Sphingosinicella Humi Sp. Nov., Isolated from Arsenic- Contaminated Farmland Soil and Emended Description of the Genus Sphingosinicella

TAXONOMIC DESCRIPTION Qiao et al., Int J Syst Evol Microbiol DOI 10.1099/ijsem.0.003186

Sphingosinicella humi sp. nov., isolated from arsenic- contaminated farmland soil and emended description of the genus Sphingosinicella

Zixu Qiao,1 Min Cao,1 Dan Wang,1 Shuijiao Liao1,2 and Gejiao Wang1,*

Abstract A Gram-stain-negative, strictly aerobic bacterium, designated strain QZX222T, was isolated from arsenic-contaminated farmland soil. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain QZX222T was clustered with Sphingosinicella vermicomposti YC7378T (97.0 %), Sphingosinicella xenopeptidilytica 3–2W4T (96.1 %), Sphingosinicella microcystinivorans Y2T (96.0 %) and Sphingosinicella soli KSL-125T (95.9 %). Compared to strain QZX222T, Spingomonas olgophenolica JCM 12082T and Sphingobium boeckii 469T had 16S rRNA gene similarities of 96.2 and 95.9 %, respectively, but they located in other phylogenetic clusters. DNA–DNA hybridization and genomic ANI values between strain QZX222T and Sphingosinicella vermicomposti DSM 21593T (KCTC 22446T) were 34.8 and 75.0 %, respectively. The genome size of strain QZX222T was 3.0 Mb including 2982 predicted genes. The strain had a DNA G+C content of 65.9 mol%. Strain QZX222T had ubiquinone Q-10 as the major respiratory quinone and homospermidine as the major polyamine. The major fatty acids T (>10 %) of strain QZX222 were C17 : 1!6c, summed feature 8 (C18 : 1!7c and/or C18 : 1!6c) and C17 : 1!8c. The polar lipids were sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and an unidentified glycolipid. Strain QZX222T could be distinguished from other Sphingosinicella strains based on the results of phylogenetic and genomic analyses, DNA–DNA hybridization, white colour colony, hydrolysis of urea, alkaline phosphatase activity, lack of phosphatidylmonomethylethanolamine, and presence of phosphatidylcholine. Therefore, strain QZX222T represents a novel species of Sphingosinicella, for which the name Sphingosinicella humi sp. nov. is proposed. The type strain is QZX222T (=KCTC 62519T=CCTCC AB 2018030T).

The genus Sphingosinicella is assigned to the family Sphin- Sphingosinicella members is 59.4–65.1 mol% and the major gomonadaceae in the class Alphaproteobacteria. It was first polar lipids are sphingoglycolipid (SGL), phosphatidyletha- proposed by Maruyama et al. with a microcystin-degrading nolamine (PE), phosphatidylglycerol (PG), diphosphatidyl- T bacterium, Sphingosinicella microcystinivorans Y2 , as the glycerol (DPG) and phosphatidylmonomethylamine (PME) type species strain [1]. So far, there are only four Sphingosi- [1–4]. In this study, a bacterial strain, QZX222T, isolated nicella species including Sphingosinicella microcystinivorans from arsenic-contaminated farmland soil was characterized [1], Sphingosinicella xenopeptidilytica [2], Sphingosinicella by polyphasic analysis. soli [3] and Sphingosinicella vermicomposti [4], represented by strains isolated from eutrophic lake, aeration tank of a During the process of isolating arsenic-resistant bacteria, the arsenic-contaminated farmland soil sample was col- wastewater, alkaline soil and vermicompost, respectively. Members of the genus Sphingosinicella are Gram-stain-neg- lected in Jiaotian village of Daye city (30 03¢ 27.13¢¢ N, 114 ative, rod-shaped and strictly aerobic. The predominant 56¢ 45.13¢¢ E), Hubei province, PR China. The soil is red respiratory quinone is ubiquinone Q-10, and homospermi- earth type, with a pH of 6.95 and an arsenic content of À1 dine is the major polyamine. The major fatty acids are C18 : 1 35.8 mg kg soil. The soil sample was gradient diluted with ’ !7c and C16 : 1!7c, the DNA G+C content range of 0.85 % NaCl, plated on Reasoner s 2A (R2A) agar medium

