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-C 16 : 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 represents 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.

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. pipeline [14]. Carbon source utilization, nitrogen source utilization The genus Amycolatopsis belongs to the family Pseudonocar- basonyms of Haloechinothrix halophila and Haloechinothrix T The 16S rRNA gene sequences of strain K81G1 , A. rhizos- and substrate degradation were assessed on basal media diaceae within the class Actinobacteria. The family currently salitolerans, respectively, and the inclusion of Yuhushiella T T phaerae TBRC 6029 , A. acidiphila JCM 30562 and A. barto- according to Flowers and Williams [24] at 28 °C for 14 days. encompasses 35 genera with Pseudonocardia as its type genus deserti as Amycolatopsis arida [2]. In general, Amycolatopsis T [1–3]. Generally, members of Pseudonocardiaceae are aerobic, strains grow well from pH 6.0 to 9.0, although some strains niae DSM 45807 extracted from the whole genome sequence Briefly, carbon source utilization was assessed on acidic Gram- stain- positive and catalase-positive. They are typically had been reported to grow below pH 6.0 albeit none are strict were compared to sequences of type strains in the EzBio- basal medium, pH 5.0, containing 1 % carbon source, 0.1 % T Cloud database [15]. Pairwise similarity was calculated by characterized by the absence of mycolic acids, the presence acidophiles [1, 5, 6]. In this study, strain K81G1 was taxo- (NH4)2SO4, 0.05 % MgSO4.7H2O, 0.05 % NaCl, 0.001 % using TaxonDC [16]. Evolutionary trees based on 16S rRNA of meso- diaminopimelic acid in the cell wall, the presence nomically characterized as representing a novel species of the FeSO4.7H2O and 0.4 % KH2PO4 (w/v). In neutral basal of arabinose or galactose in whole-cell hydrolysates, and genus Amycolatopsis. gene sequences were inferred with the neighbour- joining, medium, pH 7.0, 0.1 % l- asparagine was substituted for maximum- parsimony and maximum- likelihood tree- making the presence of tetrahydrogenated menaquinones with nine (NH4)2SO4, and 0.1 % K2HPO4 for KH2PO4. Additionally, In an effort to characterize the diversity of culturable repeat units [2, 4]. The genus Amycolatopsis is distinguished algorithms after clustal w alignment in the mega7 software acid production on neutral media was assessed by addition actinobacteria in tropical peat swamp, a soil sample (pH from members of related genera within Pseudonocardiaceae package [17]. The overall genome- related index among strain of a drop of 0.09 % phenol red as a pH indicator. Positive 4.0) was collected from Kantulee peat swamp forest, Surat T based on combinations of 16S rRNA gene phylogeny, pres- K81G1 and closely related species of the genus Amycolatopsis acid production was confirmed when phenol red turned Thani Province, Thailand (9° 41′ 15.2′′ N 99° 07′ 02.5′′ E). ence of arabinose and galactose in whole-cell hydrolysates, was calculated based on average nucleotide identity (ANI) to yellow. Nitrogen source utilization was assessed on basal Strain K81G1T was isolated using a half- strength modified and G+C content of about 65–75 mol% [2, 5–7]. via blast+ (JSpeciesWS) and in silico digital DNA–DNA medium containing 0.1 % nitrogen source, 1 % glucose, 0.05 % formulation of glycerol asparagine agar [8, 9] [containing 10 g hybridization (dDDH; Genome- to- Genome Distance Calcu- MgSO4.7H2O, 0.05 % NaCl, 0.001 % FeSO4.7H2O and either At the time of writing, the genus Amycolatopsis contains glycerol, 1.25 g asparagine, 0.5 g NaCl, 0.05 g each of CaCO , lator) [18–20]. Phylogenomic trees were inferred using the 3 0.4 % KH2PO4 (acidic medium) or 0.1 % K2HPO4 (neutral 77 recognized species following the recent proposal of FeSO4.7H2O and MgSO4.7H2O and 15 g agar in 1000 ml of autoMLST webserver [with ‘Fast alignment mode (MAFFT medium). Substrate degradation was assessed on complete Amycolatopsis halophila and Amycolatopsis salitolerans as 50 % (v/v) soil extract solution; final pH 5.0]. The medium was FFT- NS-2)’ option] [21]. basal medium containing 1 % glucose, 0.05 % MgSO4.7H2O, 0.05 % NaCl, 0.001 % FeSO .7H O together with either 0.1 % Pairwise sequence alignment of the complete 16S rRNA gene 4 2 T (NH ) SO and 0.4 % KH PO (acidic medium) or 0.1 % sequence of strain K81G1 showed closest similarity to A. 4 2 4 2 4 Author affiliations: 1Department of Microbiology, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900, Thailand. l- asparagine and 0.1 % K HPO (neutral medium), and rhizosphaerae TBRC 6029T (97.8 %), A. acidiphila JCM 30562T 2 4 *Correspondence: Kannika Duangmal, fscikkd@ ku. ac. th supplemented with substrate at