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Agromyces Insulae Sp. Nov., an Actinobacterium Isolated from a Soil

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Agromyces Insulae Sp. Nov., an Actinobacterium Isolated from a Soil International Journal of Systematic and Evolutionary Microbiology (2016), 66, 2002–2007 DOI 10.1099/ijsem.0.000978 Agromyces insulae sp. nov., an actinobacterium isolated from a soil sample Jian-Rong Huang,13 Hong Ming,2,33 Shuai Li,1 Xiao-Lin Meng,1 Jian-Xin Zhang,1 Thi-Nhan Khieu,3,4 Zhong Tang,5 Wen-Jun Li3,6 and Guo-Xing Nie1 Correspondence 1College of Fisheries, Henan Normal University, Xinxiang, 453007, PR China Wen-Jun Li 2College of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003, [email protected] PR China Guo-Xing Nie 3Yunnan Institute of Microbiology, Yunnan University, Kunming, 650091, PR China [email protected] 4Department of Food Technology, School of Biotechnology and Food Technology, Hanoi University of Science and Technology, Hanoi, Vietnam 5College of information and Management, Guangxi Medical University, Nanning, 530021, PR China 6State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, PR China A novel Gram-reaction-positive, non-motile, aerobic bacterium, designated CFH S0483T, was isolated from a soil sample collected from Catba island in Halong Bay, Vietnam. 16S rRNA gene sequence analysis showed that the strain is a member of the genus Agromyces and has highest 16S rRNA gene sequence similarities with Agromyces humatus DSM 16389T (97.3 %) and Agromyces ramosus DSM 43045T (97.1 %), and similarities ,97.0 % with type strains of other species of the genus Agromyces. Strain CFH S0483T was able to grow at 10–37 8C, at pH 7.0–9.0 and tolerated NaCl up to 2.0 % (w/v). The whole-cell sugars were mannose, galactose, glucose and ribose. The isolate contained L-2,4-diaminobutyric acid, D-alanine, T D-glutamic acid and glycine in the cell-wall peptidoglycan. Strain CFH S0483 exhibited a menaquinone system with MK-12, and the major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 T and iso-C16 : 0. The genomic DNA G+C content of strain CFH S0483 was 71.6 mol%. Based on the phylogenetic and phenotypic analysis, and low DNA–DNA hybridization values, strain CFH S0483T could not be classified into any recognized species of the genus Agromyces. Strain CFH S0483T is therefore considered to represent a novel species of the genus Agromyces, for which the name Agromyces insulae sp. nov. is proposed. The type strain is CFH S0483T (5KCTC 39117T5CCTCC AB 2014301T). The genus Agromyces was first proposed by Gledhill & fatty acids are anteiso-C15 : 0 and anteiso-C17 : 0 (Gledhill Casida (1969), with the description of newly isolated & Casida, 1969; Zgurskaya et al., 1992; Suzuki et al., strain Agromyces ramosus DSM 43045T, and the genus 1996; Li et al., 2003; Chen et al., 2011;Thawai et al., 2011; description was later emended by Zgurskaya et al. (1992). Dastager et al., 2012). At the time of writing, 28 recognized All species of the genus Agromyces are Gram-reaction-posi- species of the genus Agromyces have been described (http:// tive, non-motile and aerobic. The cell-wall peptidoglycan www.bacterio.net/agromyces.html). These species are mainly is a group B type, based on 2,4-diaminobutyric acid. The originating from soil (Gledhill & Casida, 1969; Hamada predominant menaquinone is MK-12, and the major et al., 2014), rhizosphere (Takeuchi & Hatano, 2001; Jung et al., 2007), catacombs (Jurado et al., 2005) and seafood 3 (Park et al., 2010). The objective of the present study was These authors contributed equally to this work. to determine the taxonomic position of strain CFH The GenBank/EMBL/DDBJ accession number for the 16S rRNA S0483T by using a polyphasic approach. The results of phe- gene sequence of strain CFH S0483T is KP232915. notypic, chemotaxonomic and phylogenetic analyses indi- T Four supplementary figures and a supplementary table are available cated that strain CFH S0483 represents a novel species with the online Supplementary Material. of the genus Agromyces. Downloaded from www.microbiologyresearch.org by 2002 000978 G 2016 IUMS Printed in Great Britain IP: 165.246.155.25 On: Wed, 11 May 2016 11:28:59 Agromyces insulae sp. nov. Strain CFH S0483T was obtained from a soil sample col- similarity with A. humatus DSM 16389T (97.3 %) and lected in March 2012 from Catba island (208 469 240 N A. ramosus DSM 43045T (97.1 %). However, phylogenetic 1078 079 140 E) in Halong Bay, Vietnam. The sample was analyses showed that strain CFH S0483T formed a separate spread on Reasoner’s 2A agar (R2A) medium (DSMZ branch and did not cluster with any member of the genus Medium 830; https://www.dsmz.de/?id5441) after serial Agromyces (Fig. 1). Sequence similarities between strain dilution. The purified colonies were sub-cultured on modi- CFH S0483T and all other members of the genus Agromyces fied T5 medium (Ming et al., 2014). Strain CFH S0483T ranged from 94.9–96.8 %. Similar topologies were was routinely cultured on modified T5 medium at 28 8C, obtained from the maximum-parsimony and maximum- and preserved as glycerol suspensions (20 %, v/v) likelihood phylogenetic trees (Figs S1 and S2, available in at 280 8C. the online Supplementary Material). 