International Journal of Systematic and Evolutionary Microbiology (2014), 64, 833–838
DOI 10.1099/ijs.0.057349-0
Agromyces iriomotensis sp. nov. and Agromyces subtropicus sp. nov., isolated from soil Moriyuki Hamada,1 Chiyo Shibata,1 Yuumi Ishida,1 Tomohiko Tamura,1 Hideki Yamamura,2 Masayuki Hayakawa2 and Ken-ichiro Suzuki1 1
Correspondence
Biological Resource Center, National Institute of Technology and Evaluation (NBRC), 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan
Moriyuki Hamada [email protected]
2
Division of Applied Biological Sciences, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Takeda-4, Kofu, Yamanashi 400-8511, Japan
Three novel Gram-stain-positive bacteria, designated IY07-20T, IY07-56T and IY07-113, were isolated from soil samples from Iriomote Island, Okinawa, Japan, and their taxonomic positions were investigated by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the three isolates were closely related to the members of the genus Agromyces, with similarity range of 95.6–98.7 %. The isolates contained L-2,4diaminobutylic acid, D-alanine, D-glutamic acid and glycine in their peptidoglycans. The predominant menaquinone was MK-12 and the major fatty acids were anteiso-C15 : 0 and anteisoC17 : 0. The DNA G+C contents were 70.9–72.9 mol%. The chemotaxonomic characteristics of the isolates matched those described for members of the genus Agromyces. The results of phylogenetic analysis and DNA–DNA hybridization, along with differences in phenotypic characteristics between strains IY07-20T, IY07-56T and IY07-113 and the species of the genus Agromyces with validly published names, indicate that the three isolates merit classification as representatives of two novel species of the genus Agromyces, for which the names Agromyces iriomotensis sp. nov. and Agromyces subtropica sp. nov. are proposed; the type strains are IY0720T (5NBRC 106452T5DSM 26155T) and IY07-56T (5NBRC 106454T5DSM 26153T), respectively.
The genus Agromyces proposed by Gledhill & Casida (1969) and the description of which was emended by Zgurskaya et al. (1992) currently contains 25 recognized species. This genus comprises Gram-stain-positive, nonmotile, non-spore-forming, filamentous and non-filamentous, microaerophilic to aerobic actinobacteria (Akimov & Evtushenko, 2012). The members of the genus have 2,4diaminobutyric acid (A2bu) in their peptidoglycan and MK-12 as the predominant menaquinone with smaller amounts of MK-11 and/or MK-13. Major fatty acids are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Most of the species have been isolated from soil. During the course of a study of bacterial diversity in cool-temperate and subtropical areas, three novel actinobacteria were isolated from soil samples collected from a subtropical island in Japan. Comparative 16S rRNA gene sequence analysis revealed Abbreviation: A2bu, 2,4-diaminobutyric acid. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains IY07-20T, IY07-56T and IY07-113 are AB546308, AB546310 and AB546311, respectively. A supplementary figure and a supplementary table are available with the online version of this paper.
057349 G 2014 IUMS
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that the isolates are phylogenetically related to members of the genus Agromyces. The objective of this study was to determine the taxonomic positions of the isolates by using a polyphasic approach. Strains IY07-20T, IY07-56T and IY07-113 were isolated from pineapple field soil samples (IY07-20T and IY07-56T) and a forest soil sample (IY07-113) collected on Iriomote Island, Okinawa, Japan, using the SDS/yeast extract pretreatment method (Hayakawa & Nonomura, 1989) and humic acid–vitamin (HV) agar (Hayakawa & Nonomura, 1987). NBRC medium 802 (1.0 % polypeptone, 0.2 % yeast extract, 0.1 % MgSO4 . 7H2O and 1.5 % agar if required; pH 7.0) was used as basal medium for this study. Biomass for chemotaxonomic and molecular systematic studies, except for cellular fatty acids analysis, was obtained by cultivating the strains in shake flasks for 48 h at 28 uC with shaking at 100 r.p.m. Biomass grown on trypticase soy agar (Difco) for 24 h at 28 uC was used for cellular fatty acids analysis. Agromyces flavus NBRC 109064T and Agromyces humatus NBRC 109085T were used as reference strains for comparison with the physiological, biochemical and the DNA–DNA hybridization tests. 833
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Colony appearance was examined after incubation at 28 uC for 3 days in the dark. Morphological features were observed for up to 7 days of incubation under a light microscope (BX-51; Olympus) and a scanning electron microscope (JSM-6060; JEOL). Cell motility, growth parameters (temperature, pH and NaCl tolerance), Gram staining phenotype and oxidase activity were determined using the methods described by Hamada et al. (2010). Growth under anaerobic and microaerobic conditions was determined using AnaeroPack-Anaero and AnaeroPackMicroAero (Mitsubishi Gas Chemical) with anaerobic jars, respectively. Catalase activity was determined by production of bubbles after the addition of a drop of 3.0 % H2O2. Other physiological and biochemical tests were performed using API ZYM, API Coryne and API 50 CH systems (bioMe´rieux) according to the manufacturer’s instructions. API 50 CHB/E medium was used to prepare cell suspensions for the API 50 CH test. Strain IY07-20T formed cream–yellow, circular, transparent and smooth colonies that were approximately 0.2– 0.5 mm in diameter after 3 days of cultivation, while strains IY07-56T and IY07-113 formed pale yellow, circular, transparent and smooth colonies that were approximately 0.5–1.0 mm in diameter after 3 days of cultivation. All strains developed branched vegetative hyphae (width 0.4– 0.6 mm) in the early phase of growth, but these hyphae subsequently fragmented into rod-like to coccoid fragments. The cells were Gram-stain-positive, microaerophilic to aerobic, non-motile and non-endospore-forming. Strain IY07-20T grew at 20–37 uC (optimum 25 uC) while strains IY07-56T and IY07-113 grew at 10–37 uC (optimum 28 uC). The pH range for growth of all strains was 6.0– 10.0 (optimum pH 7.0–8.0). All strains exhibited growth with NaCl concentrations of 0–3 %; no growth was observed with 5 % NaCl (w/v) or higher. No growth was observed under anaerobic conditions. The results of other physiological and biochemical analyses are summarized in the species descriptions below.