Author affiliations: 1State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China; 2College of Basic Science, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China. *Correspondence: Gejiao Wang, [email protected] Keywords: Sphingosinicella; novel species. Abbreviations: ANI, average nucleotide identity; SGL, sphingoglycolipid; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PME, phosphatidylmonomethylamine; PC, phosphatidylcholine; ML, maximum-likelihood; NJ, neighbor-joining; MP, maximum-parsimony. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain QZX222T is MG753794. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number QFFF00000000. The version described in this paper is version QFFF01000000. One supplementary table and four supplementary figures are available with the online version of this article.

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T 94 Sphingomonas glacialis C16y (GQ253122) 84 Sphingomonas psychrolutea MDB1-AT (KR258737) Sphingomonas echinoides ATCC 14820 T (JH584237) Sphingomonas oligophenolica JCM 12082 T (AB018439) Sphingomonas alpina S8-3T (GQ161989) 0.02 Sphingomonas aquatica W1-2-1T (KT309085) Sphingomonas panacis DCY99 T (CP014168) Sphingomonas koreensis NBRC 16723T (BCYW01000045) Sphingomonas asaccharolytica NBRC 15499T (BCYU01000001) 74 Sphingomonas mali NBRC 15500T (BCYX01000023) 99 97 Sphingomonas pruni NBRC 15498T (BCYZ01000050) Sphingomonas dokdonensis DS-4T (DQ178975) Sphingomonas desiccabilis CP1D T (AJ871435) Sphingomonas paucimobilis JCM 7516 T (U37337) T 100 Sphingosinicella xenopeptidilytica 3-2W4 (AY950663) 100 Sphingosinicella microcystinivorans Y2 T (AB084247) 87 Sphingosinicella soli KSL-125T (DQ087403) Sphingosincella humi QZX222T (MG753794) 75 Sphingosinicella vermicomposti YC7378 T (FJ442859) T 75 Sphingomonas faucium E62-3 (KU179043) Sphingomonas laterariae LNB2T (HM159118) Sphingobium subterraneum S-II-13T (FJ796422) Sphingobium boeckii 469T (JN591315) 89 Sphingobium yanoikuyae ATCC 51230 T (JH992904) Sphingobium qiguonii X23T (EU095328) Sphingobium xanthum NL9T (KF437579) 99 Sphingobium vulgare HU1-GD12T (FJ177535) 96 Sphingomonas sediminicola Dae 20T (AB258386) Sphingomonas jaspsi DSM 18422T (KK073876) 96 ‘Sphingomonas humi ’ PB323 (AB220146) 100 Sphingomonas kaistensis PB56T (AY769083) Sphingomonas changbaiensis NBRC 104936T (BBWU01000045) Rhodospirillum rubrum ATCC 11170 T (D30778)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship of strain QZX222T with related species.

and incubated at 28 C for 2 weeks. A strain named QZX222 performed by the thermal renaturation method [15]. To dif- was selected due to its low 16S rRNA gene sequence ferentiate strain QZX222T from Sphingosinicella microcysti- similarity. nivorans JCM 13185T, the specific gene sequences, mlrA, mlrB, mlrC and mlrD, responsible for degradation of micro- Genomic DNA was extracted and purified according to the cystin were amplified as described by Geueke et al. [2], and method described by Moore and Dowhan [5]. The 16S strain JCM 13185T was used as the positive control. rRNA gene sequence of strain QZX222T was amplified by the universal bacterial primers 27F (5¢-AGAGTTT- For genomic analysis, DNA was random fragmented by GATCCTGGCTCA-3¢) and 1492R (5¢-GGTTACCTTGT- ultrasonication and used to reconstruct a shotgun library. TACGACTT-3¢) as described by Fan et al. [6]. The purified Pair-end sequencing was carried out by Wuhan Frasergen PCR product was cloned into pGEM-T vector (Promega) Bioinformatics Company using Illumina HiSeq X. The scat- and sequenced by Tsingke Company (Beijing, China). The tered fragments were assembled using SPAdes version 3.11.1 1450 bp 16S rRNA sequence was compared with the (http://cab.spbu.ru/software/spades/). The orthologous sequences obtained in the EzTaxon-e server [7]. Multiple average nucleotide identity (OrthoANI) value between T alignments were performed using CLUSTAL_X [8]. Phyloge- strain QZX222 and the closest-related strain, Sphingosini- T netic trees were reconstructed using the maximum-likeli- cella vermicomposti KCTC 22446 (PXYJ00000000.1), was hood [9], neighbor-joining [10] and maximum-parsimony calculated by using the EzBioCloud server (www.ezbio- [11] methods by MEGA 5.0 software [12]. The bootstrap val- cloud.net/tools/ani) [16]. ues were calculated based on 1000 replications [13], and dis- Phylogenetic analysis based on 16S rRNA gene sequences tances were determined according to Kimura’s two- showed that strain QZX222T was clustered with Sphingosi- parameter method [14]. DNA–DNA hybridization was nicella vermicomposti YC7378T (97.0 %), Sphingosinicella