concentrations according Keywords: Amycolatopsis; actinomycete; peat swamp forest; polyphasic taxonomy. (97.8 %) and A. bartoniae DSM 45807T (97.6 %). Pairwise to Williams et al. [25] and Bora [26]. Additional phenotypic Abbreviations: ANI, average nucleotide identity; dDDH, digital DNA–DNA hybridization. similarities to other Amycolatopsis species ranged from 97.2 to The GenBank accession number for the genome sequence of strain K81G1T is VMNW00000000, A. rhizosphaerae TBRC 6029T is VJWX00000000, characteristics were determined using the API 20NE system 94.0 %. The phylogenomic tree (Fig. 1) as well as neighbour- A. acidiphila JCM 30562T is VJZA00000000 and A. bartoniae DSM 45807T is VJZB00000000. The GenBank/EMBL/DDBJ accession number for the (bioMérieux) with either acidic or neutral basal media. 16S rRNA gene sequence of strain K81G1T is MN399887, A. rhizosphaerae TBRC 6029T is MN399890, A. acidiphila JCM 30562T is MN399888 and A. joining (Fig. 2), maximum- likelihood (Fig. S1, available in the bartoniae DSM 45807T is MN399889. online version of this article) and maximum- parsimony (Fig. The temperature range for growth of the strains was assessed Five supplementary tables and four supplementary figures are available with the online version of this article. S2) trees based on 16S rRNA gene sequences revealed that on acidic complete basal medium using a temperature

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T 71 Amycolatopsis kentuckyensis NRRL B-24129 (MUMI00000000) 100 Amycolatopsis rifamycinica DSM 46095T (JMQI00000000) 100 Amycolatopsis tolypomycina DSM 44544T (FNSO00000000) 100 Amycolatopsis vancoresmycina NRRL B-24208T (JNYY00000000) Amycolatopsis balhimycina DSM 44591T (ARBH00000000) 100 100 Amycolatopsis vastitatis H5T (NMUL00000000) T 100 Amycolatopsis lexingtonensis NRRL B-24131 (MUMJ00000000) 100 Amycolatopsis eburnea GLM-1T (RSEC00000000) 100 Amycolatopsis pretoriensis DSM 44654T (FNUJ00000000) Amycolatopsis australiensis DSM 44671T (FPJG00000000) T 100 Amycolatopsis rubida DSM 44637 (FOWC00000000) T 100 100 Amycolatopsis circi DSM 45561 (PQIA00000000) 100 Amycolatopsis niigatensis DSM 45165T (PJMY00000000) 100 Amycolatopsis benzoatilytica AK 16-65T (ARPK00000000)

0.1 100 Amycolatopsis jejuensis NRRL B-24427T (JNYZ00000000) Amycolatopsis panacis YIM PH 21725T (QZFV00000000) 100 100 Amycolatopsis sulphurea DSM 46092T (PDJK00000000) Amycolatopsis saalfeldensis DSM 44993T (FOEF00000000) 100 T 100 Amycolatopsis keratiniphila subsp keratiniphila DSM 44409 (LQMT00000000) 100 Amycolatopsis keratiniphila subsp nogabecina FH 1893T (LT629789) 100 Amycolatopsis japonica MG417-CF17T (CP008953) 100 Amycolatopsis lurida DSM 43134T (FNTA00000000) Amycolatopsis decaplanina DSM 44594T (AOHO00000000) 75 Amycolatopsis azurea DSM 43854T (MUXN00000000) 100 100 Amycolatopsis thailandensis JCM 16380T (NMQT00000000) 100 T 100 Amycolatopsis alba DSM 44262 (ARAF00000000) Amycolatopsis regifaucium DSM 45072T (FOPQ00000000) 100 Amycolatopsis orientalis KCTC 9412T (ASJB00000000) 96 Amycolatopsis coloradensis DSM 44225T (MQUQ00000000) Amycolatopsis xylanica CPCC 202699T (FNON00000000) 51 T 100 ‘Amycolatopsis alkalitolerans’ SYSUP0005 (VDFW00000000) T 100 Amycolatopsis acidiphila JCM 3056 (VJZA00000000) Amycolatopsis sacchari DSM 44468T (FORP00000000) 100 100 Amycolatopsis bartoniae DSM 45807T (VJZB00000000) 100 Amycolatopsis acidicola K81G1T (VMNW00000000) Amycolatopsis rhizosphaerae TBRC 6029T (VJWX00000000) 100 Amycolatopsis taiwanensis DSM 45107T (JAFB00000000) 100 Amycolatopsis eurytherma DSM 44348T (RKHY00000000) 100 T 100 Amycolatopsis thermoﬂava N1165 (AXBH00000000) Amycolatopsis methanolica 239T (CP009110) 100 Amycolatopsis thermalba NRRL B-24845T (PQHW00000000) T 100 Amycolatopsis palatopharyngis DSM 44832 (PQHZ00000000) T 93 Amycolatopsis marina CGMCC 4.3568 (FOKG00000000) 100 Amycolatopsis cihanbeyliensis DSM 45679T (VFML00000000) 82 99 Amycolatopsis arida CGMCC 4.5579T (FOWW00000000) Amycolatopsis nigrescens CSC17Ta-90T (ARVW00000000) 82 Amycolatopsis albispora WP1T (CP015163) 100 Amycolatopsis suaedae 8-3EHSuT (SFCC00000000) Amycolatopsis antartica AU-G6T (NKYE00000000) Haloechinothrix (Amycolatopsis) halophila YIM 93223T (AZAK00000000) Nocardia farcinica NBRC 15532T (BDBJ00000000)

Fig. 1. Maximum- likelihood tree based on 85 concatenated gene sequences (with ‘Run ModelFinder’ and ‘Filter inconsistent MLST genes’ options) showing the relationships between strain K81G1T and Amycolatopsis species. Nocardia farcinica NBRC 15532T was used as the outgroup. Numbers on branches indicate the percentage bootstrap values of 1000 replicates (with ‘Perform IQ- TREE Ultrafast Bootstrap analysis’ option); only values >50 % are indicated. Bar, 0.1 substitutions per nucleotide position.