represented members of the genus Agromyces. However, strain IY07-20T did not form a reliable cluster with any member of the genus Agromyces (Fig. 1). The highest similarity value was observed with A. humatus CD5T (98.53 %), followed by Agromyces ramosus DSM 43045T (97.95 %), Agromyces subbeticus Z33T (97.89 %), Agromyces bauzanensis BZ41T (97.89 %), Agromyces fucosus VKM Ac1345T (97.88 %) and Agromyces neolithicus 23-23T (97.84 %). Meanwhile, strains IY07-56T and IY07-113 formed a reliable and monophyletic cluster with A. flavus in the ML tree (Fig. 1). This cluster was also recovered in the trees generated using the NJ and MP algorithms with high bootstrap values. The highest similarity value was observed with A. flavus CPCC 202695T (98.70 %), followed by Agromyces mediolanus DSM 20152T (97.67 %), Agromyces soli MJ21T (97.57 %), Agromyces ulmi XIL01T (97.54 %) and Agromyces tropicus CM9-9T (97.46 %). The similarity value between strains IY07-56T and IY07-113 was 100 % and that between strains IY07-20T and IY07-56T was 96.74 %. Amino acids and their isomers in cell-wall hydrolysates and isoprenoid quinones were analysed as described previously (Hamada et al., 2012). The acyl type of muramic acid was determined by using the method of Uchida et al. (1999). The preparation and analysis of cellular fatty acid methyl esters were performed using the protocol of the MIDI Sherlock Microbial Identification System (Sasser, 1990) and a gas chromatograph (model 6890N; Agilent Technologies) with Sherlock MIDI software (version 4.0) and a TSBA database (version 4.0). The polar lipids were analysed as described by Hamada et al. (2010), with the following chromatographic systems: chloroform/methanol/ water (65 : 25 : 4, by vol.) used in the first direction and chloroform/acetic acid/methanol/water (80 : 18 : 12 : 5, by vol.) in the second direction. The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) using a HPLC system (model LC-10A; Shimadzu).
Phylogenetic analysis, based on 16S rRNA gene sequences, showed that strains IY07-20T, IY07-56T and IY07-113
The obtained peptidoglycan samples of strains IY07-20T, IY07-56T and IY07-113 contained alanine, glutamic acid, glycine and A2bu at molar ratios of 0.8 : 1.0 : 1.2 : 1.8, 0.7 : 1.0 : 1.1 : 1.5 and 1.0 : 1.0 : 1.3 : 1.6, respectively. Enantiomeric analysis of the peptidoglycan amino acids revealed the presence of D-Ala, D-Glu, Gly and L-A2bu. Trace amounts of D-A2bu were also detected in all isolates. These data indicated that the cell-wall peptidoglycan of the three isolates is of the B1 type (Schleifer & Kandler, 1972) and contains mostly L-A2bu as the diagnostic diamino acid. This result was consistent with those described in the previous report (Sasaki et al., 1998). The acyl type of muramic acid was N-acetyl. The predominant menaquinone of the isolates was MK-12; MK-13 was present as a minor component. The major cellular fatty acids (.10 %) of strain IY07-20T were anteiso-C17 : 0 (48.2 %) and anteiso-C15 : 0 (35.6 %), while those of strains IY07-56T and IY07-113 were anteiso-C17 : 0 (34.8–36.2 %), anteisoC15 : 0 (30.4–30.5 %) and iso-C15 : 0 (12.0–12.6 %) (Table S1, available in the online Supplementary Material). The
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PCR amplification and 16S rRNA gene sequencing of strains IY07-20T, IY07-56T and IY07-113 were performed as described previously (Hamada et al., 2012). The phylogenetic neighbours were identified and pairwise 16S rRNA gene sequence similarities were calculated using EzTaxon-e (Kim et al., 2012). The almost-complete 16S rRNA gene sequences of the strains were aligned with reference sequences of members of the genus Agromyces by using the CLUSTAL X program (Thompson et al., 1997). Phylogenetic trees were reconstructed by the maximum-likelihood (ML) (Felsenstein, 1981), neighbour-joining (NJ) (Saitou & Nei, 1987) and maximum-parsimony (MP) (Fitch, 1971) algorithms using the MEGA 5.0 program (Tamura et al., 2011). A total of 1329 nucleotides present in all strains was used for tree reconstructions. The resultant tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) on the basis of 1000 replicates.
Two novel species of the genus Agromyces
88
0.01
98 Agromyces subtropicus IY07-56T (AB546310) Agromyces subtropicus IY07-113 (AB546311) Agromyces flavus CPCC 202695T (FJ529717) Agromyces tropicus CM9-9T (AB454378)
99 Agromyces soli MJ21T (GQ241325) Agromyces mediolanus DSM 20152T (X77449) Agromyces indicus NIO-1018T (HM036655) Agromyces aurantiacus YIM 21741T (AF389342)
71 83
Agromyces bracchium NBRC 16238T (AB023359) Agromyces luteolus NBRC 16235T (AB023356)
Agromyces ulmi XIL01T (AY427830) Agromyces neolithicus 23-23T (AY507128) Agromyces humatus CD5T (AY618216) Agromyces iriomotensis IY07-20T (AB546308) Agromyces bauzanensis BZ41T (FJ972171) Agromyces rhizospherae NBRC 16236T (AB023357) Agromyces subbeticus Z33T (AY737778) Agromyces ramosus DSM 43045T (X77447) Agromyces albus VKM Ac-1800T (AF503917) Agromyces fucosus NBRC 15781T (AB842300) Agromyces cerinus subsp. nitratus IMET 11532T (AY277619) 79 Agromyces cerinus subsp. cerinus JCM 9083T (D45060) Agromyces hippuratus JCM 9086T (D45061) Agromyces atrinae P27T (FJ607310) Agromyces italicus CD1T (AY618215) Agromyces lapidis CD55T (AY618217) Agromyces allii UMS-62T (DQ673873) 79
Agromyces salentinus 20-5T (AY507129) Agromyces terreus DS-10T (EF363711) Microbacterium lacticum DSM 20427T (X77441)
Fig. 1. Phylogenetic tree derived from 16S rRNA gene sequences of strains IY07-20T, IY07-56T and IY07-113 and their taxonomic neighbours, reconstructed by the maximum-likelihood method. Filled circles indicate that corresponding nodes were also recovered in the trees generated with the neighbour-joining and maximum-parsimony methods. The sequence from Microbacterium lacticum DSM 20427T (GenBank accession no. X77441) was used as the outgroup. The numbers at the branch nodes are bootstrap percentages (from 1000 replicates); only values of 70 % or above are shown. Bar, 0.01 changes per nucleotide position.
