DOI: 10.15468/dnprdh
title: Saccharopolyspora rhizosphaerae sp. nov., an actinomycete isolated from rhizosphere soil in Thailand

description: This dataset contains the digitized treatments in Plazi based on the original journal article Intra, Bungonsiri, Euanorasetr, Jirayut, Také, Akira, Inahashi, Yuki, Mori, Mihoko, Panbangred, Watanalai, Matsumoto, Atsuko (2019): Saccharopolyspora rhizosphaerae sp. nov., an actinomycete isolated from rhizosphere soil in Thailand. International Journal of Systematic and Evolutionary Microbiology 69 (5): 1299-1305, DOI: 10.1099/ijsem.0.003307, URL: http://dx.doi.org/10.1099/ijsem.0.003307AbstractA novel actinomycete, designated as strain H 219 T, was isolated from rhizosphere soil collected under an Elephant ear plant (Colocasiaesculenta) in Bangkok, Thailand. Strain H 219 T was characterised using a polyphasic approach. Phylogenetic analysis of the 16S rRNA gene sequences revealed that this isolate was most closely related to Saccharopolyspora tripterygii JCM 32123 T (97.6 %), Saccharopolyspora dendranthemae NBRC 108675 T (97.5 %) and Saccharopolyspora flava NBRC 16345 T (97.5 %). However, DNA – DNA hybridization analyses showed a low relatedness in the range of 39 – 48 % between the novel isolate and the above closely related strains. The cell-wall peptidoglycan of strain H 219 T contained meso -diaminopimelic acid. The diagnostic whole-cell sugars consisted of arabinose and galactose. The cellular fatty acid profile mainly comprised iso-C 16: 0, anteiso-C 17: 0, iso-C 15: 0, and 10-methyl C 17: 0. The major menaquinone was MK-9(H 4). The detected phospholipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylethanolaminecontaining hydroxylated fatty acids and an unknown phospholipid. The DNA G + C content was 70.6 mol%. Strain H 219 T represented chemotaxonomic and morphological characteristics that were consistent with members of the genus Saccharopolyspora. However, strain H 219 T could be distinguished from closely related strains by several phenotypic properties. Based on the data from the polyphasic studies, we propose that strain H 219 T is a novel species within the genus Saccharopolyspora, Saccharopolyspora rhizosphaerae sp. nov. The type strain is H 219 T (= TBRC 8564 T = NBRC 113388 T).The genus Saccharopolyspora was first proposed by Lacey and Goodfellow [1] as a member of the family Pseudonocardieae. The description was emended by Korn-Wendisch et al. [2]. At the time of writing, the genus Saccharopolyspora includes 30 species and three subspecies with validly published names (www.bacterio.net/saccharopolyspora. html). Members of the genus are described as aerobic, nonacid-fast organisms that form extensively branched substrate hyphae that fragment into either rod-shaped elements or aerial mycelia that in turn are transformed into chains of bead-like spores. Strains in the genus Saccharopolyspora contain meso -diaminopimelic acid (DAP) in their cell wall, and arabinose and galactose as the characteristic whole-cell sugars (wall chemotype IV). The cellular fatty acids are isobranched and anteiso-branched-chain fatty acids. The major menaquinone is MK-9(H 4) and the phospholipid pattern is type PIII, consisting of phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylmethylethanolamine. The DNA G + C content of members of this genus is in the range of 66–77 mol% [3 – 5]. Members of the genus Saccharopolyspora are potentially rich sources of bioactive compounds. Examples include erythromycin A, a Saccharopolyspora erythraea product that is used clinically as an antibiotic against pathogenic Grampositive bacteria [6]; sporaricins A, B, C, D and E, aminoglycoside products of Saccharopolyspora hirsuta subsp. kobensis