DSpace at VNU: Streptomyces catbensis sp nov., isolated from soil

IJSEM Papers in Press Published April 3, 2014 as doi:10.1099/ijs.0.048264-0 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Streptomyces catbensis sp nov isolated in Vietnam Yayoi Sakiyama1, Nguyen M Giang 2, Shinji Miyadoh1, Dao Thi Luong2, Duong Van Hop2, and Katsuhiko Ando1 NITE-Biological Resource Center, National Institute of Technology and Evaluation (NBRC-NITE), Chiba, Japan Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, Vietnam (IMBT-VNUH) Corresponding author: Yayoi Sakiyama NITE-Biological Resource Center, National Institute of Technology and Evaluation (NBRCNITE), 2-5-8 Kazusakamatarti, Kisarazu, Chiba, 292-0818, Japan Tel: +81-438-20-5763 Fax: +81-438-52-2329 E-mail: sakiyama-yayoi@nite.go.jp Running head: Streptomyces catbensis sp nov Keywords: Streptomyces, new species, Vietnam, actinomycetes DDBJ accession number for the 16S rRNA gene sequence of Streptomyces catbensis VN07A0015T (= VTCC-A-1889T = NBRC 107860T) is AB705486 Abstract Strain VN07A0015T was isolated from soil collected on Cat Ba Island, Vietnam The taxonomic position of strain VN07A0015T was near Streptomyces aomiensis (98.5% similarity) and Streptomyces scabrisporus (95.6%), and it clustered within them; however, this cluster was distant from the type strains of other Streptomyces species The aerial mycelia of strain VN07A0015T were greyish and formed imperfect spiral spore chains (retinaculiaperti type) with smooth-surfaced spores The morphological features of strain VN07A0015T were different from those of S aomiensis and S scabrisporus The chemotaxonomic characteristics of strain VN07A0015T were typical for all members of the genus Streptomyces, which possessed LL-type diaminopimelic acid, menaquinone MK-9(H6, H8) and the major fatty acids iso-C16:0 and iso-C15:0 The result of DNA relatedness between strain VN07A0015T and S aomiensis NBRC 106164T was less than 30% In addition, some physiological and biochemical traits differed from those of S aomiensis Therefore, we propose that strain VN07A0015T be classified in the genus Streptomyces as a representative of Streptomyces catbensis sp nov (Type strain VN07A0015T = VTCC-A-1889T = NBRC 107860T) 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 Strains of genus Streptomyces produce a wide variety of antibiotics and enzymes (Goodfellow et al., 2010) It is easy to screen for strains of this genus because of their good growth and ease of isolation from nature Consequently, many novel species have been proposed, and the genus Streptomyces contains about 600 validly published species at the time of writing this manuscript (List of Prokaryotic Names with Standing in Nomenclature; http://www.bacterio.cict.fr/) The genus Streptomyces also has distinctive morphological traits Cultures produce colourful substrate mycelia, aerial mycelia, and soluble pigments Sporulating aerial hyphae typically have a fibrous sheath (Wildermuth & Hopwood, 1970; Hardisson & Manzanal, 1976) Moreover, aerial mycelia have variously shaped spore chains (including short-long types, straight, rectiflexibiles, hook, loop, rectinaculiaperti, and spiral types) and spore surfaces (including smooth, warty, rugose, spiny, and hairy) (Pridham et al., 1958) Shirling & Gottlieb (1966) described the methods using the International Streptomyces Project (ISP) and introduced the above morphological characterizations This project included ISP media No 2–7, which are basic items for the observation of Streptomyces Because morphology remains important in taxonomical classification of Streptomyces species, in this study, we identify and propose strain VN07A0015T as a novel species in the genus Streptomyces Strain VN07A0015T was isolated from soil collected on Cat Ba Island in Vietnam The soil sample was dried at room temperature for 3–5 