principal polar lipids of the isolates were diphosphatidylglycerol, phosphatidylglycerol and one glycolipid. Minor or trace amounts of one unidentified phosphoglycolipid, one unidentified polar lipid and one or two unidentified phosphorus-free polar lipids were also detected (Fig. S1). The DNA G+C contents of strains IY07-20T, IY07-56T and IY07-113 were 72.9, 71.7 and 70.9 mol%, respectively. The microplate hybridization method developed by Ezaki et al. (1989) was used to determine DNA–DNA relatedness. Hybridizations were performed using five replications. After the highest and lowest values for each sample were excluded, the mean was reported as the DNA–DNA relatedness value. The DNA–DNA relatedness value between strain IY07-20T and A. humatus NBRC 109085T was 19 % (reciprocal value, 21 %). Meanwhile, strains IY07-56T and IY07-113 exhibited levels of DNA–DNA relatedness of 22 and 24 % (reciprocal values, 27 and 31 %) to A. flavus http://ijs.sgmjournals.org
NBRC 109064T, respectively. The DNA–DNA relatedness value between strains IY07-56T and IY07-113 was 71 % (reciprocal value, 74 %). The results of the phylogenetic analysis, based on the 16S rRNA gene sequences, indicated that strains IY07-20T, IY07-56T and IY07-113 were closely related to members of the genus Agromyces and their chemotaxonomic features also corresponded to those of the genus. Therefore, it is appropriate to regard strains IY07-20T, IY07-56T and IY07-113 as members of the genus Agromyces. The 16S rRNA gene sequence similarity between strains IY07-56T and IY07-113 was 100 % and the DNA–DNA relatedness between the two strains was 71–74 %. These results indicate that strains IY07-56T and IY07-113 are representatives of the same species. Meanwhile, the DNA–DNA relatedness values among strains IY07-20T and IY07-56T and related species of the genus Agromyces were low, and the results of 835
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the physiological and biochemical tests distinguished strains IY07-20T, IY07-56T and IY07-113 from related species of the genus Agromyces (Table 1). Therefore, it is proposed that strain IY07-20T and strains IY07-56T and IY07-113 be classified as representatives of two novel species of the genus Agromyces, with the names Agromyces iriomotensis sp. nov. and Agromyces subtropicus sp. nov., respectively.
Description of Agromyces iriomotensis sp. nov. Agromyces iriomotensis (i.ri.o.mot.en9sis. N.L. masc. adj. iriomotensis pertaining to Iriomote Island, Japan, where the organism was originally isolated). Cells are Gram-stain-positive, microaerophilic to aerobic, non-motile and non-endospore-forming. Young cultures
Table 1. Differential phenotypic characteristics of strains IY07-20T, IY07-56T and IY07-113 and related species of the genus Agromyces Strains: 1, IY07-20T; 2, IY07-56T; 3, IY07-113; 4, A. humatus NBRC 109085T; 5, A. flavus NBRC 109064T. Data are from this study unless indicated. +, Positive; W, weakly positive; 2, negative; ai, anteiso; i, iso. Data in parentheses were reported in the original descriptions of A. humatus (Jurado et al., 2005) and A. flavus (Chen et al., 2011). Characteristic Growth temperature (uC) Catalase Nitrate reduction Aesculin hydrolysis Gelatin hydrolysis N-Acetyl-b-glucosaminidase Chymotrypsin Esterase (C4) Esterase lipase (C8) a-Galactosidase b-Galactosidase a-Glucosidase b-Glucosidase b-Glucuronidase Pyrazinamidase Pyrrolidonyl arylamidase Trypsin Acid production from: Arbutin L-Fucose D-Galactose Gentiobiose Glycerol Inulin D-Lactose D-Mannose Melezitose Melibiose Raffinose D-Ribose Salicin Sucrose Trehalose Turanose Xylitol D-Xylose Major cellular fatty acids (.10 %) DNA G+C content (mol%)
1
2
3
4
5
20–37 2 2 + 2 + + + + + + + + 2 + + +
10–37 + + + + 2 2 + 2 2 + 2 + 2 2 2 2
10–37 + + + + 2 2 + 2 2 + 2 + 2 2 2 2
10–28 (15–37) + + 2 + 2 2 2 2 2 + 2 2 + 2 2 2
15–37 (10–40) + + + 2 2 2 + 2 2 2 2 + 2 2 2 2
+ + 2 2 2 + 2 + 2 2 + 2 + + 2 2 2 +
2 2 + + + 2 2 2 2 + + + 2 2 2 2 2 +
2 2 + + + 2 + 2 2 + + 2 2 2 2 2 + 2
ai-C17 : 0, aiC15 : 0 72.9
ai-C17 : 0, aiC15 : 0, i-C15 : 0 71.7
ai-C17 : 0, aiC15 : 0, i-C15 : 0 70.9
2 (+) 2 + 2 + 2 (+) 2 2 2 2 + 2 2 + 2 2 2 2 ai-C17 : 0, aiC15 : 0, i-C16 : 0 70.6*
2 2 (+) + 2 + 2 (+) 2 (+) + + 2 (+) 2 (W) 2 2 + + + 2 2 (+) ai-C15 : 0, aiC17 : 0, i-C16 : 0 70.9D
*Data from Jurado et al. (2005). DData from Chen et al. (2011). 836
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Two novel species of the genus Agromyces