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Table 1. Differential phenotypic characteristics among strain QZX222T and type strains of related species Strains: 1, QZX222T; 2, Sphingosinicella vermicomposti DSM 21593T; 3, Sphingosinicella microcystinivorans JCM 13185T; 4, Sphingosinicella soli KSL- 125T (data from [3]); 5, Sphingomonas paucimobilis JCM 7516T. All data are from this study unless indicated. All of the strains were positive for oxidase, hydrolysis of Tween 20, leucine arylamidase and valine arylamidase, but negative for hydrolysis of starch, gelatin, production of indole, lipase (C14), a-galactosidase, b-glucuronidase, a-mannosidase and a-fucosidase. +, Positive; À, negative; ND, no data.

Characteristic 1 2 3 4* 5

Colony colour White Yellow Yellow Yellow Yellow Growth at 40 C + ÀÀÀÀ Nitrate reduction + À + ÀÀ Catalase ÀÀ + + + Aesculin À + ÀÀ + Hydrolysis of: Tween 20 + + + + + Tween 40 ÀÀ + + + Tween 80 ÀÀ + + + Casein ÀÀ + ÀÀ L-Arginine + À + À + Urea + ÀÀÀ + D-Glucose ÀÀÀ ND + Enzyme activity (API ZYM): Alkaline phosphatase À + + + + Esterase (C4) ÀÀÀ + À Esterase lipase (C8) + + + + À Cystine arylamidase + + ÀÀ + Trypsin + + ÀÀ + a-Chymotrypsin + À + À + Acid phosphatase À + ÀÀ + Naphthol-AS-BI-phosphohydrolase + + + ÀÀ b-Galactosidase À + ÀÀ + a-Glucosidase À + ÀÀ + b-Glucosidase ÀÀÀÀ + N-acetyl-b-glucosaminidase ÀÀ + + + Acid production from: D-Galactose + + À ND W Assimilation of: D-Glucose À + ÀÀ + Propionate + À + À W Citrate ÀÀ + ÀÀ Histidine ÀÀ + ÀÀ L-Proline + À + ÀÀ Maltose À + ÀÀ + Acetate + À W À + L-Alanine ÀÀ + À + Glycogen À + ÀÀÀ L-Serine ÀÀ + ÀÀ D-Galactose + + + À + Lactose ÀÀÀÀ + L-Arabinose ÀÀÀÀ + Raffinose ÀÀÀÀ + D-Fructose ÀÀÀÀ + Melibiose ÀÀÀÀ + L-Fucose ÀÀÀÀ + L-Arabinose ÀÀÀÀ + Valerianate + + + ND À N-acetyl-D-glucosamine ÀÀÀÀ +

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Table 1. cont.