days, and used by the sodium dodecyl sulphate-yeast extract dilution method (Hayakawa & Nonomura et al., 1989) on a humic acid-vitamin medium (Hayakawa & Nonomura, 1987) containing nalidixic acid (20 mg l-1) and kabicidine (7.5 mg l-1) Strain VN07A0015T was isolated from the humic acid-vitamin medium after incubation at room temperature for approximately 10 days When strain VN07A0015T was cultured on ISP No medium at 28°C (Shirling & Gottlieb, 1966), the greyish aerial mycelia, the imperfect spiral spore chains (retinaculiaperti type), and the smooth surface spores were observed with light microscopy and scanning electron microscopy (Fig 1) The cultural characteristics of strain VN07A0015T were observed on ISP No 2–7 media (Table 1) The investigation of optimum temperature (5, 10, 15, 20, 25, 28, and 37°C), pH (3, 4, 5, 6, 7, 8, and 9), and NaCl concentration (1%, 2%, 3%, 4%, and 5%) were observed on a yeast extractsoluble starch medium (YS medium; g yeast-extract, 10 g soluble starch and 15 g agar per litre of distilled water; pH 7.3) Good growth of strain VN07A0015T was observed on ISP No medium; moderate growth on ISP No 3, 5, 6, and media; and no growth on ISP No medium Strain VN07A0015T formed greyish aerial mycelia on ISP No 2, 3, 5, and media and did not produce a soluble pigment Strain VN07A0015 T grew at temperatures of 15°C, 20°C, 25°C, and 28°C and at pH levels of 5, 6, 7, 8, and The optimal temperature was 28°C and the optimal pH was 6–7 Strain VN07A0015T grew on YS media with a NaCl concentration of 0–2% The biomass of VN07A0015T was cultured in a yeast extract-glucose medium (10 g yeast-extract, 10 g glucose per litre of distilled water; pH 7.3) for 3–5 days at 28°C, harvested by centrifugation, rinsed by sterilized water, and then lyophilized The types of diaminopimelic acids and whole-cell sugars were analysed as described by Staneck & Roberts (1974), isoprenoid quinines and phospholipids by Minnikin et al (1984), 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 cellular fatty acids by Sasser (1990) and by using the MIDI Sherlock Microbial Identification system (Microbial ID), respectively Strain VN07A0015T possessed LLdiaminopimelic acid, arabinose, glucose, rhamnose and ribose in whole cell The major isoprenoid quinones were MK-9(H6) and MK-9(H8), and the minor isoprenoid quinone was MK9-(H4) Phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), six unknown lipids and one ninhydrin-positive unknown lipid were detected (Fig 2), but not phosphatidylcholine (PC) and phosphatidylglycerol (PG) The predominant cellular fatty acids were iso-C16:0 (28.0%) and iso-C15:0 (22.2%) based on MIDI system ver 4.02 (Table 2) The major fatty acids composition of the strain VN07A0015T and Streptomyces aomiensis NBRC 106164T were branched type and pattern 2a, however Streptomyces scabrisporus NBRC 100760T were non-branched type and pattern 2c These aspects of the morphology and chemotaxonomy of strain VN07A0015T conformed to the characteristics of the genus Streptomyces DNA of strain VN07A0015T was extracted, amplified, and its 16S rRNA gene was sequenced according to the techniques of Sakiyama et al (2009) Sequence analysis was carried out with an ABI Prism BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems) and an automatic DNA sequencer (model 3130 Genetic Analyzer; PE Applied Biosystems) The 16S rRNA gene sequences were aligned by the CLUSTAL X program (Thompson et al., 1997) with corresponding sequences from some type strains of Streptomyces species available in the DDBJ/EMBL/GenBank database Subsequently, phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987), maximum-likelihood method (Felsenstein, 1981), and maximumparsimony method (Kluge & Farris, 1969) using MEGA (Tamura et al., 2011) The topology of the constructed tree was evaluated by bootstrap analysis with 1000 replicates (Felsenstein, 1985) DNA–DNA hybridization and G+C contents of the DNA were examined by the method described previously (Sakiyama et al., 2009) In the phylogenetic analysis based on the 16S rRNA gene, strain VN07A0015T was most similar to S aomiensis (98.5% similarity) and S scabrisporus (95.6%) Strain VN07A0015T was clustered with them in a phylogenetic tree (Fig 3), and the taxonomic position of this cluster was distant from that of the other type strains of the genus Streptomyces The DNA relatedness between strain VN07A0015T and S aomiensis NBRC 106164T were 29.5% (probe VN07A0015T) and 8.4% (probe S aomiensis NBRC 106164T) The values are well below the 70% cut-off point recommended for the delineation of genomic species (Wayne et al., 1987) Therefore, strain VN07A0015T was different from S aomiensis The G+C content of the DNA was 73.2% In biochemical and physiological tests, the utilization of different carbon sources and enzyme reaction by strain VN07A0015T, S aomiensis NBRC 106164T and S scabrisporus NBRC 100760T was examined using methods described previously (Sakiyama et al., 2009) and API Zym (REF 25 207, BioMérieux Inc.) and API Coryne (REF 20 907) The API examines of the strain VN07A0015T and S aomiensis NBRC 106164T were almost same results, but the results of S scabrisporus NBRC 100760T was fairly different with them (Table 3) Strain VN07A0015T showed some differences from S aomiensis NBRC 106164T in the utilization of meso-erythritol, D-raffinose, and 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 D-xylose Therefore, we propose that strain VN07A0015T is a novel species in the genus Streptomyces, which is named Streptomyces catbensis sp nov The type strain is VN07A0015T (= VTCC-A-1889T = NBRC 107860T) Description of Streptomyces catbensis sp nov Streptomyces catbensis (cat.ben’en.sis N.L masc adj., catbensis of or belonging to Cat Ba Island in Vietnam, from which the strain was isolated) The substrate and aerial mycelia are developed on ISP No medium The colour of the substrate mycelium and aerial mycelium are strong brown and grey, respectively, on ISP No medium It grows moderately on ISP Nos 3, 5, 6, and but not Aerial mycelia are found on ISP Nos 2, 3, 5, and Spore chains are imperfect spiral (retinaculiaperti type), and the spore surface is smooth It grows at 15–28°C (optimum 25–28°C) and pH 5–9 (optimum 6–8) It does not hydrolyse starch, liquefy gelatin, and coagulate milk Urease activity is negative, and nitrate reduction is positive Decomposition occurs on tyrosine but not on hypoxanthine (0.4%), adenine, and xanthine As sole carbon sources, L-arabinose, D-cellobiose, D-fructose, D-glucose, lactose, maltose, D-mannitol, melibiose, L-rhamnose, ribose, D-salicin, D-sorbitol, sucrose, and D-trehalose are utilized, but meso-erythritol, myo-inositol, D-raffinose, and D-xylose are not Menaquinones are MK-9(H6, H8), and the major fatty acids are iso-C16:0 and iso-C15:0 (pattern 2a) Phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), six unknown lipids and one ninhydrin-positive unknown lipid were detected (pattern PⅡ) The G+C contents of the DNA of the type strain are 73.2% Strain VN07A0015T (= VTCC-A-1889T = NBRC 107860T) was isolated from soil collected on Cat Ba Island in Vietnam Acknowledgements This work was conducted as a joint research project between NBRC-NITE and IMBTVNUH The authors thank Dr Tomohiko Tamura, Ms Kozue Anzai, Mr Nobuyuki Goto, Mr Shinpei Ino, Ms Ayako Hashimoto, Ms Mayuko Sukisaki (NBRC), and Ms Nguyen Thi Van (Vietnam type culture collection) for experimental assistance We are grateful to Dr Dinh Thuy Hang, Dr Nguyen Lan Dung, and all VTCC members for supporting our joint project References Felsenstein, J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach J Mol Evol 17, 368–376 Felsenstein, J (1985) Confidence limits on phylogenies: an