produce branched vegetative hyphae (width 0.4–0.6 mm) that subsequently break up into rod-like to coccoid fragments. Colonies are cream–yellow, smooth, circular and transparent. Catalase- and oxidase-negative. Growth occurs at 20–37 uC (optimum 25 uC) and pH 6.0–10.0 (optimum pH 7.0–8.0) but not at 15 or 45 uC. Growth occurs with NaCl concentrations of 0–3 % (w/v) but not with 5 %; optimal NaCl concentration is 1 %. N-Acetyl-b-glucosaminidase, acid phosphatase, a-chymotrypsin, cystine arylamidase, esterase (C4), esterase lipase (C8), a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, leucine arylamidase, pyrazinamidase, pyrrolidonyl arylamidase, trypsin and valine arylamidase are present, whereas alkaline phosphatase, a-fucosidase, b-glucuronidase, lipase (C14), a-mannosidase, phosphohydrolase and urease are not. Aesculin is hydrolysed while gelatin is not. Nitrate is not reduced. In API 50CH assays, acid is produced from N-acetylglucosamine, L-arabinose, arbutin, cellobiose, Dfructose, L-fucose, D-glucose, glycogen, inulin, maltose, D-mannitol, D-mannose, raffinose, L-rhamnose, salicin, starch, sucrose and D-xylose; no acid production occurs from D-adonitol, amygdalin, D-arabinose, D-arabitol, Larabitol, dulcitol, erythritol, D-fucose, D-galactose, gentiobiose, gluconate, glycerol, inositol, 2-ketogluconate, 5-ketogluconate, D-lactose, D-lyxose, melezitose, melibiose, methyl a-D-glucopyranoside, methyl a-D-mannopyranoside, methyl b-D-xylopyranoside, D-ribose, D-sorbitol, Lsorbose, D-tagatose, turanose, trehalose, xylitol or L-xylose. The peptidoglycan contains D-Ala, D-Glu, Gly and L-A2bu. The predominant menaquinone is MK-12; MK-13 is present as a minor component. The major cellular fatty acids (.10 %) are anteiso-C17 : 0 and anteiso-C15 : 0. The principal polar lipids are diphosphatidylglycerol, phosphatidylglycerol and one glycolipid. The type strain, IY07-20T (5NBRC 106452T5DSM 26155T), was isolated from a soil sample from a pineapple field on Iriomote Island, Okinawa, Japan. The DNA G+C content of the type strain is 72.9 mol%. Description of Agromyces subtropicus sp. nov. Agromyces subtropicus (sub.tro9pi.cus. N.L. masc. adj. subtropicus pertaining to the subtropical zone, the origin of the soil sample from which the type strain was isolated). Cells are Gram-stain-positive, microaerophilic to aerobic, non-motile and non-endospore-forming. Young cultures produce branched vegetative hyphae (width 0.4–0.6 mm) that subsequently break up into rod-like to coccoid fragments. Colonies are pale yellow, smooth, circular and transparent. Catalase-positive and oxidase-negative. Growth occurs at 10–37 uC (optimum 28 uC) and pH 6.0– 10.0 (optimum pH 7.0–8.0) but not at 5 or 45 uC. Growth occurs with NaCl concentrations of 0–3 % (w/v) but not with 5 %; optimal NaCl concentration is 0 %. Cystine arylamidase, esterase (C4), b-galactosidase, b-glucosidase, leucine arylamidase, phosphohydrolase and valine arylamidase are present, whereas N-acetyl-b-glucosaminidase, http://ijs.sgmjournals.org
acid phosphatase, alkaline phosphatase, a-chymotrypsin, esterase lipase (C8), a-fucosidase, a-galactosidase, aglucosidase, b-glucuronidase, lipase (C14), a-mannosidase, pyrazinamidase, pyrrolidonyl arylamidase, trypsin and urease are not. Aesculin and gelatin are hydrolysed. Nitrate is reduced. In API 50 CH assays, acid is produced from L-arabinose, cellobiose, D-fructose, D-galactose, gentiobiose, D-glucose, glycogen, glycerol, maltose, D-mannitol, melibiose, raffinose, L-rhamnose and starch; however, acid production from D-lactose, D-ribose, xylitol and D-xylose is variable. No acid production occurs from Nacetylglucosamine, D-adonitol, amygdalin, D-arabinose, Darabitol, L-arabitol, arbutin, dulcitol, erythritol, D-fucose, L-fucose, gluconate, inositol, inulin, 2-ketogluconate, 5ketogluconate, D-lyxose, D-mannose, melezitose, methyl aD-glucopyranoside, methyl a-D-mannopyranoside, methyl b-D-xylopyranoside, salicin, D-sorbitol, L-sorbose, sucrose, tagatose, turanose, trehalose or L-xylose. The peptidoglycan contains D-Ala, D-Glu, Gly and L-A2bu. The predominant menaquinone is MK-12; MK-13 is present as a minor component. The major cellular fatty acids (.10 %) are anteiso-C17 : 0, anteiso-C15 : 0 and iso-C15 : 0. The principal polar lipids are diphosphatidylglycerol, phosphatidylglycerol and one glycolipid. The type strain, IY07-56T (5NBRC 106454T5DSM 26153T), was isolated from a soil sample from a pineapple field on Iriomote Island, Okinawa, Japan. Strain IY07-113 (5NBRC 106455), from a forest soil sample from Iriomote Island, is a second strain of the species. The DNA G+C content of the type strain is 71.7 mol%.
Acknowledgements This study, in part, was supported by a research grant from the Institute for Fermentation, Osaka (IFO).