shown to possess antimicrobial activity [7]; cebulactams A1 and A2, new macrolactams with antioxidant activity, that have been isolated from Saccharopolyspora cebuensis [8]; and spinosyns A and D, novel compounds with a broad pesticidal/insecticidal activity that have been obtained from Saccharopolyspora spinosa [9].During a search for new bioactive compounds, many rare and slow-growing actinomycetes were isolated and identified. Strain H 219 T was isolated from a soil sample collected from the rhizosphere of an Elephant ear plant (Colocasia esculenta) in Bangmod district, Jomthong, Bangkok, Thailand. The soil sample was air-dried for 1 week and was subjected to a pretreatment using dry heat at 120 Ǫ C for 1 h [10]. Ten-fold serially diluted soil suspensions were spread on humic acid–vitamin agar [11] supplemented with 25 µg ml ―1 nalidixic acid and 50 µg ml ―1 cycloheximide. The isolation plates were incubated for 4 weeks at 28 Ǫ C. The purified culture was maintained on yeast extract–malt extract agar (International Streptomyces Project, ISP 2 medium) [12] at 20 Ǫ C and stored in 20 % (v/v) glycerol at ―80 Ǫ C. Biomass for chemical and molecular studies was harvested by centrifugation after cultivation in YD broth (1 % yeast extract, 1 % dextrose, pH 7.0) at 27 Ǫ C for 7 days.Table 1. Differential physiological and biochemical characteristics of strain H219T and its closely related type strains Strains: 1, Saccharopolyspora rhizosphaerae sp. nov. H219T; 2, S. tripterygii JCM 32123T; 3, S. dendranthemae NBRC 108675T; 4, S. flava NBRC 16345T. All tests were conducted in duplicate. +, Positive; –, negative; W, weakly positive; ND, not determined.Characteristic 1 2 3 4 Utilization of sole carbon sources: D- Arabinose + – + + Melibiose – – – + L- Rhamnose + + + + Raffinose – – + + myo -Inositol + + – + Utilization of sole nitrogen sources: Adenine – + w w Glycine – + + + L- Histidine + W W + L- Valine + W W W Acid production from: D- Arabinose + – + – D- Fructose – + + + D- Mannitol – + + + Raffinose – – – + L- Rhamnose + – + + Nitrate reduction* + – – + Gelatin liquefaction* + – + ND Starch hydrolysis + – + + Milk peptonization and coagulation* +,– +,+ +,+ ND Casein hydrolysis + – – – Growth temperature range* (Ǫ C) (optimum) 18–37 (22–36) 10–37 15–37 (28) 28–37 Growth pH range 6.0–11.0 5.0–11.0 6.0–11.0 5.0–11.0 Growth in NaCl (%, w/v) (optimum) 1–14 (1–4) 1–14 (1–6) 1–15 (1–12) 1–14 (1–12) API ZYM test results: a- Chymotrypsin – W – + a- Galactosidase – W – + a- Glucosidase – – – W b- Galactosidase – + – W b- Glucuronidase – W – W Trypsin – + + + *Data for the reference strains were taken from Li et al. [40], Zhang et al. [41] and Lu et al. [42].The cultural characteristics of strain H 219 T and three closely related type strains identified in EzBioCloud [Saccharopolyspora tripterygii JCM 32123 T (=YIM65359 T), Saccharopolyspora dendranthemae NBRC 108675 T (=KLBMP1305 T) and Saccharopolyspora flava NBRC 16345 T (= AS 4.1520 T)] were observed after cultivation at 27 Ǫ C for 14–21 days on the ISP media 2 (Difco), 3 (DAIGO, Nihon Pharmaceutical), 4 (Difco), 5 (DAIGO), 6 (DAIGO) and 7 (DAIGO) [12], and on three additional media, namely nutrient agar (Difco), Czapek’ s agar (Oxoid) and potato dextrose agar (Difco). The colours of aerial and substrate mycelia and any soluble pigments produced were determined by comparison with The Colour Harmony Manual [13]. Strain H 219 T was able to grow on all media tested with substrate mycelium colour varying from pale yellow to light-brown. Sparse aerial mycelium was produced only on ISP 2 agar. (Table S1, available in the online version of this article). Soluble pigments were not observed in any of the media tested.Table 2. Fatty