approach using the bootstrap Evolution 39, 783–791 Goodfellow, M & Fiedler, H.P (2010) A guide to success bioprospecting: informed by actinobacterial systematic Antonie van Leeuwenhoek 98, 119–142 Hardisson, C & Manzanal, M B (1976) Ultrastructural studies of sporulation in Streptomyces J Bacteriol 127, 1443–1454 Hayakawa, M & Nonomura, H (1989) A new method for the intensive isolation of Actinomycetes from soil Actinomycetologica 3, 95-104 Hayakawa, M & Nonomura, H (1987) Humic acid-vitamin agar, a new medium for 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 selective isolation of soil actinomycetes J Ferment Technol 65, 501–509 Kluge, A G & Farris, F S (1969) Quantitative phyletics and the evolution of anurans Syst Zool 18, 1–32 Minnikin, D E., O’Donnell, A G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A & Parlett, J H (1984) An integrated producer for the extraction of bacterial isoprenoid quinones and polar lipids J Microbiol Methods 2, 233–241 Ping, X., Takahashi, Y., Seino, A., Iwai, Y & Ōmura, S (2004) Streptomyces scabrisporus sp nov Int J Syst Evol Microbiol 54, 577–581 Pridham, T G., Hesseltine, C W & Benedict, R G (1958) A Guide for the Classification of Streptomycetes According to Selected Groups Placement of Strains in Morphological Sections Appl Microbiol (1), 52 Saitou, N & Nei, M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees Mol Biol Evol 4, 406–425 Sakiyama, Y., Nguyen K N T., Nguyen M G., Miyadoh, S., Duong V H & Ando, K (2009) Kineosporia babensis sp nov., isolated from plant litter in Vietnam Int J Syst Evol Microbiol 59, 550–554 Sasser, M (1990) Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101 Newark, DE: MIDI Inc Shirling, E B & Gottlieb, D (1966) Methods for characterization of Streptomyces species Int J Syst Bacteriol 16, 313–340 Staneck, J L & Roberts, G D (1974) Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography Appl Microbiol 28, 226–231 Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M & Kumar, S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods Mol Biol Evol 28, 2731– 2739 Thompson, J D., Gibson, T J., Plewniak, F., Jeanmougin, F & Higgins, D G (1997) The CLUSTAL X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 25, 4876–4882 Wayne, L G., Brenner, D J., Colwell, R R., Grimont, P A D., Kandler, O., Krichevsky, M I., Moore, W E C., Murray, R G E & other authors (1987) International Committee on Systematic Bacteriology Report of the ad hoc committee on reconciliation of approaches to bacterial systematics Int J Syst Bacteriol 37, 463-464 Wildermuth, H & Hopwood, D A (1970) Septation during sporulation in Streptomyces coelicolor J gen Microbiol 60, 51–59 225 226 227 228 229 Table Culture characteristics of S catbensis VN07A0015T, S aomiensis NBRC 106164T, and S scabrisporus NBRC 100760T The described data were placed in the order: growth, colour of aerial mycelium, and colour of substrate mycelium 230 Characteristics Spore chains Spore surface ISP medium Yeast extract/malt extract agar (ISP 2) Oatmeal agar (ISP 3) Inorganic salts/starch agar (ISP 4) Glycerol/asparagine agar (ISP 5) Peptone/yeast extract/iron agar (ISP 6) Tyrosine agar (ISP 7) 231 S catbensis VN07A0015T Retinaculiaperti Smooth S aomiensis NBRC 106164T Rectiflexibles Smooth S scabrisporus NBRC 100760T Spiral Rugose Good Light bluish grey Strong brown Moderate Light brownish grey Colourless Weak Light brownish gray Colourless Moderate Yellowish grey Colourless Moderate, Wet Moderate yellow Moderate Yellowish grey Pale yellow Good White Vivid yellow Moderate White Light yellow Moderate White Colourless Moderate White Colourless Good, Wet White Moderate yellow Moderate White Pale yellow Moderate, Wrinkled Light ivory Weak, Penetrating Colourless Weak, Penetrating Pearl Moderate, Penetrating Pearl Good, Wrinkled,Wet Pearl Moderate, Raised Bamboo 232 233 234 235 