Characteristic 1 2 3 4* 5

Sucrose ÀÀÀÀ + DNA G+C content (mol%) 65.9 59.4† 63.6–63.7‡ 65.1* 65.4§

*All data of Sphingosinicella soli KSL-125T are taken from Yoon et al. [3]. †Data from Yasir et al. [4]. ‡Data from Maruyama et al. [1]. §Data from Chen et al. [29].

xenopeptidilytica 3–2W4T (96.1 %), Sphingosinicella micro- The genome size of strain QZX222T was 3.0 Mb and com- cystinivorans Y2T (96.0 %) and Sphingosinicella soli KSL- posed of 4 contigs. It had 375Â sequencing depth of cover- 125T (95.9 %). Compared to strain QZX222T, the other two age, 2981 predicted genes (2892 protein operons, 3 rRNA strains, Spingomonas olgophenolica JCM 12082T and Sphin- operons, 45 tRNA operons, 3 other RNA operons and 38 gobium boeckii 469T had 16S rRNA gene similarities of 96.2 pseudogene operons), had an N50 length of 2 705 127 and a and 95.9 %, respectively, but they were located in other phy- G+C content of 65.9 mol%. The DNA–DNA hybridization logenetic clusters (Figs 1, S1 and S2, available in the online value between strain QZX222T and Sphingosinicella vermi- version of this article). The most closely related strain, composti DSM 21593T was 34.8 %. The OrthoANI value Sphingosinicella vermicomposti DSM 21593T, the type spe- between strain QZX222T and Sphingosinicella vermicomposti cies strain Sphingosinicella microcystinivorans JCM 13185T KCTC 22446T was 75.0 %. The mlrA, mlrB, mlrC and mlrD T and the Sphingomonas type species strain, Sphingomonas sequences were not amplified from strains QZX222 and T paucimobilis JCM 7516T, were obtained from Deutsche DSM 21 593 , but they were amplified from Sphingosinicella T Sammlung von Mikroorganismen und Zellkulturen microcystinivorans JCM 13185 , meanwhile, the putative (DSMZ) or Japan Collection of Microorganisms (JCM) and genes of mlrA, mlrB, mlrC and mlrD were not find in the T T analysed together in this study. genomes of strain QZX222 and DSM 21 593 . Gram staining was tested by using a Gram staining kit (Baso) on the basis of the manufacturer’s instructions. Cel- T Table 2. Fatty acid composition of strain QZX222 and some type lular morphology and flagella were observed by transmis- strains of related species sion electron microscope (H-7650, Hitachi). Colony T T Strains: 1, QZX222 ; 2, Sphingosinicella vermicomposti DSM 21593 ; 3, morphology was observed on R2A agar. Growth at different Sphingosinicella microcystinivorans JCM 13185T; 4, Sphingosinicella soli T T temperatures (4, 16, 28, 33, 37, 42 and 47 C) and various KSL-125 (data from [3]); 5, Sphingomonas paucimobilis JCM 7516 . – Data from this study unless indicated. –, <1 %. pH [4.0 10.0 at 0.5 pH unit intervals, adjusted by adding 0.1 M citric acid (pH 4.0–7.0) and 0.2 M Tris (pH 8.0–10.0)] Fatty acid 1 2 3 4* 5 were tested in R2A broth for 7 days. NaCl tolerance was determined in R2A broth for 7 days with NaCl concentra- C –––– 1.1 14 : 0 tions of 0–5 % (w/v, at 1 % intervals in 1–5 % and 0.2 % C ––– 4.0 – 15 : 0 intervals in 0–1 %). The motility was tested in R2A broth C 6.5 9.2 7.1 4.1 8.8 16 : 0 with 0.3 % agar. Anaerobic growth was determined by incu- C17 : 0 4.8 –––– bation in an anaerobic chamber with an O2-absorbing and C15 : 1!6c ––– 2.3 – CO2-generating agent (Anaero-Pack, Mitsubishi Gas Chem- – – C16 : 1!5c 2.7 4.0 2.2 ical) for 2 weeks. Oxidase activity was determined by 1 % C17 : 1!6c 41.1 4.3 1.4 27.7 2 (w/v) tetramethyl-p-phenylenediamine [17], and catalase –– – C17 : 1!8c 10.1 3.0 activity was obtained by assessing bubble production in 3 % C18 : 1!5c –– 1.0 – 3.1 (v/v) H2O2. Production of H2S and indole were detected – C14 : 0 2OH 9.4 8.4 6.6 4.3 according Smibert and Krieg [18]. Hydrolyses of starch, gel- –– – C15 : 0 2OH 3.5 5.6 atin, casein, cellulose and Tweens (20, 40 and 80) were ––– – iso-C16 : 0 3-OH 2.3 tested according to Cowan and Steel [19]. Nitrate reduction ––– C18 : 1!7c 11-methyl 3.5 5.6 was detected by the method described by Lanyi [20]. Antibi- Summed feature 3† 5.1 27.3 29.0 23.3 2.8 otic sensitivity tests were carried out by inoculation of the Summed feature 8† 23.9 37.9 46.7 19.0 75.6 bacterial suspension into R2A agar plates with filter-paper T discs containing different antibiotics (Hangzhou Microbial *The data of Sphingosinicella soli KSL-125 are taken from Yoon et al. [3]. Reagent), and incubation at 28 C for 7 days according to