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Amycolatopsis kentuckyensis NRRL B-24129T (MUMI00000000) Amycolatopsis keratiniphila subsp. keratiniphila DSM 44409T (LQMT01000206) 71 79* 100 Amycolatopsis rifamycinica DSM 46095T (JMQI00000000) 99* Amycolatopsis keratiniphila subsp. nogabecina DSM 44586T (AJ508238) 100 Amycolatopsis tolypomycina DSM 44544T (FNSO00000000) Amycolatopsis orientalis DSM 40040T (ASJB01000052) 100 Amycolatopsis vancoresmycina NRRL B-24208T (JNYY00000000) 74 T Amycolatopsis balhimycina DSM 44591T (ARBH00000000) Amycolatopsis minnesotensis 32U-2 (DQ076482) 100 100 Amycolatopsis vastitatis H5T (NMUL00000000) 93* Amycolatopsis nigrescens CSC17Ta-90T (ARVW01000001) T 100 Amycolatopsis lexingtonensis NRRL B-24131 (MUMJ00000000) Amycolatopsis magusensis KT2025T (HQ157190) 100 Amycolatopsis eburnea GLM-1T (RSEC00000000) 100 Amycolatopsis suaedae 8-3EHSuT (SFCC01000032) Amycolatopsis pretoriensis DSM 44654T (FNUJ00000000) 0.01 T Amycolatopsis australiensis DSM 44671T (FPJG00000000) Amycolatopsis cihanbeyliensis BNT52 (JN989302) T T 100 Amycolatopsis rubida DSM 44637 (FOWC00000000) Amycolatopsis jiangsuensis KLBMP 1262 (JQ819253) T 100 100 Amycolatopsis circi DSM 45561 (PQIA00000000) 86 Amycolatopsis sulphurea DSM 46092T (PDJK01000002) 100 Amycolatopsis niigatensis DSM 45165T (PJMY00000000) Amycolatopsis rhizosphaerae TBRC 6029T (MN399890) 100 Amycolatopsis benzoatilytica AK 16-65T (ARPK00000000) T 0.1 100 Amycolatopsis jejuensis NRRL B-24427T (JNYZ00000000) 92 Amycolatopsis dongchuanensis YIM 75904 (JN656710) 99* Amycolatopsis panacis YIM PH 21725T (QZFV00000000) Amycolatopsis sacchari DSM 44468T (jgi.1085899) 100 T 100 Amycolatopsis sulphurea DSM 46092 (PDJK00000000) Amycolatopsis acidicola K81G1T (MN399887) Amycolatopsis saalfeldensis DSM 44993T (FOEF00000000) Amycolatopsis acidiphila JCM 30562T (MN399888) 100 Amycolatopsis keratiniphila subsp keratiniphila DSM 44409T (LQMT00000000) 100 99* T T DSM 45807 (MN399889) 100 Amycolatopsis keratiniphila subsp nogabecina FH 1893 (LT629789) Amycolatopsis bartoniae T 100 Amycolatopsis japonica MG417-CF17T (CP008953) Amycolatopsis albispora WP1 (KT751086) 100 T Amycolatopsis lurida DSM 43134 (FNTA00000000) Amycolatopsis xylanica CPCC 202699T (FJ529702) Amycolatopsis decaplanina DSM 44594T (AOHO00000000) 75 Amycolatopsis marina CGMCC 4.3568T (jgi.1076316) Amycolatopsis azurea DSM 43854T (MUXN00000000) 100 100 99* Amycolatopsis palatopharyngis 1BDZT (AF479268) Amycolatopsis thailandensis JCM 16380T (NMQT00000000) 87 100 T Amycolatopsis ruanii NMG112T (HQ668524) 100 Amycolatopsis alba DSM 44262 (ARAF00000000) Amycolatopsis regifaucium DSM 45072T (FOPQ00000000) T 100 99 Haloechinothrix (Amycolatopsis) halophila YIM 93223 (KI632509) T Amycolatopsis orientalis KCTC 9412 (ASJB00000000) 99* T 96 Haloechinothrix (Amycolatopsis) salitolerans TRM F103 (HQ436534) Amycolatopsis coloradensis DSM 44225T (MQUQ00000000) Prauserella rugosa NRRL B-2295T (JOIJ01000045) Amycolatopsis xylanica CPCC 202699T (FNON00000000) 51 T Actinokineospora fastidiosa IMSNU 20054T (AJ400710) 100 ‘Amycolatopsis alkalitolerans’ SYSUP0005 (VDFW00000000) * T T 100 Amycolatopsis acidiphila JCM 3056 (VJZA00000000) Pseudonocardia thermophila DSM 43832 (jgi.1107617) Amycolatopsis sacchari DSM 44468T (FORP00000000) 100 100 T Amycolatopsis bartoniae DSM 45807 (VJZB00000000) Fig. 2. Neighbour- joining tree computed using the Maximum Composite Likelihood method based on 16S rRNA gene sequences of T 100 Amycolatopsis acidicola K81G1 (VMNW00000000) strain K81G1T and 21 Amycolatopsis species with validly published names that shared >96 % pairwise sequence similarity. All positions Amycolatopsis rhizosphaerae TBRC 6029T (VJWX00000000) containing gaps and missing data were eliminated. There were a total of 1373 positions in the final dataset. Asterisks indicate branches 100 Amycolatopsis taiwanensis DSM 45107T (JAFB00000000) that were also recovered from the maximum-parsimony and maximum- likelihood trees. Numbers on branches indicate the percentage 100 Amycolatopsis eurytherma DSM 44348T (RKHY00000000) bootstrap values of 1000 replicates; only values >50 % are indicated. Bar, 0.01 substitutions per nucleotide position. 100 T 100 Amycolatopsis thermoﬂava N1165 (AXBH00000000) Amycolatopsis methanolica 239T (CP009110) 100 Amycolatopsis thermalba NRRL B-24845T (PQHW00000000) T 100 Amycolatopsis palatopharyngis DSM 44832 (PQHZ00000000) gradient incubator (Model TN-3; Toyo Kagaku Sangyo) over were assessed for the isomer of diaminopimelic acid and sugar T 93 Amycolatopsis marina CGMCC 4.3568 (FOKG00000000) the temperature range 14–50 °C (at intervals of 1.0 °C) for compositions by TLC, and detected by 0.2 % ninhydrin in 100 Amycolatopsis cihanbeyliensis DSM 45679T (VFML00000000) 14 days. The pH tolerance (pH 3.0–10.0 at intervals of 1.0 pH acetone and aniline- phthalate reagent, respectively [28–30]. 82 99 Amycolatopsis arida CGMCC 4.5579T (FOWW00000000) unit) of the strains was assessed according to Williams et al. Mycolic acids were examined with silica TLC developed Amycolatopsis nigrescens CSC17Ta-90T (ARVW00000000) [27] at 28 °C for 14 days. Culture growth in the presence of with n- hexane/diethyl ether (4 : 1, v/v) and detected by 50 % 82 Amycolatopsis albispora WP1T (CP015163) different NaCl concentrations (0–10 %, at 1 % intervals) was sulphuric acid [31]. Polar lipid compositions of strain K81G1T 100 Amycolatopsis suaedae 8-3EHSuT (SFCC00000000) assessed on acidified ISP 2 agar, pH 5.0. Enzyme activities were examined by two- dimensional TLC and characterized Amycolatopsis antartica AU-G6T (NKYE00000000) were determined using the API ZYM system (bioMérieux) with 0.2 % ninhydrin in water- saturated n- butanol, 5 % molyb- Haloechinothrix (Amycolatopsis) halophila YIM 93223T (AZAK00000000) following the instructions of the manufacturer. Additionally, dophosphoric acid, anisaldehyde reagent, Dragendorff’s Nocardia farcinica NBRC 15532T (BDBJ00000000) catalase and oxidase activities were determined with 3 % reagent and molybdenum blue reagent [32]. Respiratory (v/v) hydrogen peroxide solution and 1 % (w/v) tetramethyl- quinones purified by silica-based solid phase extraction were p- phenylenediamine dihydrochloride solution, respectively. Fig. 1. Maximum- likelihood tree based on 85 concatenated gene sequences (with ‘Run ModelFinder’ and ‘Filter inconsistent MLST genes’ analysed by HPLC using a reversed- phase column as carried options) showing the relationships between strain K81G1T and Amycolatopsis species. Nocardia farcinica NBRC 15532T was used as the Cell biomass for chemotaxonomic analyses was prepared out by the Identification Service of the German Collection of outgroup. Numbers on branches indicate the percentage bootstrap values of 1000 replicates (with ‘Perform IQ- TREE Ultrafast Bootstrap from a culture grown in ISP 2 broth, pH 5.0, on a rotary Microorganisms and Cell Cultures. Analysis of cellular fatty analysis’ option); only values >50 % are indicated. Bar, 0.1 substitutions per nucleotide position. shaker (180 r.p.m.) at 28 °C for 5 days. Whole- cell hydrolysates acids was carried out by the Biodiversity Research Centre,

