, was isolated from the surface-sterilized stems of a coastal halophyte Tamarix chinensis Lour. collected from the city of Nantong, Jiangsu Province, east China. The strain was found to have morphological and chemotaxonomic characteristics typical of members of the genus Streptomyces. Analysis of the 16S rRNA gene sequence of strain KLBMP 1284T revealed that the strain formed a distinct clade within the phylogenetic tree based on 16S rRNA gene sequences and the highest sequence similarity (99.43 %) was to Streptomyces sulphureus NRRL B-1627T. 16S rRNA gene sequence similarity to other species of the genus Streptomyces was lower than 97 %. Based on DNA–DNA hybridization values and comparison of morphological and phenotypic data, KLBMP 1284T could be distinguished from the closest phylogenetically related species, Streptomyces sulphureus NRRL B-1627T. Thus, based on these data, it is evident that strain KLBMP 1284T represents a novel species of the genus Streptomyces, for which the name Streptomyces halophytocola sp. nov. is proposed. The type strain is KLBMP 1284T (5KCTC 19890T5NBRC 108770T).

The genus Streptomyces was proposed by Waksman & eastern China, an endophytic actinomycete strain, KLBMP Henrici (1943) to accommodate actinobacteria that 1284T, was isolated from the halophyte Tamarix chinensis develop branched substrate mycelium and aerial hyphae, Lour. The present polyphasic study was designed to establish present LL-diaminopimedic acid without characteristic the taxonomic status of this strain. sugars in the cell wall (wall chemotype I; Lechevalier & Healthy samples of the halophyte Tamarix chinensis Lechevalier, 1970). At the time of writing, the genus Strep- Lour., collected from Nantong coast (121u 09 38.960 E tomyces contains a large number of described species 32u 349 24.910 N), Jiangsu Province, east of China in and more than 600 have validly published names (http:// October 2010, were used for isolation. In accordance with www.bacterio.cict.fr/s/streptomycesa.html). Endophytic the surface-sterilized method described previously (Qin et Streptomyces have been isolated from many higher plants al., 2009), the actinobacteria strains were isolated from the in recent years and many of them have been demonstrated stems on modified tap water-yeast extract agar [3 % (w/v) to have capacity to produce a vast array of secondary NaCl added to this agar] (TWYE, Crawford et al., 1993) metabolites exhibiting a wide range of biological activity, after a 3-week incubation at 28 uC. Strain KLBMP 1284T such as antibiotic anti-tumour and anti-infection, plant was picked, purified and then maintained on yeast extract- growth promoters and enzymes (Strobel et al., 2004; malt extract agar [International Streptomyces Project Hasegawa et al., 2006; Qin et al., 2011). In an effort to medium 2 (ISP 2); Shirling & Gottlieb, 1966] slants at discover novel actinomycetes from coastal halophytes in 4 uC and as glycerol suspensions (20 %, v/v) at –80 uC. Abbreviation: ISP, International Streptomyces Project. Genomic DNA was extracted from the biomass prepara- The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene tions and PCR amplification and 16S rRNA gene sequen- sequence of strain KLBMP 1284T is JQ819259. cing was performed using procedures described by Li et al. A supplementary table and a supplementary figure are available with the (2007). The PCR product was purified and sequenced online version of this paper. directly by an automated DNA sequencing system (ABI

2770 047456 G 2013 IUMS Printed in Great Britain Streptomyces halophytocola sp. nov.

3730XL). The resultant sequences were first aligned via the algorithms, neighbour-joining (Saitou & Nei, 1987), max- BLAST search program of NCBI (http://blast.ncbi.nlm.nih. imum-likelihood (Felsenstein, 1981) and maximum- gov/). Phylogenetic neighbours were identified and pair- parsimony (Kluge & Farris, 1969). The stability of the wise 16S rRNA gene sequence similarities were calculated clades in the trees was appraised using a bootstrap value with using the EzTaxon-e database (http://eztaxon-e.ezbiocloud. 1000 repeats (Felsenstein, 1985). A distance matrix was net/; Kim et al., 2012). Multiple alignments with sequences generated using Kimura’s two-parameter model (Kimura, of the most closely related taxa and the construction of the 1980). All positions containing gaps and missing data were phylogenetic trees were carried out using MEGA software eliminated from the dataset (complete deletion option). The version 5.0 (Tamura et al., 2011), and three treeing G+C content of the genomic DNA was determined using