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Emended description of the genus Agromyces and description of Agromyces cerinus subsp. cerinus sp. nov., subsp. nov., Agromyces cerinus subsp. nitratus sp. nov., subsp. nov., Agromyces fucosus subsp. fucosus sp. nov., subsp. nov., and Agromyces fucosus subsp. hippuratus sp. nov., subsp. nov. Int J Syst Bacteriol 42, 635–641.
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DOI 10.1099/ijs.0.057349-0
Agromyces iriomotensis sp. nov. and Agromyces subtropicus sp. nov., isolated from soil Moriyuki Hamada,1 Chiyo Shibata,1 Yuumi Ishida,1 Tomohiko Tamura,1 Hideki Yamamura,2 Masayuki Hayakawa2 and Ken-ichiro Suzuki1 1
Correspondence
Biological Resource Center, National Institute of Technology and Evaluation (NBRC), 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan
Moriyuki Hamada [email protected]
2
Division of Applied Biological Sciences, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Takeda-4, Kofu, Yamanashi 400-8511, Japan
Three novel Gram-stain-positive bacteria, designated IY07-20T, IY07-56T and IY07-113, were isolated from soil samples from Iriomote Island, Okinawa, Japan, and their taxonomic positions were investigated by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the three isolates were closely related to the members of the genus Agromyces, with similarity range of 95.6–98.7 %. The isolates contained L-2,4diaminobutylic acid, D-alanine, D-glutamic acid and glycine in their peptidoglycans. The predominant menaquinone was MK-12 and the major fatty acids were anteiso-C15 : 0 and anteisoC17 : 0. The DNA G+C contents were 70.9–72.9 mol%. The chemotaxonomic characteristics of the isolates matched those described for members of the genus Agromyces. The results of phylogenetic analysis and DNA–DNA hybridization, along with differences in phenotypic characteristics between strains IY07-20T, IY07-56T and IY07-113 and the species of the genus Agromyces with validly published names, indicate that the three isolates merit classification as representatives of two novel species of the genus Agromyces, for which the names Agromyces iriomotensis sp. nov. and Agromyces subtropica sp. nov. are proposed; the type strains are IY0720T (5NBRC 106452T5DSM 26155T) and IY07-56T (5NBRC 106454T5DSM 26153T), respectively.
The genus Agromyces proposed by Gledhill & Casida (1969) and the description of which was emended by Zgurskaya et al. (1992) currently contains 25 recognized species. This genus comprises Gram-stain-positive, nonmotile, non-spore-forming, filamentous and non-filamentous, microaerophilic to aerobic actinobacteria (Akimov & Evtushenko, 2012). The members of the genus have 2,4diaminobutyric acid (A2bu) in their peptidoglycan and MK-12 as the predominant menaquinone with smaller amounts of MK-11 and/or MK-13. Major fatty acids are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Most of the species have been isolated from soil. During the course of a study of bacterial diversity in cool-temperate and subtropical areas, three novel actinobacteria were isolated from soil samples collected from a subtropical island in Japan. Comparative 16S rRNA gene sequence analysis revealed Abbreviation: A2bu, 2,4-diaminobutyric acid. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains IY07-20T, IY07-56T and IY07-113 are AB546308, AB546310 and AB546311, respectively. A supplementary figure and a supplementary table are available with the online version of this paper.
057349 G 2014 IUMS
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that the isolates are phylogenetically related to members of the genus Agromyces. The objective of this study was to determine the taxonomic positions of the isolates by using a polyphasic approach. Strains IY07-20T, IY07-56T and IY07-113 were isolated from pineapple field soil samples (IY07-20T and IY07-56T) and a forest soil sample (IY07-113) collected on Iriomote Island, Okinawa, Japan, using the SDS/yeast extract pretreatment method (Hayakawa & Nonomura, 1989) and humic acid–vitamin (HV) agar (Hayakawa & Nonomura, 1987). NBRC medium 802 (1.0 % polypeptone, 0.2 % yeast extract, 0.1 % MgSO4 . 7H2O and 1.5 % agar if required; pH 7.0) was used as basal medium for this study. Biomass for chemotaxonomic and molecular systematic studies, except for cellular fatty acids analysis, was obtained by cultivating the strains in shake flasks for 48 h at 28 uC with shaking at 100 r.p.m. Biomass grown on trypticase soy agar (Difco) for 24 h at 28 uC was used for cellular fatty acids analysis. Agromyces flavus NBRC 109064T and Agromyces humatus NBRC 109085T were used as reference strains for comparison with the physiological, biochemical and the DNA–DNA hybridization tests. 833
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Colony appearance was examined after incubation at 28 uC for 3 days in the dark. Morphological features were observed for up to 7 days of incubation under a light microscope (BX-51; Olympus) and a scanning electron microscope (JSM-6060; JEOL). Cell motility, growth parameters (temperature, pH and NaCl tolerance), Gram staining phenotype and oxidase activity were determined using the methods described by Hamada et al. (2010). Growth under anaerobic and microaerobic conditions was determined using AnaeroPack-Anaero and AnaeroPackMicroAero (Mitsubishi Gas Chemical) with anaerobic jars, respectively. Catalase activity was determined by production of bubbles after the addition of a drop of 3.0 % H2O2. Other physiological and biochemical tests were performed using API ZYM, API Coryne and API 50 CH systems (bioMe´rieux) according to the manufacturer’s instructions. API 50 CHB/E medium was used to prepare cell suspensions for the API 50 CH test. Strain IY07-20T formed cream–yellow, circular, transparent and smooth colonies that were approximately 0.2– 0.5 mm in diameter after 3 days of cultivation, while strains IY07-56T and IY07-113 formed pale yellow, circular, transparent and smooth colonies that were approximately 0.5–1.0 mm in diameter after 3 days of cultivation. All strains developed branched vegetative hyphae (width 0.4– 0.6 mm) in the early phase of growth, but these hyphae subsequently fragmented into rod-like to coccoid fragments. The cells were Gram-stain-positive, microaerophilic to aerobic, non-motile and non-endospore-forming. Strain IY07-20T grew at 20–37 uC (optimum 25 uC) while strains IY07-56T and IY07-113 grew at 10–37 uC (optimum 28 uC). The pH range for growth of all strains was 6.0– 10.0 (optimum pH 7.0–8.0). All strains exhibited growth with NaCl concentrations of 0–3 %; no growth was observed with 5 % NaCl (w/v) or higher. No growth was observed under anaerobic conditions. The results of other physiological and biochemical analyses are summarized in the species descriptions below.