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45807T showed complex mixtures of saturated and branched

chain fatty acids, with 14-methylpentadecanoic (iso- C16 : 0) as one of the major components (Table S3). Furthermore, strain K81G1T could be distinguished from A. rhizosphaerae TBRC 6029T, A. acidiphila JCM 30562T and A. bartoniae DSM 45807T based on its phenotypic profile at pH 5.0 (Table 1) and pH 7.0 (Table S4) as well as physiological and chemotaxonomic patterns (Table S5). These Amycolatopsis strains can be clearly distinguished from each other based on carbon utilization and acid production when grown at pH 5.0 and 7.0. For example, when grown on fructose as sole carbon source, strain K81G1T grew at both pH 5.0 and 7.0 and produced acid. However, A. rhizosphaerae TBRC 6029T grew at both pH 5.0 and 7.0 without acid production, A. acidiphila JCM 30562T could not grow at pH 5.0 but grew at pH 7.0 and produced acid, while A. bartoniae DSM 45807T grew at pH 7.0 without acid production and showed no growth at pH 5.0. In contrast, when grown on mannitol as sole carbon source, strain K81G1T grew at both pH 5.0 and 7.0 without acid production, A. rhizosphaerae TBRC 6029T grew at both pH 5.0 and 7.0 and produced acid, A. acidiphila JCM 30562T could not grow at either pH 5.0 or 7.0, while A. bartoniae Fig. 3. Scanning electron micrograph showing rod-shaped fragments T of strain K81G1T when grown on ISP 2 agar, pH 5.0, at 28 °C for 7 days. DSM 45807 could not grow at pH 5.0 but grew at pH 7.0 Bar, 3 µm. and produced acid. Based on the data presented, strain K81G1T represents a novel species of the genus Amycolatopsis, for which the name Thailand Institute of Scientific and Technological Research, Amycolatopsis acidicola sp. nov. is proposed. using the Sherlock Microbial Identification System (version 6.2B, MIDI database: RTSBA6). DESCRIPTION Of AmycolAtopsis The cultural characteristics and appearance of strain K81G1T, A. rhizosphaerae TBRC 6029T, A. acidiphila JCM 30562T AcidicolA SP. NOv. and A. bartoniae DSM 45807T on various agar media are Amycolatopsis acidicola (a. ci. di′ co. la. N.L. neut. n. acidum shown in Table S2. These Amycolatopsis strains can be clearly an acid; L. suff. cola- from L. n. incola an inhabitant; N.L. n. distinguished from each other based on colony morphology acidicola an inhabitant of an acidic environment). (colour of aerial spore mass, substrate mycelia and diffusible Aerobic, Gram-stain- positive, filamentous actinobacte- pigment) when grown on acidified ISP 5 and ISP 7. Addi- rium. Growth occurs on neutral and acidified ISP 1–5, ISP tionally, distinct growth of each strain was observed when 7 and MBA media but not on acidified ISP 6. The aerial simultaneously grown on ISP 4 at pH 5.0 and 7.0. Generally, spore mass is white, and the substrate mycelial colour acidification with 6 M HCl or 0.4 % (w/v) KH PO to achieve 2 4 ranges from greenish yellow (ISP 1) to yellowish white (ISP pH 5.0 showed no adverse effect on growth and morphology. 5). Pale yellowish brown diffusible pigments are produced However, no growth was observed for strain K81G1T while on MBA. Growth occurs at 14–41 °C, at pH 4.0–7.0 and in scant growth was observed for A. rhizosphaerae TBRC 6029T, the presence of 0–4 % (w/v) NaCl, with optimum growth A. acidiphila JCM 30562T and A. bartoniae DSM 45807T on at 28–30 °C, at pH 5.0–6.0 and in the absence of NaCl. acidified ISP 6. Aesculin, gelatin, hypoxanthine, l- tyrosine and Tween 80 Strain K81G1T was Gram-stain- positive and formed exten- are not degraded at pH 5.0 but are degraded at pH 7.0. sively branching mycelium (Fig. S3) that fragmented into Casein (skimmed milk), but not adenine, starch or xylan, rod- shaped elements (Fig. 3). The strain contained meso- is degraded at pH 5.0 and 7.0. Growth occurs at pH 5.0 diaminopimelic acid, arabinose, galactose and ribose in the with adonitol, ammonium nitrate, ammonium sulfate, whole-cell hydrolysates and lacked mycolic acids. Respiratory asparagine, cellobiose, fructose, galactose, glucose, glyc- quinones consisted of MK-9(H4) (75 %), MK-9(H2) (10 %) erol, mannitol, mannose, potassium nitrate, sodium citrate, T and MK-9(H6) (10 %). Strain K81G1 exhibited a type II sodium nitrate, sucrose, xylitol and xylose as sole carbon phospholipid pattern containing phosphatidylethanolamine or nitrogen sources, but not with ammonium chloride, but not phosphatidylcholine (Fig. S4). The chemotaxonomic arabinose, maltose, melibiose, myo- inositol, raffinose, characteristics of strain K81G1T are typical of Amycolatopsis. rhamnose, sodium acetate, sodium propionate or trehalose. The fatty acid profiles of strain K81G1T, A. rhizosphaerae Growth occurs at pH 7.0 with adonitol, ammonium chlo- TBRC 6029T, A. acidiphila JCM 30562T and A. bartoniae DSM ride, ammonium nitrate, ammonium sulfate, asparagine,

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45807T showed complex mixtures of saturated and branched Table 1. Differential characteristics between strain K81G1T and related members of the genus Amycolatopsis T T T T chain fatty acids, with 14-methylpentadecanoic (iso- C16 : 0) as Strains: 1, K81G1 ; 2, A. rhizosphaerae TBRC 6029 ; 3, A. acidiphila JCM 30562 ; 4, A. bartoniae DSM 45807 . Growth was tested at pH 5.0.* +, Positive; one of the major components (Table S3). –, negative.