Streptomyces lilacinus NBRC 3944T (AB184819) 0.005 Streptomyces coerulescens NBRC 12758T (AB184122) Streptomyces abikoensis NBRC 13860T (AB184537) Streptomyces varsoviensis NRRL B-3589T (DQ026653) 93* Streptomyces blastmyceticus NRRL B-5480T (AY999802) Streptomyces ardus NBRC 13430T (AB184864) 67* Streptomyces cinnamoneus NBRC 12852T (AB184850) Streptomyces hiroshimensis NBRC 3839T (AB184802) Streptomyces lacticiproducens GIMN4.001T (GQ184344) LMG 20074T (AJ781349) 60* Streptomyces morookaense 55 Streptomyces thioluteus LMG 20253T (AJ781360) Streptomyces kasugaensis M338-M1T (AB024441) Streptomyces youssoufiensis X4T (FN421338) 65* Streptomyces yatensis NBRC 101000T (AB249962) ‘Streptomyces bingchenggensis’ BCW-1 (CP002047) Streptomyces sclerotialus DSM 43032T (AJ621608) Streptomyces ascomycinicus DSM 40822T (EU170121) Streptomyces ramulosus NRRL B-2714- T (DQ026662) 79* Streptomyces platensis JCM 4662T (AB045882) 93* Streptomyces fulvissimus NBRC 3717T (AB184787) Streptomyces variegatus LMG 20315T (AJ781371) 75* 99* Streptomyces spectabilis NBRC 13424T (AB184393) DSM 44293T(U94490) 65* Streptomyces thermocarboxydus T 55* Streptomyces albogriseolus NRRL B-1305- (AJ494865) 100* Streptomyces viridodiastaticus NBRC 13106T (AB184317) 100* Streptomyces halophytocola KLBMP 1284T (JQ819259 ) 64* Streptomyces sulphureus NRRL B-1627-1627T (DQ442546) Streptomyces gibsonii NBRC 15415T (AB184663) Streptomyces almquistii NBRC 13015T (AB184258) 100* Streptomyces rangoonensis LMG 20295T (AJ781366) 98* Streptomyces megasporus NBRC 14749T (AB184617) Streptomyces macrosporus NBRC 14748T (AB184616) 99* Streptomyces radiopugnans R97T (DQ912930) GIMN4.003T (GU356598) 85* Streptomyces fenghuangensis 69 Streptomyces nanhaiensis SCSIO 01248T (GQ871748) Streptomyces thermolineatus DSM 41451T (Z68097) Kitasatospora setae KM-6054- T (AB022868)

Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic positions of strain KLBMP 1284T and some related species of the genus Streptomyces. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branch points. Asterisks indicate branches of the trees that were also found using the maximum- parsimony and maximum-likelihood tree-making algorithms. Bar, 0.005 substitutions per nucleotide position. http://ijs.sgmjournals.org 2771 S. Qin and others the method of Mesbah et al. (1989). DNA–DNA hybridiza- DNA–DNA hybridization revealed 46±1.4 % relatedness tions were performed using the fluorometric micro-well between strain KLBMP 1284T and S. sulphureus NRRL B- method (Ezaki et al., 1989; He et al., 2005). 1627T, which is below the commonly accepted threshold value for the phylogenetic definition of different species as The morphology of the spore chain and the spore surface recognized by Wayne et al. (1987). ornamentation of strain KLBMP 1284T were observed by light microscopy (SA3300-PL) and scanning electron Morphological observation of a 21-day culture of strain microscopy (S-3400N; Hitachi) using cultures grown on KLBMP 1284T grown on ISP 2 medium revealed that strain ISP 2 agar for 21 days at 28 uC. Cultural characteristics KLBMP 1284T had the typical characteristics of members were observed on a number of standard media (Table S1, of the genus Streptomyces. The isolate formed a highly available in IJSEM Online) after 2 weeks at 28 uC. Colours branched substrate mycelium and aerial hyphae which were determined by the methods described by Kelly (1964). differentiated into spiral spore chains with smooth spores The growth temperature (4, 10, 15, 20, 28, 37, 45 and (Fig. 2). Growth of the organism was good on ISP 2, ISP 4, 55 uC) and NaCl tolerance [0–15 % (w/v), at intervals of nutrient agar (NA) and potatoe-dextrose agar (PDA) 1%] was determined on ISP 2 agar at 28 uC for 14 days. media, moderate on ISP media 3 and 5, but poor on The pH range for growth (pH 4.0–13.0, at intervals of 0.5 Czapek’s agar. The colour of the aerial mycelium was pH units) was determined using ISP 2 medium broth adjusted with 1 M HCl, 20 % (w/v) Na2CO3 and 1 M u NaOH solution after sterilization, and incubated at 28 C (a) for 14 days. Carbon-source utilization was tested by using ISP 9 medium (Shirling & Gottlieb, 1966) supplemented with 1 % (final concentration) carbon sources. The utilization of amino acids as sole nitrogen sources was tested as described by Williams et al. (1983). Production of acid and other physiological and biochemical character- istics were tested by using the well-established procedures described by Gordon et al. (1974). Biomass for molecular systematic and chemotaxonomic studies was obtained by cultivation in shake flasks on a rotary shaker (150 r.p.m.) using Trypticase Soy Broth at 28 uC for 6 days. The isomer of diaminopimelic acid and whole-cell sugar compositions were analysed using TLC according to the procedures described by Lechevalier & Lechevalier (1980). Polar lipids were examined by two- dimensional TLC and identified using the method of Minnikin et al. (1984). Menaquinones were extracted and purified as described by Collins et al. (1977) and analysed by HPLC (Groth et al., 1997). Fatty acids were analysed (b) using the standard MIDI (Microbial Identification, Sherlock version 6.0) procedure (Sasser, 1990) and the Agilent GC 6850 gas chromatograph. The resulting fatty acid profiles were identified using the database library TSBA6 version 6.0. The almost-complete 16S rRNA gene sequence (1453 bp) of strain KLBMP 1284T was compared with sequences in the GenBank database. The results indicated that the isolate belonged to the genus Streptomyces. Strain KLBMP 1284T showed the highest 16S rRNA gene sequence similarity with Streptomyces sulphureus NRRL B-1627T (99.43 %). The 16S rRNA gene sequence similarity to other species of the genus Streptomyces was less than 97 %. It is evident from the phylogenetic tree (Fig. 1) that strain KLBMP 1284T was clustered to S. sulphureus NRRL B-1627T, and they formed a distinct subclade with a high bootstrap value of 100 % by neighbour-joining analysis. This relationship Fig. 2. Scanning electron micrographs of strain KLBMP 1284T was also found in trees constructed using the maximum- grown on ISP 2 medium for 21 days at 28 6C. Bars, 10 mm (a) and parsimony and maximum-likelihood algorithms. However, 5 mm (b).

2772 International Journal of Systematic and Evolutionary Microbiology 63 Streptomyces halophytocola sp. nov. yellowish to orange–yellow and the substrate mycelium was that strain KLBMP 1284T should be assigned to the genus yellowish-white to orange–yellow. Diffusible pigments were Streptomyces. not produced on any of the media tested. (Table S1). Cultural characteristics of strain KLBMP 1284T clearly Growth was observed at 15–37 uC, pH 6.0–9.0 and in showed that it differed from the most closely related species presence of 0–10 % (w/v) NaCl (optimum, 3 %) on ISP 2 S. sulphureus NRRL B-1627T (Table S1). Moreover, a agar. Detailed physiological characteristics are presented in combination of physiological and biochemical character- the species description and in Table 1. istics enabled strain KLBMP 1284T to be distinguished from T Strain KLBMP 1284 contained LL-diaminopimelic acid as its closest phylogenetic neighbour (Table 1). In addition, the the diamino acid. Whole-cell hydrolysates comprised phospholipid profile and the major fatty acid contents of arabinose, galactose and glucose. The major menaquinones strain KLBMP 1284T are clearly different from S. sulphureus T were MK-9(H6) (67 %), MK-9(H8) (26 %) and MK-9(H4) NRRL B-1627 (Table 1-2). Together with the low DNA– T (7 %). The major cellular fatty acids were anteiso-C15 : 0 DNA relatedness value, strain KLBMP 1284 represents a (30.36 %), iso-C16 : 0 (16.52 %), C16 : 0 (10.65 %) and anteiso- novel species of the genus Streptomyces, for which the name C17 : 0 (9.35 %).. A detailed fatty acid profile comparison of Streptomyces halophytocola sp. nov. is proposed. strain KLBMP 1284T with its nearest neighbour species, S. sulphureus NRRL B-1627T, is given in Table 2. The T Description of Streptomyces halophytocola sp. phospholipid profile of strain KLBMP 1284 consisted of nov. diphosphatidylglycerol, phosphatidylglycerol, phosphatidy- linositol, phosphatidylinositol mannosides, phosphatidy- Streptomyces halophytocola (ha.lo.phy.to9co.la. Gr. n. hal, lethanolamine, an unknown aminophospholipid and several halos salt; Gr. n. phyton a plant; L. suff. -cola inhabitant, unknown glycolipids (Fig. S1). The G+C content of the dweller; N.L. n. halophytocola inhabitant of a halophyte, DNA was 71.6 mol%. These chemotaxonomic data showed Tamarix chinensis Lour.).

