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Amycolatopsis Acidicola Sp. Nov., Isolated from Peat Swamp Forest Soil

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Amycolatopsis Acidicola Sp. Nov., Isolated from Peat Swamp Forest Soil TAXONOMIC DESCRIPTION Teo et al., Int. J. Syst. Evol. Microbiol. 2020;70:1547–1554 DOI 10.1099/ijsem.0.003933 Amycolatopsis acidicola sp. nov., isolated from peat swamp forest soil Wee Fei Aaron Teo, Nantana Srisuk and Kannika Duangmal* Abstract A novel actinobacterial strain, designated K81G1T, was isolated from a soil sample collected in Kantulee peat swamp forest, Surat Thani Province, Thailand, and its taxonomic position was determined using a polyphasic approach. Optimal growth of strain K81G1T occurred at 28–30 °C, at pH 5.0–6.0 and without NaCl. Strain K81G1T had cell- wall chemotype IV (meso- diaminopimelic acid as the diagnostic diamino acid, and arabinose and galactose as diagnostic sugars) and phospholipid pattern type II, characteristic of the genus Amycolatopsis. It contained MK-9(H4) as the predominant menaquinone, iso- C16 : 0, C17 : 0 cyclo and C16 : 0 as the major cellular fatty acids, and phospholipids consisting of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol and two unidentified phospholipids. Based on 16S rRNA gene sequence similarity and phylogenetic analyses, strain K81G1T was most closely related to Amycolatopsis rhizosphaerae TBRC 6029T (97.8 % similarity), Amycolatopsis acidiphila JCM 30562T (97.8 %) and Amycolatopsis bartoniae DSM 45807T (97.6 %). Strain K81G1T exhibited low average nucleotide identity and digital DNA–DNA hybridization values with A. rhizosphaerae TBRC 6029T (76.4 %, 23.0 %), A. acidiphila JCM 30562T (77.9 %, 24.6 %) and A. bartoniae DSM 45807T (77.8 %, 24.3 %). The DNA G+C content of strain K81G1T was 69.7 mol%. Based on data from this polyphasic study, strain K81G1T rep- resents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis acidicola sp. nov. is proposed. The type strain is K81G1T (=TBRC 10047T=NBRC 113896T). The genus Amycolatopsis belongs to the family Pseudonocar- basonyms of Haloechinothrix halophila and Haloechinothrix diaceae within the class Actinobacteria. The family currently salitolerans, respectively, and the inclusion of Yuhushiella encompasses 35 genera with Pseudonocardia as its type genus deserti as Amycolatopsis arida [2]. In general, Amycolatopsis [1–3]. Generally, members of Pseudonocardiaceae are aerobic, strains grow well from pH 6.0 to 9.0, although some strains Gram- stain- positive and catalase-positive. They are typically had been reported to grow below pH 6.0 albeit none are strict characterized by the absence of mycolic acids, the presence acidophiles [1, 5, 6]. In this study, strain K81G1T was taxo- of meso- diaminopimelic acid in the cell wall, the presence nomically characterized as representing a novel species of the of arabinose or galactose in whole-cell hydrolysates, and genus Amycolatopsis. the presence of tetrahydrogenated menaquinones with nine In an effort to characterize the diversity of culturable repeat units [2, 4]. The genus Amycolatopsis is distinguished actinobacteria in tropical peat swamp, a soil sample (pH from members of related genera within Pseudonocardiaceae 4.0) was collected from Kantulee peat swamp forest, Surat based on combinations of 16S rRNA gene phylogeny, pres- Thani Province, Thailand (9° 41′ 15.2′′ N 99° 07′ 02.5′′ E). ence of arabinose and galactose in whole-cell hydrolysates, Strain K81G1T was isolated using a half- strength modified and G+C content of about 65–75 mol% [2, 5–7]. formulation of glycerol asparagine agar [8, 9] [containing 10 g At the time of writing, the genus Amycolatopsis contains glycerol, 1.25 g asparagine, 0.5 g NaCl, 0.05 g each of CaCO3, 77 recognized species following the recent proposal of FeSO4.7H2O and MgSO4.7H2O and 15 g agar in 1000 ml of Amycolatopsis halophila and Amycolatopsis salitolerans as 50 % (v/v) soil extract solution; final pH 5.0]. The medium was Author affiliations: 1Department of Microbiology, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900, Thailand. *Correspondence: Kannika Duangmal, fscikkd@ ku. ac. th Keywords: Amycolatopsis; actinomycete; peat swamp forest; polyphasic taxonomy. Abbreviations: ANI, average nucleotide identity; dDDH, digital DNA–DNA hybridization. The GenBank accession number for the genome sequence of strain K81G1T is VMNW00000000, A. rhizosphaerae TBRC 6029T is VJWX00000000, A. acidiphila JCM 30562T is VJZA00000000 and A. bartoniae DSM 45807T is VJZB00000000. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain K81G1T is MN399887, A. rhizosphaerae TBRC 6029T is MN399890, A. acidiphila JCM 30562T is MN399888 and A. bartoniae DSM 45807T is MN399889. Five supplementary tables and four supplementary figures are available with the online version of this article. 003933 © 2020 The Authors 1547 Reproduced from International Journal of Systematic and Evolutionary Microbiology 70: 1547-1554 (2020). 242 243 TAXONOMIC DESCRIPTION Teo et al., Int. J. Syst. Evol. Microbiol. 2020;70:1547–1554 Teo et al., Int. J. Syst. Evol. Microbiol. 