acid compositions (%) of strain H219 T and the closely related strainsStrains: 1, Saccharopolyspora rhizosphaerae sp. nov. H219 T; 2, S. tripterygii JCM 32123 T; 3, S. dendranthemae NBRC 108675 T; 4, S. flava NBRC 16345 T. All data were obtained from this study. Fatty acid contents of &lt;1.0 % are omitted. –, Not detected or less than 1.0 %.Fatty acid 1 2 3 4 C15:0 2.2 – – – C17:0 2.0 1.3 1.0 1.4 C16:1 2-OH – – – 1.0 C16:1 Ɯ 9 c 1.4 2.0 1.7 – C17:1 Ɯ 9 c 6.4 5.2 5.5 – iso-C14: 0 1.5 1.0 – 1.1 iso-C15: 0 10.6 8.7 6.5 10.4 iso-C16: 0 28.3 30.3 23.3 20.7 iso-C17: 0 5.6 6.4 5.9 11.0 iso-C16: 1 h 1.4 2.0 3.5 2.8 anteiso-C15: 0 2.6 3.0 3.0 3.6 anteiso-C17: 0 16.1 26.8 33.0 27.8 10-Methyl C16: 0 3.2 3.8 4.6 5.0 10-Methyl C17: 0 14.1 5.0 5.0 7.3Morphological characteristics of strain H 219 T were observed under light microscopy (Model Labophot-2, Nikon) and scanning electron microscopy (JSM-5600, JEOL) after incubation on ISP 2 at 27 Ǫ C for 28 days. Samples for scanning electron microscopy were prepared using the previously described methods [14]. Strain H 219 T produced short spore chains comprising three to eight spores each. Spores were spherical in shape with a warty surface and diameter of approximately 0.8–1 µm (Fig. S1).Test of growth at various temperatures (4–50 Ǫ C; temperature gradient incubator TN-2148, Advantec), pH (pH 3.0– 11.0, at 1 pH unit intervals) and NaCl concentrations (1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15 and 20 %, w/v) were determined on ISP 2 basal media after 21 days of incubation. The capacity to use each of several substrates as carbon and nitrogen sources was examined by the methods described by Shirling and Gottlieb [15] and Williams et al. [16], respectively. Tests also were performed to investigate the strains’ activities for starch hydrolysis, hydrogen sulfide production, melanin production, nitrate reduction, gelatin degradation and casein hydrolysis using the following media: ISP 4, ISP 6, ISP 7, ISP 8 (0.5 % peptone, 0.3 % beef extract, 0.5 % KNO 3, pH 7.0), peptone–glucose–gelatin (2.0 % glucose, 0.5 % peptone, 20 % gelatin, pH 7.0) and skim milk agar [10 % skim milk (Difco), 1.5 % agar, pH 7.0], respectively. For coagulation and peptonization of milk, 10 % skim milk (Difco) was used. Catalase activity was detected based on the production of bubbles after the addition of a drop of 3 % (v/v) H 2 O 2. Oxidase activity was detected based on the oxidation of tetramethyl- p -phenylenediamine [17]. Enzyme activities were determined with the commercial API ZYM System kit (bio- Ḿerieux), according to the manufacturer’ s instructions. Strain H 219 T was able to grow at 18–37 Ǫ C with an optimal temperature range of 22–36 Ǫ C and at pH 6.0–11.0 with optimal growth at pH 7.0. In addition, strain H 219 T grew in the presence of NaCl concentrations of up to 14 %, with optimal growth observed at 1–4 % NaCl concentration. Enzyme activity test results, investigated by using the API ZYM system, were positive for acid phosphatase, alkaline phosphatase, a- mannosidase, b- glucosidase, cystine arylamidase, esterase (C 4), esterase lipase (C 8), leucine arylamidase, N -acetyl-b- glucosaminidase, naphthol-AS-BIphosphohydrolase, lipase and valine arylamidase; and negative for a- chymotrypsin, a- fucosidase, a- galactosidase, a- glucosidase, b- galactosidase, b- glucuronidase and trypsin. As for nitrate reduction, both strain H 219 T and S. flava NBRC 16345 T had positive nitrate reductase activity results, whereas the other two strains had negative results. The ability to hydrolyse casein was one of the properties distinguishing the strain H 219 T from the reference strains, which showed positive results. Detailed physiological characteristics