†Summed feature 3 comprises C16 : 1!7c and/or C16 : 1!6c Chen et al. [21]. Acid production from various carbohy- drates was tested using traditional methods [22]. API 20NE, ‡Summed feature 8 comprises C18 : 1!7c and/or C18 : 1!6c API ID 32GN and API ZYM kits (bioMerieux) were used to

Downloaded from www.microbiologyresearch.org by IP: 153.90.184.104 On: Mon, 14 Jan 2019 21:10:14 Qiao et al., Int J Syst Evol Microbiol ascertain the carbon-source assimilations and enzyme activ- suggest that strain QZX222T represents a novel species of ities on the basis of the manufacturer’s instructions. In some the genus Sphingosinicella, for which the name Sphingosini- cases, the traditional carbon-source assimilation tests were cella humi. nov. is proposed. -1 also performed in basal medium [ g l (NH4)2SO4, 0.5 g l-1 K2HPO4, 0.2 g l-1 MgSO4Á7H2O, 0.5 g l-1 NaH2PO4 and EMENDED DESCRIPTION OF Á 0.1 g l-1 CaCl2 2H2O] as described by Dong and Cai [22]. SPHINGOSINICELLA MARUYAMA ET AL. 2006 To determine the arsenite resistance level of strain QZX222T AND GEUEKE ET AL. 2007 , NaAsO2 (1, 2 and 3 mM) was added in R2A broth and incubated for 7 days to detect the minimum inhibitory con- The genus is described as Maruyama et al. 2006 and Geueke centration (MIC). et al. 2007, with the following emendations and modifica- T tions. Colonies are yellow or white. Catalase activity and Strain QZX222 was Gram-stain-negative and aerobic. Cells nitrate reduction are variable. The common polar lipids are – Â – were rod-shaped (0.3 0.4 1 1.2 µm), motile and sphingoglycolipid, phosphatidylethanolamine, phosphati- had a flagellum (Fig. S3). The strain grew on R2A medium dylglycerol and diphosphatidylglycerol. and 1/10 tryptic soy broth (TSB). Colonies were smooth, white, circular, convex and slightly transparent after incuba- SPHINGOSINICELLA HUMI tion on R2A agar at 28 C for 7 days. The temperature range DESCRIPTION OF for growth was 16–42 C and the optimum temperature was SP. NOV. 28 C. The pH range for growth was 6.5–9.0. The strain Sphingosinicella humi (hu¢mi. L. gen. n. humi of soil). could grow with 0–0.4 % NaCl and grew best without the T – Â – addition of NaCl. Strain QZX222 could grow in up to Cells are Gram-stain-negative, rod-shaped (0.3 0.4 1 1.2 µm), strictly aerobic and motile by means of a flagellum. 1 mM NaAsO2. The main differences between strain QZX222T and the other strains of Sphingosinicella were col- Colonies are white, smooth, circular, convex and slightly transparent on R2A medium. Growth occurs at 16–42 C ony colour, hydrolysis of urea, alkaline phosphatase activity (Table 1) and resistance to ciprofloxacin (Table S1). and the optimum temperature is 28 C. The pH range for growth is 6.5–9.0. Growth occurs with NaCl concentrations T In order to analyse the cellular fatty acids of strain QZX222 of 0–0.4 % (w/v; optimum without the addition of NaCl). and the reference strains, cells were harvested in the expo- Oxidase-positive, but catalase-negative. Can hydrolyse nential growth phase and analysed by GC (6890, Hewlett Tween 20, L-arginine and urea, but starch, gelatin, cellulose, Packard) according to the standard protocol of the Sherlock aesculin and Tweens (40 and 80) are not. Nitrate reduction Microbial Identification