represented members of the genus Agromyces. However, strain IY07-20T did not form a reliable cluster with any member of the genus Agromyces (Fig. 1). The highest similarity value was observed with A. humatus CD5T (98.53 %), followed by Agromyces ramosus DSM 43045T (97.95 %), Agromyces subbeticus Z33T (97.89 %), Agromyces bauzanensis BZ41T (97.89 %), Agromyces fucosus VKM Ac1345T (97.88 %) and Agromyces neolithicus 23-23T (97.84 %). Meanwhile, strains IY07-56T and IY07-113 formed a reliable and monophyletic cluster with A. flavus in the ML tree (Fig. 1). This cluster was also recovered in the trees generated using the NJ and MP algorithms with high bootstrap values. The highest similarity value was observed with A. flavus CPCC 202695T (98.70 %), followed by Agromyces mediolanus DSM 20152T (97.67 %), Agromyces soli MJ21T (97.57 %), Agromyces ulmi XIL01T (97.54 %) and Agromyces tropicus CM9-9T (97.46 %). The similarity value between strains IY07-56T and IY07-113 was 100 % and that between strains IY07-20T and IY07-56T was 96.74 %. Amino acids and their isomers in cell-wall hydrolysates and isoprenoid quinones were analysed as described previously (Hamada et al., 2012). The acyl type of muramic acid was determined by using the method of Uchida et al. (1999). The preparation and analysis of cellular fatty acid methyl esters were performed using the protocol of the MIDI Sherlock Microbial Identification System (Sasser, 1990) and a gas chromatograph (model 6890N; Agilent Technologies) with Sherlock MIDI software (version 4.0) and a TSBA database (version 4.0). The polar lipids were analysed as described by Hamada et al. (2010), with the following chromatographic systems: chloroform/methanol/ water (65 : 25 : 4, by vol.) used in the first direction and chloroform/acetic acid/methanol/water (80 : 18 : 12 : 5, by vol.) in the second direction. The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) using a HPLC system (model LC-10A; Shimadzu).
Phylogenetic analysis, based on 16S rRNA gene sequences, showed that strains IY07-20T, IY07-56T and IY07-113
The obtained peptidoglycan samples of strains IY07-20T, IY07-56T and IY07-113 contained alanine, glutamic acid, glycine and A2bu at molar ratios of 0.8 : 1.0 : 1.2 : 1.8, 0.7 : 1.0 : 1.1 : 1.5 and 1.0 : 1.0 : 1.3 : 1.6, respectively. Enantiomeric analysis of the peptidoglycan amino acids revealed the presence of D-Ala, D-Glu, Gly and L-A2bu. Trace amounts of D-A2bu were also detected in all isolates. These data indicated that the cell-wall peptidoglycan of the three isolates is of the B1 type (Schleifer & Kandler, 1972) and contains mostly L-A2bu as the diagnostic diamino acid. This result was consistent with those described in the previous report (Sasaki et al., 1998). The acyl type of muramic acid was N-acetyl. The predominant menaquinone of the isolates was MK-12; MK-13 was present as a minor component. The major cellular fatty acids (.10 %) of strain IY07-20T were anteiso-C17 : 0 (48.2 %) and anteiso-C15 : 0 (35.6 %), while those of strains IY07-56T and IY07-113 were anteiso-C17 : 0 (34.8–36.2 %), anteisoC15 : 0 (30.4–30.5 %) and iso-C15 : 0 (12.0–12.6 %) (Table S1, available in the online Supplementary Material). The
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PCR amplification and 16S rRNA gene sequencing of strains IY07-20T, IY07-56T and IY07-113 were performed as described previously (Hamada et al., 2012). The phylogenetic neighbours were identified and pairwise 16S rRNA gene sequence similarities were calculated using EzTaxon-e (Kim et al., 2012). The almost-complete 16S rRNA gene sequences of the strains were aligned with reference sequences of members of the genus Agromyces by using the CLUSTAL X program (Thompson et al., 1997). Phylogenetic trees were reconstructed by the maximum-likelihood (ML) (Felsenstein, 1981), neighbour-joining (NJ) (Saitou & Nei, 1987) and maximum-parsimony (MP) (Fitch, 1971) algorithms using the MEGA 5.0 program (Tamura et al., 2011). A total of 1329 nucleotides present in all strains was used for tree reconstructions. The resultant tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) on the basis of 1000 replicates.
Two novel species of the genus Agromyces
88
0.01
98 Agromyces subtropicus IY07-56T (AB546310) Agromyces subtropicus IY07-113 (AB546311) Agromyces flavus CPCC 202695T (FJ529717) Agromyces tropicus CM9-9T (AB454378)
99 Agromyces soli MJ21T (GQ241325) Agromyces mediolanus DSM 20152T (X77449) Agromyces indicus NIO-1018T (HM036655) Agromyces aurantiacus YIM 21741T (AF389342)
71 83
Agromyces bracchium NBRC 16238T (AB023359) Agromyces luteolus NBRC 16235T (AB023356)
Agromyces ulmi XIL01T (AY427830) Agromyces neolithicus 23-23T (AY507128) Agromyces humatus CD5T (AY618216) Agromyces iriomotensis IY07-20T (AB546308) Agromyces bauzanensis BZ41T (FJ972171) Agromyces rhizospherae NBRC 16236T (AB023357) Agromyces subbeticus Z33T (AY737778) Agromyces ramosus DSM 43045T (X77447) Agromyces albus VKM Ac-1800T (AF503917) Agromyces fucosus NBRC 15781T (AB842300) Agromyces cerinus subsp. nitratus IMET 11532T (AY277619) 79 Agromyces cerinus subsp. cerinus JCM 9083T (D45060) Agromyces hippuratus JCM 9086T (D45061) Agromyces atrinae P27T (FJ607310) Agromyces italicus CD1T (AY618215) Agromyces lapidis CD55T (AY618217) Agromyces allii UMS-62T (DQ673873) 79
Agromyces salentinus 20-5T (AY507129) Agromyces terreus DS-10T (EF363711) Microbacterium lacticum DSM 20427T (X77441)
Fig. 1. Phylogenetic tree derived from 16S rRNA gene sequences of strains IY07-20T, IY07-56T and IY07-113 and their taxonomic neighbours, reconstructed by the maximum-likelihood method. Filled circles indicate that corresponding nodes were also recovered in the trees generated with the neighbour-joining and maximum-parsimony methods. The sequence from Microbacterium lacticum DSM 20427T (GenBank accession no. X77441) was used as the outgroup. The numbers at the branch nodes are bootstrap percentages (from 1000 replicates); only values of 70 % or above are shown. Bar, 0.01 changes per nucleotide position.