are provided in Table 1 and the species description.The isomers of DAP and sugars within the whole-cell hydrolysate were detected on cellulose plates by thin-layer chromatography (TLC), which was performed as described by Staneck and Roberts [18]. The N -acyl types of muramic acid in peptidoglycan were determined as described by Uchida and Aida [19]. The presence of mycolic acid was determined by TLC following the method of Tomiyasu [20]. Isoprenoid quinones from freeze-dried biomass were extracted [21] and subsequently analysed by liquid chromatography/mass spectrometry (JMS-T100LP, JEOL) with a CAPCELL PAK C 18 UG120 column (Shiseido) using methanol/isopropanol (7: 3, v/v) and UV detection at 270 nm. The phospholipids were extracted and analysed by two-dimensional TLC according to Minnikin et al. [22]. Methyl esters of cellular fatty acids were prepared by direct transmethylation with methanolic hydrochloride. Identification of cellular fatty acid composition was performed on a Hewlett Packard 6890 (Agilent Technologies) gas chromatograph assisted by the ACTIN6 database, according to guidelines of the Sherlock Microbial Identification System (Microbial ID; MIDI, version 6.0).The whole-organism hydrolysate of strain H 219 T contained meso -DAP as the cell-wall diamino acid, and arabinose and galactose as diagnostic whole-cell sugars, corresponding to wall chemotype IV [23]. The type of muramic acid was acetyl and mycolic acids were not detected. The menaquinones found in strain H 219 T were 97.5 % MK-9(H 4) and 2.5 % MK- 9(H 6). Phospholipids consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylethanolamine-containing hydroxylated fatty acids and an unknown phospholipid (Fig. S2). The predominant fatty acids (&gt;10 %) were iso-C 16: 0 (28.3 %), anteiso-C 17: 0 (16.1 %), iso-C 15: 0 (10.6 %), and 10- methyl C 17: 0 (14.1 %) (Table 2). All these properties are consistent with the general chemotaxonomic features of the genus Saccharopolyspor a [3 – 5].Fig. 1. Neighbour-joining phylogenetic tree based on the 16S rRNA gene sequences, showing the genetic relatedness between strain H219 T and other type strains in the genus Saccharopolyspora. Streptosporangium roseum DSM 43021 T (NR074558) was used as the outgroup. Bootstrap values above 50 % or higher (percentages of 1000 replications) are shown. Bar, 0.01 substitutions per nucleotide position. Filled circles indicate branches that are also recovered in the maximum-parsimony and the maximum-likelihood trees (see Figs S3 and S4).Total genomic DNA of strain H 219 T was extracted using a sonication method [24]. PCR amplification and sequencing of the 16S rRNA gene were carried out as previously described [25], using an automated sequencer (31030 Genetic Analyser, Applied Biosystems) according to the manufacturer’ s instructions. The sequence similarities between the new isolate and available reference strains were computed using the EzBioCloud server (https://www.ezbiocloud.net) [26]. Using CLUSTAL_X software [27], an almostcomplete 16S rRNA gene sequence (1441 bp) from strain H 219 T was aligned with multiple sequences of available Saccharopolyspora type strains. Phylogenetic trees were reconstructed by using the neighbour-joining [28], maximum-parsimony [29], and maximum-likelihood [30] treemaking algorithms using MEGA 7 software [31]. Evolutionary distance matrices were calculated according to Kimura’ s two-parameter model [32]. The robustness of the tree topologies was evaluated by bootstrap analysis with 1000 replicates [33]. Fluorometric DNA–DNA hybridization (DDH) was performed in microdilution wells using a photobiotin-labelling approach according to the method of Ezaki et al. [34]. The extraction and