System (MIDI Sherlock version 6.1, is positive. Can produce H2S, but not indole. Acid is pro- MIDI database TSA40 4.10) [23, 24]. Respiratory quinones duced from D-galactose, but not from D-ribose, lactose, D- were extracted and analysed by HPLC as described by Minni- xylose, D-sorbitol, D-rhamnose, L-arabinose, sucrose, myo- kin et al. [25]. Polyamines were analysed as described by inositol, raffinose, D-fructose, maltose, D-glucose, L-sorbose, Busse and Auling [26], and Busse et al. [27]. Polar lipids mannitol or D-mannose. Assimilates propionate, L-proline, were extracted and determined by following the method acetate, D-galactose and valerianate, but not mannitol, D- described by Collins and Jones [28]. sorbitol, rhamnose, myo-inositol, itaconic acid, malonic T acid, D-glucose, citrate, histidine, maltose, L-alanine, glyco- The major cellar fatty acids of strain QZX222 were C17 : 1 gen, L-serine, lactose, L-arabinose, raffinose, D-fructose, !6c (41.1 %), summed feature 8 (C18 : 1!7c and/or C18 : 1!6c; melibiose, L-fucose, L-arabinose, N-acetyl-D-glucosamine or 23.9 %) and C !8c (10.0 %). The major cellar fatty acids 17 : 1 sucrose. The activities of esterase lipase (C8), leucine aryla- were generally similar among the strains of the genus Sphin- T midase, valine arylamidase, cystine arylamidase, trypsin, a- gosinicella, but strain QZX222 and Sphingosinicella soli T chymotrypsin and naphthol-AS-BI-phosphohydrolase are KSL-125 [3] had high amounts of C17 : 1!6c (Table 2). The T present, but alkaline phosphatase, esterase (C4), lipase polar lipids of strain QZX222 were SGL, PE, PG, DPG, (C14), acid phosphatase, a-galactosidase, b-galactosidase, phosphatidylcholine (PC) and an unidentified glycolipid b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b- (GL; Fig. S4). The four Sphingosinicella strains all had SGL, glucosaminidase, a-mannosidase and a-fucosidase are PE, PG, DPG and PME, and some had GL [1–4]. Strain absent. The major quinone is Q-10, and the major poly- QZX222T specifically had PC and no PME, which could be amine is homospermidine. The major fatty acids are C used to distinguish it from the other Sphingosinicella strains 17 : 1 !6c, summed feature 8 (C !7c and/or C !6c) and [1–4]. The major polyamine was homospermidine [28.9 18 : 1 18 : 1 À C !8c. Sphingoglycolipid, phosphatidylethanolamine, mol (g dry weight) 1] and ubiquinone Q-10 was the major 17 : 1 phosphatidylglycerol, diphosphatidylglycerol, phosphatidyl- respiratory quinone, these results were in agreement with choline and an unidentified glycolipid are the polar lipids. the Sphingosinicella strains [1–4]. In summary, strain QZX222T had typical characters of Sphingosinicella, but it The type strain is QZX222T (=KCTC 62519T=CCTCC AB could be distinguished from the others by the results of phy- 2018030T), isolated from arsenic-contained farmland soil logenetic and genomic analyses, DNA–DNA hybridization, sampled at Jiaotian village, Daye city, Hubei province, PR white colour colony, hydrolyses of urea, alkaline phospha- China. The DNA G+C content of the type strain is 65.9 mol tase activity, and having PC and no PME. Therefore, we %. The GenBank accession numbers of the 16S rRNA gene

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