T Furthermore, strain K81G1 could be distinguished from A. Characteristic 1 2 3 4 rhizosphaerae TBRC 6029T, A. acidiphila JCM 30562T and A. bartoniae DSM 45807T based on its phenotypic profile at pH Growth on sole carbon source 5.0 (Table 1) and pH 7.0 (Table S4) as well as physiological and Fructose + + – – chemotaxonomic patterns (Table S5). These Amycolatopsis strains can be clearly distinguished from each other based Mannose + + – – on carbon utilization and acid production when grown at pH Raffinose – + – – 5.0 and 7.0. For example, when grown on fructose as sole Rhamnose – + + + carbon source, strain K81G1T grew at both pH 5.0 and 7.0 and produced acid. However, A. rhizosphaerae TBRC 6029T grew Sucrose + + – – at both pH 5.0 and 7.0 without acid production, A. acidiphila Trehalose – + – – JCM 30562T could not grow at pH 5.0 but grew at pH 7.0 and produced acid, while A. bartoniae DSM 45807T grew at Xylose + – – – pH 7.0 without acid production and showed no growth at Adonitol + – – – pH 5.0. In contrast, when grown on mannitol as sole carbon source, strain K81G1T grew at both pH 5.0 and 7.0 without Glycerol + + – + acid production, A. rhizosphaerae TBRC 6029T grew at both myo- Inositol – + – – pH 5.0 and 7.0 and produced acid, A. acidiphila JCM 30562T Mannitol + + – – could not grow at either pH 5.0 or 7.0, while A. bartoniae Scanning electron micrograph showing rod-shaped fragments Fig. 3. T Xylitol + – – – of strain K81G1T when grown on ISP 2 agar, pH 5.0, at 28 °C for 7 days. DSM 45807 could not grow at pH 5.0 but grew at pH 7.0 Bar, 3 µm. and produced acid. Growth on sole nitrogen source T Based on the data presented, strain K81G1 represents a Ammonium chloride – + + + novel species of the genus Amycolatopsis, for which the name Thailand Institute of Scientific and Technological Research, Amycolatopsis acidicola sp. nov. is proposed. Substrate degradation using the Sherlock Microbial Identification System (version Aesculin – + – + 6.2B, MIDI database: RTSBA6). Gelatin – + + + T DESCRIPTION Of AmycolAtopsis The cultural characteristics and appearance of strain K81G1 , Hypoxanthine – – + – A. rhizosphaerae TBRC 6029T, A. acidiphila JCM 30562T AcidicolA SP. NOv. and A. bartoniae DSM 45807T on various agar media are Amycolatopsis acidicola (a. ci. di′ co. la. N.L. neut. n. acidum l- Tyrosine – – – + shown in Table S2. These Amycolatopsis strains can be clearly an acid; L. suff. cola- from L. n. incola an inhabitant; N.L. n. Tween 80 – – + + distinguished from each other based on colony morphology acidicola an inhabitant of an acidic environment). (colour of aerial spore mass, substrate mycelia and diffusible API 20NE system results Aerobic, Gram-stain- positive, filamentous actinobacte- pigment) when grown on acidified ISP 5 and ISP 7. Addi- rium. Growth occurs on neutral and acidified ISP 1–5, ISP Reduction of nitrates to nitrites – + – – tionally, distinct growth of each strain was observed when 7 and MBA media but not on acidified ISP 6. The aerial Urease + + – – simultaneously grown on ISP 4 at pH 5.0 and 7.0. Generally, spore mass is white, and the substrate mycelial colour acidification with 6 M HCl or 0.4 % (w/v) KH PO to achieve Protease (gelatinase) – + + + 2 4 ranges from greenish yellow (ISP 1) to yellowish white (ISP pH 5.0 showed no adverse effect on growth and morphology. 5). Pale yellowish brown diffusible pigments are produced β-Galactosidase + – – + However, no growth was observed for strain K81G1T while on MBA. Growth occurs at 14–41 °C, at pH 4.0–7.0 and in scant growth was observed for A. rhizosphaerae TBRC 6029T, Assimilation of d- mannose + + – + the presence of 0–4 % (w/v) NaCl, with optimum growth A. acidiphila JCM 30562T and A. bartoniae DSM 45807T on at 28–30 °C, at pH 5.0–6.0 and in the absence of NaCl. Assimilation of potassium gluconate – – – + acidified ISP 6. Aesculin, gelatin, hypoxanthine, l- tyrosine and Tween 80 Growth temperature range (°C) 14–41 24–41 14–41 14–46 Strain K81G1T was Gram-stain- positive and formed exten- are not degraded at pH 5.0 but are degraded at pH 7.0. sively branching mycelium (Fig. S3) that fragmented into Casein (skimmed milk), but not adenine, starch or xylan, Growth pH range 4–7 4–9 5–8 4–8 rod- shaped elements (Fig. 3). The strain contained meso- is degraded at pH 5.0 and 7.0. Growth occurs at pH 5.0 Growth with NaCl (%) 0–4 0–2 0–3 0–3 diaminopimelic acid, arabinose, galactose and ribose in the with adonitol, ammonium nitrate, ammonium sulfate, *Growth of all strains on cellobiose, galactose and glucose was positive, but negative on arabinose, maltose, melibiose, sodium acetate, sodium whole- cell hydrolysates and lacked mycolic acids. Respiratory asparagine, cellobiose, fructose, galactose, glucose, glyc- citrate and sodium propionate, as sole carbon sources. Growth of all strains on ammonium nitrate, ammonium sulfate, asparagine, potassium quinones consisted of MK-9(H4) (75 %), MK-9(H2) (10 %) erol, mannitol, mannose, potassium nitrate, sodium citrate, nitrate and sodium nitrate was positive, as sole nitrogen sources. All strains degraded casein (skimmed milk), but not adenine, starch or xylan. T and MK-9(H6) (10 %). Strain K81G1 exhibited a type II sodium nitrate, sucrose, xylitol and xylose as sole carbon Results based on API 20NE system showed that all strains were positive for arginine dihydrolase, β-glucosidase, assimilation of d-glucose, d- phospholipid pattern containing phosphatidylethanolamine or nitrogen sources, but not with ammonium chloride, mannitol, maltose, malic acid, trisodium citrate and N-acetyl- glucosamine, but negative for reduction of nitrates to nitrogen, indole production, but not phosphatidylcholine (Fig. S4). The chemotaxonomic arabinose, maltose, melibiose, myo- inositol, raffinose, and assimilation of l-arabinose, adipic acid, capric acid and phenylacetic acid. characteristics of strain K81G1T are typical of Amycolatopsis. rhamnose, sodium acetate, sodium propionate or trehalose. The fatty acid profiles of strain K81G1T, A. rhizosphaerae Growth occurs at pH 7.0 with adonitol, ammonium chlo- TBRC 6029T, A. acidiphila JCM 30562T and A. bartoniae DSM ride, ammonium nitrate, ammonium sulfate, asparagine,

