TAXONOMIC DESCRIPTION Wang et al., Int J Syst Evol Microbiol 2019;69:909–913 DOI 10.1099/ijsem.0.003212

Chitinophaga silvisoli sp. nov., isolated from forest soil

Chunling Wang,1,2 Yingying Lv,2 Anzhang Li,2 Guangda Feng,2 Gegen Bao,1 Honghui Zhu2,* and Zhiyuan Tan1,*

Abstract A Gram-stain-negative, rod-shaped and aerobic bacterium, designated K20C18050901T, was isolated from forest soil collected on 11 September 2017 from Dinghushan Biosphere Reserve, Guangdong Province, PR China (23 10¢ 24¢¢ N; 112 32¢ 10¢¢ E). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain K20C18050901T belongs to the genus Chitinophaga, and showed the highest similarities to Chitinophaga sancti NBRC 15057T (98.6 %) and Chitinophaga T oryziterrae JCM 16595 (96.9 %). The major fatty acids (>10 %) were iso-C15 : 0,C16 : 1!5c, summed feature 3 (C16 : 1!6c and/or

C16 : 1!7c) and iso-C17 : 0 3-OH. The predominant respiratory quinone was menaquinone-7. The major polar lipid was phosphatidylethanolamine. The draft genome size of strain K20C18050901T was 8.36 Mb with a DNA G+C content of 44.7 mol%. The digital DNA–DNA hybridization and average nucleotide identity values between strain K20C18050901T and C. sancti NBRC 15057T were 31.40 and 85.82 %, respectively. On the basis of phenotypic, genotypic and phylogenetic analysis, strain K20C18050901T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga silvisoli sp. nov. is proposed. The type strain is K20C18050901T (=GDMCC 1.1411T=KCTC 62860T).

Chitinophaga Sangkhobol and Skerman 1981 emend. of 1972 mm. It is a hot spot area for the study of tropical K€ampfer et al. 2006, as the type genus of the family Chiti- and subtropical forest ecosystems. During our investigations nophagaceae K€ampfer et al. [1], was first described by of biodiversity of myxobacteria in forest soil of Dinghushan Sangkhobol and Skerman with Chitinophaga pinensis Biosphere Reserve, a yellow-coloured strain, designated Sangkhobol and Skerman 1981 as the type species [2]. K20C18050901T, was isolated from a forest soil sample   Members of the genus Chitinophaga are Gram-stain-nega- (23 10¢ 24¢¢ N; 112 32¢ 10¢¢ E) collected on 11 tive, filamentous or rod-shaped and oxidase-variable. The September 2017. major respiratory quinone is menaquinone-7 (MK-7), the The bacterium was isolated according to the following predominant polar lipids are phosphatidylethanolamine method: 1 g air-dried soil sample was soaked for 12 h with À and unidentified polar lipids, and the main fatty acid pro- 100 µg ml 1 actidione to avoid fungicontamination. The ! files include iso-C15 : 0, iso-C17 : 0 3-OH and C16 : 1 5c [3]. standard filter paper-baiting method was adopted to induce Species of the genus Chitinophaga have been isolated from the motile cellulolytic strains on mineral salt agar (CNST) various habitats, such as soil [4, 5], sediment [6], activated [13], and the colonies of strain K20C18050901T were found sludge [7], rock surface [8, 9], vermicompost [10] and as fruiting body-like aggregates on filter paper. Then the bark [11]. At the time of writing (20 August 2018), the strain was purified by repeated transfer of the colony edge genus Chitinophaga consists of 30 species listed in LPSN onto fresh bakers’ yeast agar (VY/2) [14], and preserved in  (www.bacterio.net/-allnamesac.html) and a validly pub- 20 % (v/v) glycerol solution under À80 C. lished species C. parva Ke et al. [12]. The genomic DNA of strain K20C18050901T was extracted The Dinghushan Biosphere Reserve was established in 1950 with the improved CTAB method [15] and sequenced on the and accepted in the first batch of Man and Biosphere Illumina HiSeq platform. A draft genome with a coverage of Reserves in China in 1978. The reserve has a south subtropi- 165Â was de novo assembled by using A5-MiSeq v20150522 cal monsoon climate with an annual average precipitation [16] and has been submitted to the NCBI database (www.ncbi.

