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Chitinophaga Silvisoli Sp. Nov., Isolated from Forest Soil

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Chitinophaga Silvisoli Sp. Nov., Isolated from Forest Soil 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: Bacteroidetes; 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. 003212 ã 2019 IUMS Downloaded from www.microbiologyresearch.org by IP: 124.16.161.13909 On: Wed, 19 Jun 2019 01:47:56 Wang et al., Int J Syst Evol Microbiol 2019;69:909–913 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 Filimonas aurantiibacter 1458T (KR780052) 100 Filimonas lacunae YT21T (AB352776) Flavihumibacter 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- Downloaded from www.microbiologyresearch.org by IP: 124.16.161.13910 On: Wed, 19 Jun 2019 01:47:56 Wang et al., Int J Syst Evol Microbiol 2019;69:909–913 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
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