2020;70:1547–1554 DOI 10.1099/ijsem.0.003933 autoclaved prior to incorporation of nalidixic acid, nystatin strain K81G1T formed a distinct lineage that differentiates the and ketaconozole at final concentrations of 25, 50 and 100 µg strain from other related members of the genus Amycolatopsis. ml−1, respectively, to suppress the proliferation of fungal and The general properties of the sequenced genomes of strain non- target bacterial colonies. An air-dried peat soil sample K81G1T, A. rhizosphaerae TBRC 6029T, A. acidiphila JCM Amycolatopsis acidicola sp. nov., isolated from peat swamp (1 g) was suspended in 9 ml of sterilized soil extract solution 30562T and A. bartoniae DSM 45807T are summarized and pretreated in a water bath at 55 °C for 10 min. The suspen- forest soil in Table S1. The genome comparison revealed that strain sion was then serially diluted to 10−3 prior to spread- plating K81G1T had low ANI values to A. rhizosphaerae TBRC 6029T (100 µl) on the modified glycerol asparagine agar. One colony T Wee Fei Aaron Teo, Nantana Srisuk and Kannika Duangmal* T (76.4 %), A. acidiphila JCM 30562 (77.9 %) and A. bartoniae of strain K81G1 was isolated from the plate after incubation DSM 45807T (77.8 %), well below the threshold of 95–96 % at 28 °C for 2 weeks. The colony was then purified on glucose- for species demarcation. Strain K81G1T showed 23.0, 24.6 and yeast extract (GYE) agar [10], acidified to pH 5.0 with 6 M T T 24.3 % dDDH relatedness to A. rhizosphaerae TBRC 6029 , HCl [11]. Strain K81G1 was subsequently maintained and T T Abstract A. acidiphila JCM 30562 and A. bartoniae DSM 45807 , preserved on acidified GYE agar slants at room temperature respectively, values that are below the proposed criterion for A novel actinobacterial strain, designated K81G1T, was isolated from a soil sample collected in Kantulee peat swamp forest, and in a 20 % (v/v) glycerol suspension at –20 °C. bacterial species delineation. Surat Thani Province, Thailand, and its taxonomic position was determined using a polyphasic approach. Optimal growth T Amycolatopsis rhizosphaerae TBRC 6029 , Amycolatopsis T of strain K81G1T occurred at 28–30 °C, at pH 5.0–6.0 and without NaCl. Strain K81G1T had cell- wall chemotype IV (meso- Cell morphology of strain K81G1 was observed under light acidiphila JCM 30562T and Amycolatopsis bartoniae DSM diaminopimelic acid as the diagnostic diamino acid, and arabinose and galactose as diagnostic sugars) and phospholipid microscopy (Eclipse E100, Nikon) and scanning electron 45807T were obtained from the respective culture collec- pattern type II, characteristic of the genus Amycolatopsis. It contained MK-9(H ) as the predominant menaquinone, iso- C , C microscopy (Quanta 450; FEI) from cultures grown on acidi- 4 16 : 0 17 : 0 tions. Genomic DNAs of strain K81G1T, A. rhizosphaerae cyclo and C as the major cellular fatty acids, and phospholipids consisting of phosphatidylglycerol, diphosphatidylglycerol, T T fied International Streptomyces Project (ISP) 2 agar at 28 °C 16 : 0 TBRC 6029 , A. acidiphila JCM 30562 and A. bartoniae for 7 days. The physiological characteristics of strain K81G1T phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol and two unidentified phospholipids. Based DSM 45807T were extracted and purified according to T were compared to those of closely related type strains, namely on 16S rRNA gene sequence similarity and phylogenetic analyses, strain K81G1 was most closely related to Amycolatopsis T Kieser et al. [12]. The whole genomes of strain K81G1 , A. A. rhizosphaerae TBRC 6029T, A. acidiphila JCM 30562T and rhizosphaerae TBRC 6029T (97.8 % similarity), Amycolatopsis acidiphila JCM 30562T (97.8 %) and Amycolatopsis bartoniae DSM T T rhizosphaerae TBRC 6029 , A. acidiphila JCM 30562 and A. bartoniae DSM 45807T. Cultural characteristics on neutral 45807T (97.6 %). Strain K81G1T exhibited low average nucleotide identity and digital DNA–DNA hybridization values with A. T A. bartoniae DSM 45807 were sequenced on an Illumina and acidified ISP media 1–7 [22], and on modified Bennett’s rhizosphaerae TBRC 6029T (76.4 %, 23.0 %), A. acidiphila JCM 30562T (77.9 %, 24.6 %) and A. bartoniae DSM 45807T (77.8 %, HiSeq platform and assembled using SPAdes version 3.7 agar supplemented with mannitol and soybean flour (MBA) 24.3 %). The DNA G+C content of strain K81G1T was 69.7 mol%. Based on data from this polyphasic study, strain K81G1T rep- by MicrobesNG. The final assembled genomes were evalu- [23], were assessed. Generally, media were acidified to pH 5.0 resents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis acidicola sp. nov. is proposed. The type ated with quast version 4.6.3 [13]. The genome annota- with 6 M HCl [11]. Media incorporating 0.1 % (w/v) K HPO T T T T T 2 4 strain is K81G1 (=TBRC 10047 =NBRC 113896 ). tion of strain K81G1 , A. rhizosphaerae TBRC 6029 , A. were substituted with 0.4 % KH PO to achieve pH 5.0 [24]. T T 2 4 acidiphila JCM 30562 and A. bartoniae DSM 45807 was A suspension of each strain (10 µl) was then streaked or spot- implemented in the NCBI prokaryotic genome annotation dropped onto media and incubated for 14 days at 28 °C.
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