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fructose, glucose, glycerol, mannitol, mannose, potassium 5. Oyuntsetseg B, Cho S- H, Jeon SJ, Lee HB, Shin K- S et al. Amyco- latopsis acidiphila sp. nov., a moderately acidophilic species isolated nitrate, rhamnose, sodium nitrate and xylitol as sole carbon from coal mine soil. Int J Syst Evol Microbiol 2017;67:3387–3392. or nitrogen sources but not arabinose, cellobiose, galac- 6. Thawai C. Amycolatopsis rhizosphaerae sp. nov., isolated from rice tose, maltose, melibiose, myo- inositol, raffinose, sodium rhizosphere soil. Int J Syst Evol Microbiol 2018;68:1546–1551. acetate, sodium citrate, sodium propionate, sucrose, 7. Wang J, Leiva S, Huang J, Huang Y. Amycolatopsis antarctica sp. trehalose or xylose. Acid is produced from adonitol, fruc- nov., isolated from the surface of an Antarctic brown macroalga. tose, glucose, mannose and rhamnose. Positive for acid Int J Syst Evol Microbiol 2018;68:2348–2356. phosphatase, catalase, esterase (C4), esterase lipase (C8), 8. Porter JN, Wilhelm JJ, Tresner HD. Method for the prefer- ential isolation of actinomycetes from soils. Appl Microbiol N- acetyl-β-glucosaminidase and β-glucosidase but nega- 1960;8:174–178. tive for alkaline phosphatase, α-chymotrypsin, cystine 9. Tan GYA, Robinson S, Lacey E, Brown R, Kim W et al. Amyco- arylamidase, α-fucosidase, α-galactosidase, β-galactosidase, latopsis regifaucium sp. nov., a novel actinomycete that produces α-glucosidase, β-glucuronidase, leucine arylamidase, lipase kigamicins. Int J Syst Evol Microbiol 2007;57:2562–2567. (C14), α-mannosidase, naphthol- AS- BI- phosphohydrolase, 10. Waksman SA. The Actinomycetes: Their Nature, Occurrence, Activi- oxidase, trypsin and valine arylamidase. The diagnostic ties, and Importance, 70. Waltham, Massachusetts: Chronica Botanica Company; 1950. p. 161. diamino acid is meso- diaminopimelic acid. Mycolic acids 11. Donadio S. Genus I. Actinospica Cavaletti, Monciardini, Schumann, are absent. Whole-cell sugars consist of arabinose, galactose Rohde, Bamonte, Busti, Sosio and Donadio 2006, 1751VP. In: Good- and ribose. The polar lipid profile contains phosphatidylg- fellow M, Kämpfer P and Busse H- J (editors). Bergey's Manual lycerol, diphosphatidylglycerol, phosphatidylethanolamine, of Systematic Bacteriology, 2012. New York: Springer; 2006. pp. hydroxyphosphatidylethanolamine, phosphatidylinositol, 232–234. 12. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical two unidentified phospholipids, one ninhydrin-positive Streptomyces Genetics. Norwich: John Innes Foundation; 2000. lipid and two unidentified lipids. The predominant 13. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality menaquinone is MK-9(H4). The major fatty acids (>10 %) assessment tool for genome assemblies. Bioinformatics 2013;29:1072–1075. are iso- C16 : 0, C17 : 0 cyclo and C16 : 0. T T T 14. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP The type strain, K81G1 (=TBRC 10047 =NBRC 113896 ), et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids was isolated from a soil sample of Kantulee peat swamp forest, Res 2016;44:6614–6624. Surat Thani Province, Thailand. The DNA G+C content of 15. Yoon S- H, Ha S- M, Kwon S, Lim J, Kim Y et al. Introducing EzBi- the type strain is 69.7 mol%. The GenBank genome accession oCloud: a taxonomically united database of 16S rRNA gene T sequences and whole- genome assemblies. Int J Syst Evol Microbiol number of strain K81G1 is VMNW00000000. The GenBank 2017;67:1613–1617. accession number for the 16S rRNA gene sequence of strain T 16. Tarlachkov SV, Starodumova IP. TaxonDC: calculating the simi- K81G1 is MN399887. larity value of the 16S rRNA gene sequences of prokaryotes or ITS regions of fungi. J Bioinf Genom 2017;3:1–4. 17. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary Funding information genetics analysis version 7.0 for bigger datasets. Mol Biol Evol This research was supported by the Centre of Excellence on Biodiver - 2016;33:1870–1874. sity (BDC), Office of Higher Education Commission (Project Code BDC- 18. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR et al. Proposed PG1-160003), and was supported in part by the Graduate Program minimal standards for the use of genome data for the taxonomy of Scholarship from The Graduate School, Kasetsart University to prokaryotes. Int J Syst Evol Microbiol 2018;68:461–466. T.W.F.A. 19. Meier- Kolthoff JP, Auch AF, Klenk H- P, Göker M. Genome sequence- Acknowledgements based species delimitation with confidence intervals and improved The authors are grateful to Professor Dr Savitree Limtong of Kasetsart distance functions. BMC Bioinformatics 2013;14:1–14. University, the director of the research programme. 20. Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. JSpe- ciesWS: a web server for prokaryotic species circumscription based Conflicts of interest on pairwise genome comparison. Bioinformatics 2016;32:929–931. The authors declare that there are no conflicts of interest. 21. Alanjary M, Steinke K, Ziemert N. AutoMLST: an automated web References server for generating multi- locus species trees highlighting natural product potential. Nucleic Acids Res 2019;47:W276–W282. 1. GYA T, Goodfellow M. Amycolatopsis. In: Whitman WB, Rainey F, Kämpfer P, Trujillo M and Chun J (editors). Bergey's Manual of 22. Shirling EB, Gottlieb D. Methods for characterization of Strepto- Systematics of Archaea and Bacteria. John Wiley & Sons, Inc; 2015. myces species. Int J Syst Bacteriol 1966;16:313–340. 2. Nouioui I, Carro L, García- López M, Meier- Kolthoff JP, Woyke T 23. GYA T, Ward AC, Goodfellow M. Exploration of Amycolatopsis diver- et al. Genome- based taxonomic classification of the phylum Actin- sity in soil using genus-specific primers and novel selective media. obacteria. Front Microbiol 2018;9:2007. Syst Appl Microbiol 2006;29:557–569. 3. Ningsih F, Yokota A, Sakai Y, Nanatani K, Yabe S et al. Gandjariella 24. Flowers TH, Williams ST. Nutritional requirements of acidophilic thermophila gen. nov., sp. nov., a new member of the family Pseu- streptomycetes. Soil Biol Biochem 1977;9:225–226. donocardiaceae, isolated from forest soil in a geothermal area. Int J 25. Williams ST, Goodfellow M, Alderson G, Wellington EMH, Syst Evol Microbiol 2019;69:3080–3086. Sneath PHA et al. Numerical classification of Streptomyces and 4. Labeda DP, Goodfellow M, Chun J, Zhi X- Y, Li W- J. Reassessment related genera. Microbiology 1983;129:1743–1813. of the systematics of the suborder Pseudonocardineae: transfer 26. Bora N. Characterization of actinomycetes from smear ripened of the genera within the family Actinosynnemataceae Labeda and cheeses – A polyphasic approach. In: Bora N, Dodd C and Desmas- Kroppenstedt 2000 emend. Zhi et al. 2009 into an emended family ures N (editors). Diversity, Dynamics and Functional Role of Actino- Pseudonocardiaceae Embley et al. 1989 emend. Zhi et al. 2009. Int J mycetes on European Smear Ripened Cheeses. Cham: Springer Syst Evol Microbiol 2011;61:1259–1264. International Publishing; 2015. pp. 51–101.

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fructose, glucose, glycerol, mannitol, mannose, potassium 5. Oyuntsetseg B, Cho S- H, Jeon SJ, Lee HB, Shin K- S et al. Amyco- 27. Williams S, Davies F, Mayfield C, Khan M. Studies on the ecology 30. Hasegawa T, Takizawa M, Tanida S. A rapid analysis for chem- latopsis acidiphila sp. nov., a moderately acidophilic species isolated of actinomycetes in soil II. The pH requirements of streptomycetes ical grouping of aerobic actinomycetes. J Gen Appl Microbiol nitrate, rhamnose, sodium nitrate and xylitol as sole carbon from coal mine soil. Int J Syst Evol Microbiol 2017;67:3387–3392. from two acid soils. Soil Biol Biochem 1971;3:187–195. or nitrogen sources but not arabinose, cellobiose, galac- 1983;29:319–322. 6. Thawai C. Amycolatopsis rhizosphaerae sp. nov., isolated from rice 28. Becker B, Lechevalier MP, Gordon RE, Lechevalier HA. Rapid differ- 31. Tomiyasu I. Mycolic acid composition and thermally adaptative tose, maltose, melibiose, myo- inositol, raffinose, sodium rhizosphere soil. Int J Syst Evol Microbiol 2018;68:1546–1551. entiation between Nocardia and Streptomyces by paper chromatog- changes in Nocardia asteroides. J Bacteriol 1982;151:828–837. acetate, sodium citrate, sodium propionate, sucrose, 7. Wang J, Leiva S, Huang J, Huang Y. Amycolatopsis antarctica sp. raphy of whole- cell hydrolysates. Appl Microbiol 1964;12:421–423. trehalose or xylose. Acid is produced from adonitol, fruc- nov., isolated from the surface of an Antarctic brown macroalga. 29. Staneck JL, Roberts GD. Simplified approach to identification of 32. Minnikin DE, Patel PV, ALSHAMAONY L, Goodfellow M. Polar lipid tose, glucose, mannose and rhamnose. Positive for acid Int J Syst Evol Microbiol 2018;68:2348–2356. aerobic actinomycetes by thin- layer chromatography. Appl Micro- composition in the classification of Nocardia and related bacteria. biol 1974;28:226–231. Int J Syst Bacteriol 1977;27:104–117. phosphatase, catalase, esterase (C4), esterase lipase (C8), 8. Porter JN, Wilhelm JJ, Tresner HD. Method for the prefer- ential isolation of actinomycetes from soils. Appl Microbiol N- acetyl-β-glucosaminidase and β-glucosidase but nega- 1960;8:174–178. tive for alkaline phosphatase, α-chymotrypsin, cystine 9. Tan GYA, Robinson S, Lacey E, Brown R, Kim W et al. Amyco- arylamidase, α-fucosidase, α-galactosidase, β-galactosidase, latopsis regifaucium sp. nov., a novel actinomycete that produces α-glucosidase, β-glucuronidase, leucine arylamidase, lipase kigamicins. Int J Syst Evol Microbiol 2007;57:2562–2567. (C14), α-mannosidase, naphthol- AS- BI- phosphohydrolase, 10. Waksman SA. The Actinomycetes: Their Nature, Occurrence, Activi- oxidase, trypsin and valine arylamidase. The diagnostic ties, and Importance, 70. Waltham, Massachusetts: Chronica Botanica Company; 1950. p. 161. diamino acid is meso- diaminopimelic acid. Mycolic acids 11. Donadio S. Genus I. Actinospica Cavaletti, Monciardini, Schumann, are absent. Whole-cell sugars consist of arabinose, galactose Rohde, Bamonte, Busti, Sosio and Donadio 2006, 1751VP. In: Good- and ribose. The polar lipid profile contains phosphatidylg- fellow M, Kämpfer P and Busse H- J (editors). Bergey's Manual lycerol, diphosphatidylglycerol, phosphatidylethanolamine, of Systematic Bacteriology, 2012. New York: Springer; 2006. pp. hydroxyphosphatidylethanolamine, phosphatidylinositol, 232–234. 12. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical two unidentified phospholipids, one ninhydrin-positive Streptomyces Genetics. Norwich: John Innes Foundation; 2000. lipid and two unidentified lipids. The predominant 13. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality menaquinone is MK-9(H4). The major fatty acids (>10 %) assessment tool for genome assemblies. Bioinformatics 2013;29:1072–1075. are iso- C16 : 0, C17 : 0 cyclo and C16 : 0. T T T 14. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP The type strain, K81G1 (=TBRC 10047 =NBRC 113896 ), et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids was isolated from a soil sample of Kantulee peat swamp forest, Res 2016;44:6614–6624. Surat Thani Province, Thailand. The DNA G+C content of 15. Yoon S- H, Ha S- M, Kwon S, Lim J, Kim Y et al. Introducing EzBi- the type strain is 69.7 mol%. The GenBank genome accession oCloud: a taxonomically united database of 16S rRNA gene T sequences and whole- genome assemblies. Int J Syst Evol Microbiol number of strain K81G1 is VMNW00000000. The GenBank 2017;67:1613–1617. accession number for the 16S rRNA gene sequence of strain T 16. Tarlachkov SV, Starodumova IP. TaxonDC: calculating the simi- K81G1 is MN399887. larity value of the 16S rRNA gene sequences of prokaryotes or ITS regions of fungi. J Bioinf Genom 2017;3:1–4. 17. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary Funding information genetics analysis version 7.0 for bigger datasets. Mol Biol Evol This research was supported by the Centre of Excellence on Biodiver - 2016;33:1870–1874. sity (BDC), Office of Higher Education Commission (Project Code BDC- 18. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR et al. Proposed PG1-160003), and was supported in part by the Graduate Program minimal standards for the use of genome data for the taxonomy of Scholarship from The Graduate School, Kasetsart University to prokaryotes. Int J Syst Evol Microbiol 2018;68:461–466. T.W.F.A. 19. Meier- Kolthoff JP, Auch AF, Klenk H- P, Göker M. Genome sequence- Acknowledgements based species delimitation with confidence intervals and improved The authors are grateful to Professor Dr Savitree Limtong of Kasetsart distance functions. BMC Bioinformatics 2013;14:1–14. University, the director of the research programme. 20. Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. JSpe- ciesWS: a web server for prokaryotic species circumscription based Conflicts of interest on pairwise genome comparison. Bioinformatics 2016;32:929–931. The authors declare that there are no conflicts of interest. 21. Alanjary M, Steinke K, Ziemert N. AutoMLST: an automated web References server for generating multi-locus species trees highlighting natural product potential. Nucleic Acids Res 2019;47:W276–W282. 1. GYA T, Goodfellow M. Amycolatopsis. In: Whitman WB, Rainey F, Kämpfer P, Trujillo M and Chun J (editors). Bergey's Manual of 22. Shirling EB, Gottlieb D. Methods for characterization of Strepto- Systematics of Archaea and Bacteria. John Wiley & Sons, Inc; 2015. myces species. Int J Syst Bacteriol 1966;16:313–340. 2. Nouioui I, Carro L, García- López M, Meier- Kolthoff JP, Woyke T 23. GYA T, Ward AC, Goodfellow M. Exploration of Amycolatopsis diver- et al. Genome- based taxonomic classification of the phylum Actin- sity in soil using genus-specific primers and novel selective media. obacteria. Front Microbiol 2018;9:2007. Syst Appl Microbiol 2006;29:557–569. five reasons to publish your next article with a Microbiology Society journal 3. Ningsih F, Yokota A, Sakai Y, Nanatani K, Yabe S et al. Gandjariella 24. Flowers TH, Williams ST. Nutritional requirements of acidophilic 1. The Microbiology Society is a not-for-profit organization. thermophila gen. nov., sp. nov., a new member of the family Pseu- streptomycetes. Soil Biol Biochem 1977;9:225–226. 2. We offer fast and rigorous peer review – average time to first decision is 4–6 weeks. donocardiaceae, isolated from forest soil in a geothermal area. Int J 25. Williams ST, Goodfellow M, Alderson G, Wellington EMH, Syst Evol Microbiol 2019;69:3080–3086. Sneath PHA et al. Numerical classification of Streptomyces and 3. Our journals have a global readership with subscriptions held in research institutions around the world. 4. Labeda DP, Goodfellow M, Chun J, Zhi X- Y, Li W- J. Reassessment related genera. Microbiology 1983;129:1743–1813. 4. 80% of our authors rate our submission process as ‘excellent’ or ‘very good’. of the systematics of the suborder Pseudonocardineae: transfer 26. Bora N. Characterization of actinomycetes from smear ripened of the genera within the family Actinosynnemataceae Labeda and cheeses – A polyphasic approach. In: Bora N, Dodd C and Desmas- 5. Your article will be published on an interactive journal platform with advanced metrics. Kroppenstedt 2000 emend. Zhi et al. 2009 into an emended family ures N (editors). Diversity, Dynamics and Functional Role of Actino- find out more and submit your article at microbiologyresearch.org. Pseudonocardiaceae Embley et al. 1989 emend. Zhi et al. 2009. Int J mycetes on European Smear Ripened Cheeses. 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