purification of chromosomal DNA for genome sequence analysis were performed according to the method of Saito and Miura [35]. The draft genome of strain H 219 T was sequenced by following the paired-end method and using the Illumina HiSeq platform. The G + C content of the strain H 219 T genomic DNA was determined according to the genomic DNA sequences. The genome of S. flava NBRC 16345 T (GenBank accession no. FOZX00000000) was obtained from NCBI. The genomic similarities between strain H 219 T and S. flava NBRC 16345 T were analysed with the average nucleotide identity (ANI) algorithm using OrthoANIu tool [36], and digital DDH (dDDH) analysis using the Genome-to-Genome Distance Calculator (GGDC) 2.1 platform [37].Based on pairwise comparison of the nearly complete 16S rRNA gene sequences, the strains that displayed the highest similarity to strain H 219 T were S. tripterygii JCM 32123 T (97.6 %), S. dendranthemae NBRC 108675 T (97.5 %) and S. flava NBRC 16345 T (97.5 %). Sequence similarity values of strain H 219 T to the type strains of members of the genus ranged from 94–97 %. Furthermore, phylogenetic analysis showed that strain H 219 T formed a clade with other members of the genus Saccharopolyspora (Fig. 1). However, strain H 219 T exhibited a distinct phyletic relationship with the closely related strains: S. tripterygii JCM 32123 T, S. dendranthemae NBRC 108675 T and S. flava NBRC 16345 T at a high bootstrap value of 94 % from the neighbour-joining tree. The phylogenetic relationships also were supported by the maximum-parsimony and maximum-likelihood trees, as shown in Figs S3 and S4, respectively. In contrast to the similarity observed in the 16S rRNA gene, the DDH relatedness values from comparisons of strain H 219 T to S. tripterygii JCM 32123 T, S. dendranthemae NBRC 108675 T and S. flava NBRC 16345 T (via two independent experiments each) were 39–40 %, 39–41 % and 43–48 %, respectively. All of the scores were clearly below the cut-off level of 70 % for the delineation of species [38], indicating that strain H 219 T is distinct from the other closely related species. Draft genome sequencing of strain H 219 T yielded a genome of 5 824 817 bp after assembly, which produced 36 scaffolds with an N 50 value of 345 543 bp. The G + C content of strain H 219 T, calculated from the draft genome sequence, was 70.6 mol%. The ANI value for the comparison of strain H 219 T to S. flava NBRC 16345 T was 87.49 %. The H 219 T dDDH value was 52 % compared to S. flava NBRC 16345 T. This value was below the bacterial species threshold demarcation [39].According to the morphological characteristics, phylogenetic data and chemotaxonomic results, the new isolate belongs to the genus Saccharopolyspora. However, the results of DNA–DNA relatedness data, together with culture differences, clearly distinguished the isolate from other closely related members of this genus. The major distinctions observed between strain H 219 T and S. tripterygii JCM 32123 T, S. dendranthemae NBRC 108675 T and S. flava NBRC 16345 T include carbon and nitrogen utilisation, degradative properties and enzyme activities, gelatin liquefaction, growth temperature and pH, nitrate reduction, and tolerance to NaCl. Strain H 219 T showed differential biochemical and physiological properties compared to the reference strains; these properties are summarised in Table 1. In addition, the culture characteristics of strain H 219 T (e.g. colour of both vegetative and aerial mycelia on standard media, and growth on various substrates) differed from those of the reference strains. Therefore, strain H 219 T should be classified as a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora rhizosphaerae sp. nov. is proposed.