principal polar lipids of the isolates were diphosphatidylglycerol, phosphatidylglycerol and one glycolipid. Minor or trace amounts of one unidentified phosphoglycolipid, one unidentified polar lipid and one or two unidentified phosphorus-free polar lipids were also detected (Fig. S1). The DNA G+C contents of strains IY07-20T, IY07-56T and IY07-113 were 72.9, 71.7 and 70.9 mol%, respectively. The microplate hybridization method developed by Ezaki et al. (1989) was used to determine DNA–DNA relatedness. Hybridizations were performed using five replications. After the highest and lowest values for each sample were excluded, the mean was reported as the DNA–DNA relatedness value. The DNA–DNA relatedness value between strain IY07-20T and A. humatus NBRC 109085T was 19 % (reciprocal value, 21 %). Meanwhile, strains IY07-56T and IY07-113 exhibited levels of DNA–DNA relatedness of 22 and 24 % (reciprocal values, 27 and 31 %) to A. flavus http://ijs.sgmjournals.org
NBRC 109064T, respectively. The DNA–DNA relatedness value between strains IY07-56T and IY07-113 was 71 % (reciprocal value, 74 %). The results of the phylogenetic analysis, based on the 16S rRNA gene sequences, indicated that strains IY07-20T, IY07-56T and IY07-113 were closely related to members of the genus Agromyces and their chemotaxonomic features also corresponded to those of the genus. Therefore, it is appropriate to regard strains IY07-20T, IY07-56T and IY07-113 as members of the genus Agromyces. The 16S rRNA gene sequence similarity between strains IY07-56T and IY07-113 was 100 % and the DNA–DNA relatedness between the two strains was 71–74 %. These results indicate that strains IY07-56T and IY07-113 are representatives of the same species. Meanwhile, the DNA–DNA relatedness values among strains IY07-20T and IY07-56T and related species of the genus Agromyces were low, and the results of 835
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the physiological and biochemical tests distinguished strains IY07-20T, IY07-56T and IY07-113 from related species of the genus Agromyces (Table 1). Therefore, it is proposed that strain IY07-20T and strains IY07-56T and IY07-113 be classified as representatives of two novel species of the genus Agromyces, with the names Agromyces iriomotensis sp. nov. and Agromyces subtropicus sp. nov., respectively.
Description of Agromyces iriomotensis sp. nov. Agromyces iriomotensis (i.ri.o.mot.en9sis. N.L. masc. adj. iriomotensis pertaining to Iriomote Island, Japan, where the organism was originally isolated). Cells are Gram-stain-positive, microaerophilic to aerobic, non-motile and non-endospore-forming. Young cultures
Table 1. Differential phenotypic characteristics of strains IY07-20T, IY07-56T and IY07-113 and related species of the genus Agromyces Strains: 1, IY07-20T; 2, IY07-56T; 3, IY07-113; 4, A. humatus NBRC 109085T; 5, A. flavus NBRC 109064T. Data are from this study unless indicated. +, Positive; W, weakly positive; 2, negative; ai, anteiso; i, iso. Data in parentheses were reported in the original descriptions of A. humatus (Jurado et al., 2005) and A. flavus (Chen et al., 2011). Characteristic Growth temperature (uC) Catalase Nitrate reduction Aesculin hydrolysis Gelatin hydrolysis N-Acetyl-b-glucosaminidase Chymotrypsin Esterase (C4) Esterase lipase (C8) a-Galactosidase b-Galactosidase a-Glucosidase b-Glucosidase b-Glucuronidase Pyrazinamidase Pyrrolidonyl arylamidase Trypsin Acid production from: Arbutin L-Fucose D-Galactose Gentiobiose Glycerol Inulin D-Lactose D-Mannose Melezitose Melibiose Raffinose D-Ribose Salicin Sucrose Trehalose Turanose Xylitol D-Xylose Major cellular fatty acids (.10 %) DNA G+C content (mol%)
1
2
3
4
5
20–37 2 2 + 2 + + + + + + + + 2 + + +
10–37 + + + + 2 2 + 2 2 + 2 + 2 2 2 2
10–37 + + + + 2 2 + 2 2 + 2 + 2 2 2 2
10–28 (15–37) + + 2 + 2 2 2 2 2 + 2 2 + 2 2 2
15–37 (10–40) + + + 2 2 2 + 2 2 2 2 + 2 2 2 2
+ + 2 2 2 + 2 + 2 2 + 2 + + 2 2 2 +
2 2 + + + 2 2 2 2 + + + 2 2 2 2 2 +
2 2 + + + 2 + 2 2 + + 2 2 2 2 2 + 2
ai-C17 : 0, aiC15 : 0 72.9
ai-C17 : 0, aiC15 : 0, i-C15 : 0 71.7
ai-C17 : 0, aiC15 : 0, i-C15 : 0 70.9
2 (+) 2 + 2 + 2 (+) 2 2 2 2 + 2 2 + 2 2 2 2 ai-C17 : 0, aiC15 : 0, i-C16 : 0 70.6*
2 2 (+) + 2 + 2 (+) 2 (+) + + 2 (+) 2 (W) 2 2 + + + 2 2 (+) ai-C15 : 0, aiC17 : 0, i-C16 : 0 70.9D
*Data from Jurado et al. (2005). DData from Chen et al. (2011). 836