Author affiliations: 1Guangdong Province Key Laboratory of Plant Molecular Breeding, College of Agriculture, South China Agricultural University, Guangzhou 510642, PR China; 2State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Microbial Culture Collection Center (GDMCC), Guangdong Institute of Microbiology, Guangzhou 510070, PR China. *Correspondence: Honghui Zhu, [email protected]; Zhiyuan Tan, [email protected] Keywords: ; Chitinophaga; phylogenetic tree. Abbreviations: ANI, average nucleotide identity; dDDH, digital DNA–DNA hybridization; ML, maximum-likelihood; MP, maximum-parsimony; NA, nutri- ent agar; NJ, neighbour-joining; R2A, Reasoner’s 2A; TSA, trypticase soy agar. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain K20C18050901T is MH734907. The GenBank accession number for the whole genome sequence is QTJV00000000. Four supplementary figures are available with the online version of this article.

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Chitinophaga longshanensis Z29T (KJ579707) Chitinophaga polysaccharea MRP-15T (KC430923) 0.0100 Chitinophaga ginsengisegetis Gsoil 040T (AB264798) T 81 Chitinophaga arvensicola DSM 3695 (AM237311) Chitinophaga taiwanensis CC-ALB-1 T (KC479802) Chitinophaga niastensis JS16-4T (EU714260) 91 Chitinophaga dinghuensis DHOC24 T (KM389531) 84 Chitinophaga terrae KP01T (AB278570) Chitinophaga jiangningensis JN53T (KF150362) 89 T 89 Chitinophaga eiseniae YC6729 (FJ750951) Chitinophaga varians 10-7W-9003T (MF685226) 100 Chitinophaga qingshengii JN246T (KF150484) 87 Chitinophaga japonensis IFO 16041T (AB078055) Chitinophaga ginsengihumi SR18T (FJ772016) 99 Chitinophaga rupis CS5-B1T (FM865977) 93 Chitinophaga rhizosphaerae T16R-86T (KX417377) Chitinophaga caseinilytica S-52T (KY117468) Chitinophaga barathri YLT18T (KP013246) Chitinophaga humicola Ktm-2T (MF405103) Chitinophaga cymbidii R156-2T (JN680880) Chitinophaga costaii A37T2T (KC922450) Chitinophaga niabensis JS13-10T (EU714259) Chitinophaga sedimenti TFL-3T (KX962169) 98 Chitinophaga silvisoli K20C18050901T (MF734907) 96 Chitinophaga sancti NBRC 15057T (AB078066) Chitinophaga oryziterrae YC7001 T (JF710262) 99 Chitinophaga ginsengisoli Gsoil 052T (AB245374) Chitinophaga filiformis IFO 15056T (AB078049) Chitinophaga pinensis DSM 2588T (CP001699) Chitinophaga skermanii CC-SG1B T (DQ062743) 100 Arvibacter flaviflagrans C-1-16 T (KU312691) 100 aurantiibacter 1458T (KR780052) 100 Filimonas lacunae YT21T (AB352776) petaseus T41T (EU854577)

Fig. 1. Maximum- likelihood tree based on 16S rRNA gene sequences showing the relationship between strain K20C18050901T and other related taxa. GenBank accession numbers are shown in parentheses. Bootstrap values >80 % are shown. Bar, 0.01 substitutions per nucleotide position.

nlm.nih.gov/genome) under the accession QTJV00000000. JCM 16595T (96.9 %), and it exhibited 95.6 % similarity to The genome size of strain K20C18050901T was 8.36 Mb and C. pinensis KACC 12763T (type species of the genus Chiti- the genomic DNA G+C content was 44.7 mol%. nophaga). Phylogenetic analysis based on the ML tree T A full-length 16S rRNA gene sequence (1523 nt) was extracted revealed that strain K20C18050901 belongs to the genus from the draft genome and showed one nucleotide difference Chitinophaga and formed a stable cluster with C. sancti T T from the PCR-amplified 16S rRNA gene sequence (1382 nt) NBRC 15057 and C. oryziterrae JCM 16595 with a boot- using the universal primers 27F and 1492R. The full-length strap value of 98 % (Fig. 1). Similar results were also sequence was searched against all the available sequences in obtained from the NJ and MP phylogenetic trees (Figs S1 EzBioCloud (http://eztaxon-e.ezbiocloud.net/) [17] and Gen- and S2, available in the online version of this article). Based Bank (www.ncbi.nlm.nih.gov). The neighbour-joining (NJ) on 16S rRNA gene sequence similarity and phylogenetic [18], maximum-likelihood (ML) [19] and maximum-parsi- analysis, the two most closely related type strains, C. sancti mony (MP) [20] methods were used to reconstruct phyloge- NBRC 15057T and C. oryziterrae JCM 16595T, were used as netic trees in MEGA 6.0 software [21] with bootstrap analysis references for further study. (1000 resamplings). Evolutionary distances were calculated The whole genome sequence of C. sancti NBRC 15057T was using Kimura’s two-parameter model [22]. retrieved from NCBI (FPIZ00000000). Digital DNA–DNA According to 16S rRNA gene sequence analysis, strain hybridization (dDDH) and average nucleotide identity K20C18050901T was most closely related to Chitinophaga (ANI) analyses between strain K20C18050901T and C. sancti NBRC 15057T (98.6 %) and Chitinophaga oryziterrae sancti NBRC 15057T were performed by using Genome-to-