16S rRNA gene sequence similarity analysis indicated that Growth at different pH (4.0–10.0, at intervals of 1.0 pH strain CFH S0483T was most closely related to Agromyces units) and temperatures (4, 10, 20, 28, 37, 45 and 50 8C) humatus DSM 16389T and Agromyces ramosus DSM was tested on the modified T5 medium agar. Salt tolerance 43045T with 97.3 % and 97.1 % 16S rRNA gene sequence tests with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 5.0 % similarity, respectively, and ,97.0 % similarity with type (w/v) NaCl were carried out on modified T5 agar at strains of other species of the genus Agromyces. Thus, Agro- 28 8C for 7 days. Microscopic observation was performed myces humatus DSM 16389T and Agromyces ramosus DSM after incubation at 28 8C for 3 days on modified T5 T 43045T were obtained from the DSMZ (German Collection medium. Cell morphology of strain CFH S0483 was of Microorganisms and Cell Cultures, Germany), and used examined by a light microscope (BX-51; Olympus) and a as reference strains in this study. All the strains were routi- scanning electron microscope (XL30 ESEM-TMP; Philips). nely grown on modified T5 medium at 28 8C for 3 days. Colony appearance was examined after growing at 28 8C Biomass for chemical and molecular studies, except cellular for 3 days. The Gram reaction was examined according fatty acids analysis, was obtained by cultivation on modi- to Buck (1982). Catalase activity was detected by the pro- fied T5 medium at 28 8C for 3 days. Biomass of strain duction of bubbles after the addition of a drop of 3 % T CFH S0483 for cellular fatty acids analysis was obtained (v/v) H2O2. Determination of oxidase activity was carried from cultures grown on tryptic soy agar (TSA; Becton out by using 1 % (w/v) tetramethyl-p-phenylenediamine Dickinson) for 3 days at 28 8C. Cells were checked for as described by Kovacs (1956). Cell motility was examined purity, harvested and washed twice with distilled water by the development of turbidity through a tube of semi- and then freeze-dried. solid medium as described by Leifson (1960). The ability to utilize sole carbon sources was tested with Pridham Genomic DNA was extracted and the 16S rRNA gene was and Gottlieb’s basal mineral salts medium supplemented PCR-amplified and sequenced as described by Li et al. with test carbon sources and by using the Biolog GIII (2007). The almost-complete 16S rRNA gene sequence MicroPlate system according to the manufacturer’s instruc- was submitted to the NCBI database and compared with tions. Nitrogen source utilization was observed in a basal the reference sequences of cultured species by using the liquid medium (Nie et al., 2012). API galleries (API EzTaxon-e server (http://www.ezbiocloud.net/eztaxon; Kim 20NE, API ZYM and API 50 CHB; bioMe´rieux) were et al., 2012) to determine the phylogenetic relationships used to determine some carbon source assimilation, acid T of strain CFH S0483 . The CLUSTAL X software package production and enzyme activities according to the manu- was used for multiple alignments of the 16S rRNA gene facturer’s instructions. Other physiological and biochemi- sequences with reference type strains of species of the cal tests were examined on modified T5 agar at 28 8Cfor genus Agromyces (Thompson et al., 1997). Phylogenetic 3 days as described by Chung et al. (2000), Manaia & da trees were reconstructed with neighbour-joining (Saitou Costa (1991) and Hudson et al. (1986). & Nei, 1987), maximum-likelihood (Felsenstein, 1981) T and maximum-parsimony (Fitch, 1971) algorithms by Cells of strain CFH S0483 were Gram-reaction-positive, using the MEGA version 6.0 software package (Tamura aerobic and produced short, branched vegetative hyphae, et al., 2013). The resultant tree topologies were evaluated while aerial hyphae were absent. The cells were by bootstrap analysis with 1000 replications (Felsenstein, 0.2–0.3 mm in diameter and 0.5–2.7 mm in length. The bac- 1985). The16S rRNA gene sequence of Microbacterium terium was non-motile and did not form spores (Fig. S3). lacticum DSM 20427T (GenBank accession no. X77441) Colonies were circular, cream–white, smooth and transpar- was used as an outgroup. ent when grown on modified T5 and R2A media. Growth of strain CFH S0483T occurred at 10–37 8C (optimum The almost-complete 16S rRNA gene sequence of strain 28 8C), at pH 7.0–9.0 (optimum pH 7.0) and with CFH S0483T (1494 nt) was determined, and comparison 0–2.0 % NaCl (optimum 0 %, w/v).
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