Table 1. Different characteristics of strain KLBMP 1284T and its closely related neighbour Streptomyces sulphureus NRRL B-1627T

All data were obtained during this study under identical growth conditions. Both strains were positive for the assimilation of cellobiose, D-fructose, D-galactose, maltose, D-mannose, D-ribose, D-xylose, L-alanine, L-glycine and L-proline. Both strains degraded Tweens 20 and 40 and grew with 10 % NaCl. +, Positive or present; W, weakly positive; 2, negative or absent.

Characteristic KLBMP 1284T S. sulphureus NRRL B-1627T

Aerial spore mass colour on TSA and NA media Orange–yellow Grey–white Spore-chain morphology Flexuous to spiral Spiral Growth on Czapek’s medium Poor Moderate Hydrolysis of: Casein + 2 Starch 2 + Tween 80 + 2

H2S production 2 + Growth at 15 uC W 2 Growth at pH 10.0 2 + Assimilation of sole carbon sources Erythritol + 2 D-Glucose + 2 Raffinose 2 + D-Rhamnose + 2 Trehalose – + Acid produced from: Cellobiose 2 W Raffinose 2 + Assimilation of sole nitrogen sources L-Arginine + 2 L-Histidine 2 + Polar lipids* DPG, PG, PE, PIM, PI, APL, 3GL DPG, PE, 2GL

*Polar lipids: DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; PE, phosphatidylethanolamine; APL, unknown aminophospholipid; GL, unknown glycolipid. http://ijs.sgmjournals.org 2773 S. Qin and others

Table 2. Fatty acid content (%) of strain KLBMP 1284T and its phosphatidylinositol, phosphatidylinositol mannosides, closely related neighbour Streptomyces sulphureus NRRL phosphatidylethanolamine, an unknown aminophospholipid B-1627T and several unknown glycolipids. T T All data are from this study. Values are percentages of total fatty acids; The type strain KLBMP 1284 (5KCTC 19890 5NBRC T fatty acids amounting to less than 0.5 % in both species are not 108770 ) was isolated from surface-sterilized stems of a shown. 2, Not detected. coastal halophyte Tamarix chinensis Lour. collected from the city of Nantong, Jiangsu Province, east China. The Fatty acid KLBMP 1284T S. sulphureus DNA G+C content of the type strain is 71.6 mol%. NRRL B-1627T