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Table 1. Differential characteristics of strain K20C18050901T and Based on 16S rRNA gene similarity and phylogenetic analy- other related species of the genus Chitinophaga sis, strain K20C18050901T was a member of the genus Chi- Strains: 1, K20C18050901T; 2, C. sancti NBRC 15057T; 3, C. oryziterrae tinophaga. While the ANI and dDDH values, especially the T JCM 16595 . All data are from this study unless indicated otherwise. higher genomic G+C content (44.7 mol%) of strain All strains are negative for flexirubin-type pigments, catalase, indole K20C18050901T than C. sancti NBRC 15057T (43.3 mol%) production and hydrolyses of starch, arginine dihydrolase and urease. T All strains are positive for hydrolysis of gelatin, CM-cellulose and and C. oryziterrae JCM 16595 (41.3 mol%) (Table 1), T Tween 80. According to API 20NE and Biolog GENIII MicroPlate assays, clearly distinguished strain K20C18050901 from the estab- all strains assimilate glucose, arabinose, mannose, maltose, dextrin, lished species of the genus Chitinophaga. maltose, cellobiose, gentiobiose, sucrose, turanose, lactose, melibiose, D-salicin, a-D-glucose, D-mannose D-fructose, D-galactose, L-fucose, L- Phenotypic characteristics were tested under the same con- rhamnose, glycerol and pectin. For the API ZYM test, all strains are ditions with cells incubated on Reasoner’s 2A (R2A) agar positive for alkaline phosphatase, esterase, lipase, leucine arylami- and 272 agar (3 g bacto casitone, 1 g yeast extract, 1.36 g dase, valine arylamidase, cystine arylamidase, trypsin, acid phospha- Á CaCl2 2H2O, 15 g agar in 1.0 l distilled water, pH 7.2). Mor- tase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b- phology was observed by light microscopy (DM6/MC190, galactosidase, a-glucosidase, a-glucosidase, N-acetyl-b-glucosamini- Leica) and transmission electron microscopy (H7650, Hita- dase and b-fucosidase. All strains are negative for b-glucuronidase.  +, Positive; À, negative; ND, no data available. Data from: a, [30]; b, [4]; chi) with cells incubated on R2A agar at 30 C for 5 days. c, NCBI under the accession number FPIZ00000000. Gram-staining reaction was conducted using a Gram-stain kit (bioMerieux) according to the manufacturer’s instruc- Characteristic 1 2 3 tion. Growth at different temperature (10, 15, 20, 25, 30, 35,  Oxidase + + À 37, 40 and 42 C) was tested on 272 agar for 5 days. Salt tol-  Growth at 37 C + À + erance was evaluated in 272 broth with 0–2.5 % (w/v) NaCl Temperature range for growth 15–40 15–35 15–40 at intervals of 0.5 %. pH range for growth was tested from  Max. NaCl for growth (% w/v) 2.0 1.5 0.5 pH 4.0 to 10.0 at intervals of 0.5 units for 5 days at 30 C as pH range for growth 5.0–9.0 5.0–9.0 4.5–9.0 previously described [26]. Oxidase activity was determined Growth on Reasoner’s 2A agar + À + by using oxidase test strips [1 % (w/v) tetramethyl-p-phe- Nitrate reduction + + À nylenediamine, HKM]. Catalase activity was determined by Glucose fermentation + + À bubble production after mixing cells with 3 % H2O2. Flexir- Hydrolysis of: ubin-type pigments were detected by 20 % (w/v) KOH solu- Tween 40 À + + tion, with a colour change from yellow to red indicative of a Gelatin + À + positive result. Motility test was performed by the hanging- Assimilation of: drop method on 272 broth with 0.4 % (w/v) agar. Growth À on R2A agar, nutrient agar (NA), trypticase soy agar (TSA) Methyl b-D-glucoside + +  N-Acetyl-D-glucosamine + À + and MacConkey agar was tested at 30 C for 5 days. Hydrol- N-Acetyl-b-dmannosamine ÀÀ + yses of CM-cellulose, chitin, casein, Tween 40 and Tween 80 were examined on 272 agar with cellulose (1 %, w/v), chi- N-Acetyl-D-galactosamine + À + tin (1 %, w/v), casein (1 %, w/v), Tween 40 (1 %, w/v) and 3-Methyl-D-glucoside ÀÀ + ÀÀ Tween 80 (1 %, w/v), respectively. Various other physiologi- D-Glucose-6- PO4 + Trehalose + À + cal and biochemical properties were tested using the API 20NE and API ZYM kits (bioMerieux), and the Biolog Raffinose + À + GENIII MicroPlate according to the manufacturers’ instruc- Glucuronamide + ÀÀ tions. All experiments were prepared in duplicate. Glycyl-L-prolin + À + T L-Serine + ÀÀ Colonies of strain K20C18050901 were yellow-pigmented, Enzyme activities: circular, sticky and convex with extracellular secretions. a-Chymotrypsin + À + Cells were Gram-stain-negative, aerobic, rod-shaped, 3.5– a-Mannosidase ÀÀ + 7.1 µm long and 0.4–0.5 µm in diameter after incubation  DNA G+C content (mol%) 44.7 43.3a 41.3b on R2A agar for 5 days at 30 C (Fig. S3). The strain was c Genome size (Mb) 8.36 8.24 ND oxidase-positive, catalase-negative and grew well on R2A, TSA and NA agar, but not on MacConkey agar. The dif- T Genome Distance Calculator (GGDC; version 2.1)(http:// ferent characteristics between strain K20C18050901 and ggdc.dsmz.de/) [23] and the OrthANIu algorithm (www. the two closely related type strains (C. sancti NBRC T T ezbiocloud.net/tools/ani) [24], respectively. The dDDH 15057 and C. oryziterrae JCM 16595 ) are listed in value between K20C18050901T and C. sancti NBRC 15057T Table 1. Strain K20C18050901T could be clearly distin- was 31.4 % (Table 1), which was lower than the 70 % cut-off guished from its closely related type species by growth value for species boundaries. The ANI value between them conditions (pH, temperature, tolerance to NaCl), enzyme was only 85.82 %, which was also lower than the threshold activities (oxidase, a-chymotrypsin and a-mannosidase) value of 95–96 % for species discrimination [25]. and substrate assimilation (Table 1).