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produce branched vegetative hyphae (width 0.4–0.6 mm) that subsequently break up into rod-like to coccoid fragments. Colonies are cream–yellow, smooth, circular and transparent. Catalase- and oxidase-negative. Growth occurs at 20–37 uC (optimum 25 uC) and pH 6.0–10.0 (optimum pH 7.0–8.0) but not at 15 or 45 uC. Growth occurs with NaCl concentrations of 0–3 % (w/v) but not with 5 %; optimal NaCl concentration is 1 %. N-Acetyl-b-glucosaminidase, acid phosphatase, a-chymotrypsin, cystine arylamidase, esterase (C4), esterase lipase (C8), a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, leucine arylamidase, pyrazinamidase, pyrrolidonyl arylamidase, trypsin and valine arylamidase are present, whereas alkaline phosphatase, a-fucosidase, b-glucuronidase, lipase (C14), a-mannosidase, phosphohydrolase and urease are not. Aesculin is hydrolysed while gelatin is not. Nitrate is not reduced. In API 50CH assays, acid is produced from N-acetylglucosamine, L-arabinose, arbutin, cellobiose, Dfructose, L-fucose, D-glucose, glycogen, inulin, maltose, D-mannitol, D-mannose, raffinose, L-rhamnose, salicin, starch, sucrose and D-xylose; no acid production occurs from D-adonitol, amygdalin, D-arabinose, D-arabitol, Larabitol, dulcitol, erythritol, D-fucose, D-galactose, gentiobiose, gluconate, glycerol, inositol, 2-ketogluconate, 5-ketogluconate, D-lactose, D-lyxose, melezitose, melibiose, methyl a-D-glucopyranoside, methyl a-D-mannopyranoside, methyl b-D-xylopyranoside, D-ribose, D-sorbitol, Lsorbose, D-tagatose, turanose, trehalose, xylitol or L-xylose. The peptidoglycan contains D-Ala, D-Glu, Gly and L-A2bu. The predominant menaquinone is MK-12; MK-13 is present as a minor component. The major cellular fatty acids (.10 %) are anteiso-C17 : 0 and anteiso-C15 : 0. The principal polar lipids are diphosphatidylglycerol, phosphatidylglycerol and one glycolipid. The type strain, IY07-20T (5NBRC 106452T5DSM 26155T), was isolated from a soil sample from a pineapple field on Iriomote Island, Okinawa, Japan. The DNA G+C content of the type strain is 72.9 mol%. Description of Agromyces subtropicus sp. nov. Agromyces subtropicus (sub.tro9pi.cus. N.L. masc. adj. subtropicus pertaining to the subtropical zone, the origin of the soil sample from which the type strain was isolated). Cells are Gram-stain-positive, microaerophilic to aerobic, non-motile and non-endospore-forming. Young cultures produce branched vegetative hyphae (width 0.4–0.6 mm) that subsequently break up into rod-like to coccoid fragments. Colonies are pale yellow, smooth, circular and transparent. Catalase-positive and oxidase-negative. Growth occurs at 10–37 uC (optimum 28 uC) and pH 6.0– 10.0 (optimum pH 7.0–8.0) but not at 5 or 45 uC. Growth occurs with NaCl concentrations of 0–3 % (w/v) but not with 5 %; optimal NaCl concentration is 0 %. Cystine arylamidase, esterase (C4), b-galactosidase, b-glucosidase, leucine arylamidase, phosphohydrolase and valine arylamidase are present, whereas N-acetyl-b-glucosaminidase, http://ijs.sgmjournals.org
acid phosphatase, alkaline phosphatase, a-chymotrypsin, esterase lipase (C8), a-fucosidase, a-galactosidase, aglucosidase, b-glucuronidase, lipase (C14), a-mannosidase, pyrazinamidase, pyrrolidonyl arylamidase, trypsin and urease are not. Aesculin and gelatin are hydrolysed. Nitrate is reduced. In API 50 CH assays, acid is produced from L-arabinose, cellobiose, D-fructose, D-galactose, gentiobiose, D-glucose, glycogen, glycerol, maltose, D-mannitol, melibiose, raffinose, L-rhamnose and starch; however, acid production from D-lactose, D-ribose, xylitol and D-xylose is variable. No acid production occurs from Nacetylglucosamine, D-adonitol, amygdalin, D-arabinose, Darabitol, L-arabitol, arbutin, dulcitol, erythritol, D-fucose, L-fucose, gluconate, inositol, inulin, 2-ketogluconate, 5ketogluconate, D-lyxose, D-mannose, melezitose, methyl aD-glucopyranoside, methyl a-D-mannopyranoside, methyl b-D-xylopyranoside, salicin, D-sorbitol, L-sorbose, sucrose, tagatose, turanose, trehalose or L-xylose. The peptidoglycan contains D-Ala, D-Glu, Gly and L-A2bu. The predominant menaquinone is MK-12; MK-13 is present as a minor component. The major cellular fatty acids (.10 %) are anteiso-C17 : 0, anteiso-C15 : 0 and iso-C15 : 0. The principal polar lipids are diphosphatidylglycerol, phosphatidylglycerol and one glycolipid. The type strain, IY07-56T (5NBRC 106454T5DSM 26153T), was isolated from a soil sample from a pineapple field on Iriomote Island, Okinawa, Japan. Strain IY07-113 (5NBRC 106455), from a forest soil sample from Iriomote Island, is a second strain of the species. The DNA G+C content of the type strain is 71.7 mol%.
Acknowledgements This study, in part, was supported by a research grant from the Institute for Fermentation, Osaka (IFO).
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