C14 : 0 0.93 1.23 Acknowledgements C16 : 0 10.65 8.67 C 0.78 0.90 This research was partially supported by National Natural Science 17 : 0 Foundation of China (31000005, 31101502, 31100009), the Program C 2.19 2 18 : 0 of Natural Science Foundation of the Jiangsu Higher Education C v5c 2 1.16 16 : 1 Institutions of China (10 kJB180008, 11 kJD210002), the project C17 : 1v8c 22 funded by the Priority Academic Program Development of Jiangsu C18 : 1v9c 1.86 2 Higher Education Institutions (PAPD) and Natural Science iso-C14 : 0 2 6.45 Foundation by Xuzhou City (No. XZZD1004). iso-C15 : 0 7.14 16.41 anteiso-C15 : 0 30.36 24.55 anteiso-C15 : 0 A 2.58 2 References iso-C16 : 0 16.52 21.03 Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). iso-C16 : 1 H 2 0.55 iso-C 2.05 3.25 Distribution of menaquinones in actinomycetes and corynebacteria. 17 : 0 J Gen Microbiol 100, 221–230. cyclo C17 : 0 2.45 3.95 Crawford, D. L., Lynch, J. M., Whipps, J. M. & Ousley, M. A. (1993). anteiso-C17 : 0 9.35 5.39 anteiso-C v9c 0.65 0.53 Isolation and characterization of actinomycete antagonists of a fungal 17 : 1 root pathogen. Appl Environ Microbiol 59, 3899–3905. Summed features* 1 6.57 2 Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric 3 1.43 2.01 deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro- dilution wells as an alternative to membrane filter hybridization in 5 2.35 2 which radioisotopes are used to determine genetic relatedness among 8 2 0.55 bacterial strains. Int J Syst Bacteriol 39, 224–229. 9 0.88 2 Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376. *Summed features represent groups of two or three fatty acids that Felsenstein, J. (1985). Confidence limits on phylogenies: an approach cannot be separated by GC with the MIDI system. Summed feature using the bootstrap. Evolution 39, 783–791. 1comprised C15 : 1 iso H/C13 : 0 3-OH; summed feature 3 comprised C v7c /C v6c; summed feature 5 comprised C v6, 9c /C Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. 16 : 1 16 : 1 18 : 2 18 : 0 (1974). Nocardia coeliaca, Nocardia autotrophica, and the nocardin ante; summed feature 8 comprised C v7c /C v6c; summed 18 : 1 18 : 1 strain. Int J Syst Bacteriol 24, 54–63. feature 9 comprised C17 : 1 iso v9c /C16 : 0 10-methyl. Groth, I., Schumann, P., Rainey, F. A., Martin, K., Schuetze, B. & Augsten, K. (1997). Demetria terragena gen. nov., sp. nov., a new genus of actinomycetes isolated from compost soil. Int J Syst Bacteriol Aerobic, Gram-stain-positive actinomycete that develops 47, 1129–1133. well-branched yellowish-white to orange–yellow substrate Hasegawa, S., Meguro, A., Shimizu, M., Nishimura, T. & Kunoh, H. mycelium and yellowish to orange–yellow aerial mycelium. (2006). Endophytic actinomycetes and their interactions with host Produces long spiral spore chains with smooth spores. No plants. Actinomycetologica 20, 72–81. diffusible pigment is formed on any of the tested media. He, L., Li, W., Huang, Y., Wang, L., Liu, Z., Lanoot, B., Vancanneyt, M. Growth occurs at 15–37 uC (optimum, 28 uC). Optimal & Swings, J. (2005). Streptomyces jietaisiensis sp. nov., isolated from growth occurs at pH 7.0–7.5. NaCl tolerance on ISP 2 agar soil in northern China. Int J Syst Evol Microbiol 55, 1939–1944. medium is 10 % (w/v) (optimum, 3 %). No reduction of Kelly, K. L. (1964). Inter-Society Color Council-National Bureau of nitrate to nitrite is observed. Degrades casein and Tweens 20, standards color-name charts illustrated with centroid colors published in 40 and 80, but not starch, cellulose or gelatin. Most sugars US. Washington, DC: US Government Printing Office. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., can be utilized as sole carbon sources for growth, but not D- Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: arabinose, inositol, lactose, raffinose, D-sorbitol or trehalose. a prokaryotic 16S rRNA gene sequence database with phylotypes Acid is produced on D-galactose, D-glucose, maltose and that represent uncultured species. Int J Syst Evol Microbiol 62, xylose. The major cellular fatty acids are anteiso-C15 : 0,iso- 716–721. C16 : 0,C16 : 0 and anteiso-C17 : 0.Themajormenaquinonesare Kimura, M. (1980). A simple method for estimating evolutionary rates MK-9(H6), MK-9(H8)andMK-9(H4). The polar lipid pro- of base substitutions through comparative studies of nucleotide file consists of diphosphatidylglycerol, phosphatidylglycerol, sequences. J Mol Evol 16, 111–120.

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