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Table 2. Cellular fatty acid profiles of strain K20C18050901T and other proportion of iso-C15 : 0 3-OH, C16 : 0 2-OH and summed related species of the genus Chitinophaga feature 3, and the lower proportion of C16 : 0 in strain Strains: 1, K20C18050901T; 2, C. sancti NBRC 15057T; 3, C. oryziterrae K20C18050901Tdiffered from the related type strains JCM 16595T. All data are from this study under the same conditions. (Table 2). The predominant respiratory quinone of strain Values are percentages of the total fatty acids. –, Not detected K20C18050901T was MK-7, which was in accordance with or <1.0 %. the typical characteristic of the genus Chitinophaga. Strain T Fatty acid 1 2 3 K20C18050901 contained phosphatidylethanolamine as the major polar lipid, together with unidentified aminolipids – – C12 : 0 1.0 and unidentified lipids (Fig. S4), which were similar to – C14 : 0 1.0 1.1 those of members of the genus Chitinophaga. C15 : 0 1.7 1.0 1.0 Based on the consensus of phylogenetic, genotypic and phe- iso-C15 : 0 3-OH 5.0 3.6 3.6 notypic analyses, strain K20C18050901T represents a novel iso-C15 : 0 35.3 35.0 34.7 species of the genus Chitinophaga, for which the name Chi- C16 : 1!5c 16.2 14.8 25.9 – – tinophaga silvisoli is proposed. C16 : 1!11c 1.0 C16 : 0 2.9 3.9 4.6 DESCRIPTION OF CHITINOPHAGA SILVISOLI C16 : 0 2-OH 5.8 2.1 3.3 – SP. NOV. C16 : 0 3-OH 1.6 1.0 – – C17 : 0 3-OH 1.5 Chitinophaga silvisoli (sil.vi.so¢li. L. fem. n. silva forest; L. iso-C17 : 0 3-OH 10.2 11.2 9.7 neut. n. solum soil; N.L. gen. n. silvisoli of forest soil, the –– C18 : 0 1.5 source of isolation of the type strain). C 2-OH – 1.1 – 18 : 1 Cells are aerobic, Gram-stain-negative, gliding-motile, rod- C !9c – 1.2 – 18 : 1 shaped, 3.5–7.1 µm long and 0.4–0.5 µm in diameter after Summed features:* incubation on R2A agar for 5 days. Colonies on R2A agar 3 14.1 8.3 8.1 are yellow, circular, sticky and convex with extracellular – –   9 1.0 secretions. Growth occurs at 15–40 C (optimum, 30 C) *Summed features are groups of two or three fatty acids that are and at a pH range from 5.0 to 9.0 (optimum, pH 7.0). Cells treated together for the purpose of evaluation in the MIDI system and can tolerate 2.0 % (w/v) NaCl. Flexirubin-type pigments are include both peaks with discrete ECLs as well as those where the absent. Oxidase-positive and catalase-negative. It can ECLs are not reported separately. Summed feature 3 comprises C16 : 1 hydrolyse CM-cellulose, chitin and Tween 80, but not ! ! ! 6c and/or C16 : 1 7c. Summed feature 9 comprises iso-C17 : 1 9c and/ Tween 40 and casein. The major fatty acids (>10 %) include or 10-methyl C16 : 0. iso-C15 : 0,C16 : 1!5c, summed feature 3 (C16 : 1!6c and/or C16 : 1!7c) and iso-C17 : 0 3-OH. The predominant polar lipid is phosphatidylethanolamine. The main respiratory quinone T Cellular fatty acids of strain K20C18050901 and its related is MK-7. In the API ZYM test, positive for alkaline phos- type strains, C. sancti NBRC 15057T and C. oryziterrae JCM phatase, esterase, lipase, leucine arylamidase, valine arylami-  16595T, were cultured on 272 agar for 2 days at 30 C. Bio- dase, cystine arylamidase, trypsin, acid phosphatase, mass of all strains were harvested at the logarithmic phase naphthol-AS-BI-phosphohydrolase, a-galactosidase, b- (2 days) and subjected to saponification and methylation. galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glu- Fatty acids were extracted using the Sherlock Microbial cosaminidase and a-fucosidase; but negative for b-glucu- Identification System (MIDI) protocol (version 6.1), and ana- ronidase and a-mannosidase. In the API 20NE tests, lysed by gas chromatography (Agilent 7890A) using the positive for nitrate reduction, glucose fermentation, b-glu- Microbial Identification software package with the Sherlock cosidase, b-galactosidase, gelatin hydrolysis, assimilating MIDI 6.1 system and the Sherlock Aerobic Bacterial Database glucose, arabinose, mannose, N-acetyl-D-glucosamine and (TSBA 6.1) [27]. The menaquinones were extracted and puri- maltose; negative for indole production, arginine dihydro- fied according to the method of Minnikin et al. [28] and lase and urease. Does not assimilate mannitol, gluconate, then analysed by HPLC (UltiMate 3000, Dionex). The polar capric acid, adipate, malic acid, citric acid and phenylacetic lipids were extracted and analysed by two-dimensional TLC acid. According to the Biolog GENIII MicroPlate assay, pos- based on the method of Tindall et al. [29]. itive for dextrin, maltose, trehalose, cellobiose, gentiobiose, D T sucrose, turanose, raffinose, lactose, melibiose, methyl b- - The fatty acid profiles of strain K20C18050901 and its glucoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-D- related type strains of the genus Chitinophaga are shown in a D D D D T galactosamine, - -glucose, -mannose, -fructose, - Table 2. The major fatty acids of strain K20C18050901 galactose, L-fucose, L-rhamnose, glycerol, L-serine, D-galac- ! (>10 %) included iso-C15 : 0 (35.3 %), C16 : 1 5c (16.2 %), turonic acid, L-galactonic acid lactone, D-gluconic acid, glu- ! ! summed feature 3 (C16 : 1 6c and/or C16 : 1 7c) (14.1 %) and curonamide, nalidixic acid and aztreonam; negative for iso-C17 : 0 3-OH (10.2 %), which are consistent with other stachyose, N-acetyl-b-dmannosamine, N-acetylneuraminic species of the genus Chitinophaga. However, the higher acid, 3-methyl glucose, D-fucose, inosine, fusidic acid, D-

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