236 Table Cellular fatty acid composition (%) of S catbensis VN07A0015T, S aomiensis NBRC 106164T and S scabrisporus NBRC 100760T Values less than 1% were omitted from this table S catbensis VN07A0015T S aomiensis NBRC 106164T S scabrisporus NBRC 100760T 11.0 22.2 4.1 12.4 28.0 9.0 19.0 16.0 4.9 25.6 6.3 5.2 6.3 14.8 13.1 4.3 6.1 7.4 24.9 18.7 4.9 5.8 3.7 5.8 Branched type iso-C14:0 iso-C15:0 anteiso-C15:0 iso-C16:1 H iso-C16:0 anteiso-C17:0 Non-branched type C16:1 cis C16:0 Methyl type C16:0 9?Methyl Sum In Feature 237 238 239 240 241 242 243 244 245 246 247 10.9 6.1 Table Biochemical and physiological characteristics of S catbensis sp nov VN07A0015T, S aomiensis NBRC 106164T, and S scabrisporus NBRC 100760T Characteristics S catbensis VN07A0015T S aomiensis NBRC 106164T S scabrisporus NBRC 100760T ＋ - ＋ - + - - + + + + - + + API ZYM Esterase C4 α-cymotrypsin α-glucosidase α-fucosidase API Coryne Pyrrolidonyl arylamidase Pyrrolidonyl arylamidase α-glucosidase Utilization of D-xylose D-raffinose + + + inositol 248 249 250 + - - 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 Figure legends Figure Scanning electron micrograph of strain VN07A0015T grown on humic acid-vitamin medium for weeks at 28°C Bar is 10 µm Figure Phospholipids analysis of S catbensis sp nov VN07A0015T The detected regents are Molybdatophosphoric acid (plate 1), Dittmer-Lester (plate 2), Schiff (plate 3), and Anisaldehyde (plate 4) Phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), unknown lipid (PL), and ninhydrin-positive unknown lipid (NL) are abbreviated Figure Phylogenetic tree of strain VN07A0015T with related type strains of Streptomyces species based on 16S rRNA gene sequences The tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) Scale bar = 0.005 Knuc in nucleotide sequences The numbers on the branches are the confidence limits estimated by bootstrap analysis with 1,000 replicates (only values above 500 are presented) A closed circle indicates a branch that was identical in both maximum-likelihood method and maximum-parsimony method analyses; open circles indicate maximum-likelihood method analysis only 275 0.005 Knuc Streptomyces cacaoi subsp cacaoi NBRC 12748T (AB184115) Streptomyces sodiiphilus YIM 80305T (AY236339) Streptomyces gibsonii NBRC 15415T (AB184663) 928 577 Streptomyces rangoonensis LMG 20295T (AJ781366) Streptomyces almquistii NBRC 13015T (AB184258) 999 576 Streptomyces albus subsp albus NRRL B-2365T (DQ026669) Streptomyces flocculus NBRC 13041T (AB184272) Streptomyces rimosus subsp rimosus JCM 4667T (AB045883) 727 1000 Streptomyces chrestomyceticus DSM 40545T (AJ621609) Streptomyces rimosus subsp paromomycinus DSM 41429T (AJ621610) 616 Streptomyces catenulae ISP 5258T (AY999778) Streptomyces mashuensis DSM 40221T (X79323) 994 1000 613 707 Streptomyces sparsogenes NBRC 13086T (AB184301) Streptomyces cuspidosporus NBRC 12378T (AB184090) Streptomyces hiroshimensis NBRC 3839T (AB184802) Streptomyces iranensis HM 35T (FJ472862) Streptomyces mobaraensis NBRC 13819T (AB184870) Streptomyces xinghaiensis CCTCC AA 208049T (EF577247) Streptomyces scabrisporus NBRC 100760T (AB249946) 965 Streptomyces aomiensis M24DS4T (AB522686) 1000 VN07A0015T (AB705486) 276 Nocardioides albus KCTC9186T (AF004988) ... Streptomyces catbensis sp nov Streptomyces catbensis (cat.ben’en.sis N.L masc adj., catbensis of or belonging to Cat Ba Island in Vietnam, from which the strain was isolated) The substrate and aerial... classification of Streptomyces species, in this study, we identify and propose strain VN07A0015T as a novel species in the genus Streptomyces Strain VN07A0015T was isolated from soil collected on Cat... extract agar (ISP 2) Oatmeal agar (ISP 3) Inorganic salts/starch agar (ISP 4) Glycerol/asparagine agar (ISP 5) Peptone/yeast extract/iron agar (ISP 6) Tyrosine agar (ISP 7) 231 S catbensis VN07A0015T