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.

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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). Strain CFH S0483T of the sequence with the corresponding 16S rRNA gene was negative for catalase, oxidase and urease activities sequences from the GenBank/EMBL/DDBJ databases and could not degrade Tweens 20, 40, 60 and 80. Negative clearly demonstrated that strain CFH S0483T represented results were detected in tests for hydrolysis of casein, a member of the genus Agromyces. 16S rRNA gene analysis CM-cellulose, chitin, starch, tyrosine and xylan, and nitrate showed that strain CFH S0483T had highest sequence reduction. Positive results were detected for hydrolysis of

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99* ‘Agromyces arachidis’ AK-1 (FN868445) 54* Agromyces indicus NIO-1018T (HM036655) 56* YIM 21741T (AF389342) 0.01 Agromyces aurantiacus Agromyces mediolanus DSM 20152T (X77449) 100* T Agromyces soli MJ21 (GQ241325) Agromyces marinus H23-8T (AB847104) Agromyces tropicus CM9-9T (AB454378) Agromyces ulmi XIL01T (AY427830) 65 Agromyces bracchium NBRC 16238T (AB023359) 82* Agromyces luteolus NBRC 16235T (AB023356) Agromyces flavus CPCC 202695T (FJ529717) 94* T Agromyces subtropicus IY07-56 (AB546310) Agromyces bauzanensis BZ41T (FJ972171) Agromyces iriomotensis IY07-20T (AB546308) Agromyces humatus DSM 16389T (AY618216) 97* Agromyces neolithicus 23-23T (AY507128) Agromyces insulae CFH S0483T (KP232915) Agromyces rhizospherae NBRC 16236T (AB023357) 72 Agromyces albus VKM Ac-1800T (AF503917) Agromyces ramosus DSM 43045T (X77447) Agromyces subbeticus DSM 16689T (ATXG01000026) 80 Agromyces fucosus VKM Ac-1345T (AY158025) subsp. DSM 8595T (X77448) 75* Agromyces cerinus cerinus 99 Agromyces hippuratus JCM 9086T (D45061) Agromyces cerinus subsp. nitratus ATCC 51763T (AY277619) Agromyces atrinae P27T (FJ607310) 72* Agromyces lapidis CD55T (AY618217) Agromyces italicus DSM 16388T (ATXF01000012) 93 Agromyces allii UMS-62T (DQ673873) Agromyces salentinus 20-5T (AY507129) 52 Agromyces terreus DS-10T (EF363711) Microbacterium lacticum DSM 20427T (X77441)

Fig. 1. Neighbour-joining tree showing the phylogenetic relationship of strain CFH S0483T and related species of the genus Agromyces based on 16S rRNA gene sequences. Asterisks denote nodes that were also recovered using the maximum-likeli- hood and maximum-parsimony methods. Bootstrap values .50 % are shown at the branch points. Microbacterium lacticum DSM 20427T (GenBank accession no. X77441) was used as the outgroup. Bar, 0.01 substitutions per nucleotide position.

2 2 aesculin and gelatin. Phenotypic properties useful for buffer (1 mg ml 1 trypsin, 0.1 mol l 1 PBS, pH 7.9). distinguishing strain CFH S0483T from A. humatus DSM This suspension was incubated at 37 8C on a shaker for 16389T and A. ramosus DSM 43045T are shown in about 3 h, followed by centrifugation, and the pellet was Table 1. The detailed physiological characteristics of then washed three times with distilled water. The cell- strain CFH S0483T are given in the species description. wall preparation was hydrolysed with 6 M HCL at 120 8C for 6 h and the amino acid compositions were determined Biomasses used for chemotaxonomic characteristics of by using TLC and HPLC analysis (Tang et al., 2009). Mena- strain CFH S0483T and the reference strains A. humatus quinones were extracted according to Collins et al. (1977), DSM 16389T and A. ramosus DSM 43045T were obtained and analysed by HPLC (Kroppenstedt, 1982; Tamaoka using several standard methods under identical conditions. et al., 1983). For analysis of fatty acids, strains CFH Sugars of whole-cell hydrolysates were analysed as S0483T, A. humatus DSM 16389T and A. ramosus DSM described by Komagata & Suzuki (1987). To prepare the 43045T were grown on TSA (Becton Dickinson) medium purified cell-wall peptidoglycan, a modified method was at 28 8C for 3 days. The cellular fatty acids were extracted, used as described by Schleifer & Kandler (1972). The cell methylated and analysed according to the instructions of sediment was resuspended in 10 % trichloroacetic acid after being harvested, was placed in a boiling water bath the Microbial Identification System (MIDI) (Sherlock ver- for 45 min and then centrifuged for 10 min. The sediment sion 6. 1; MIDI database: TSBA6) (Sasser, 1990). For polar material was carefully rinsed with distilled water three lipid analysis, cells of strain CFH S0483T were extracted times and resuspended carefully in trypsin-phosphate and examined by two-dimensional TLC and the profile

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Table 1. Differential phenotypic characteristics of strain CFH MK-11 (11 %) and MK-13 (4 %) were found as minor S0483T and its closest phylogenetic neighbours components, which were common to the genus Agromyces. The major fatty acids of strain CFH S0483T (.5 %) were Strains: 1, CFH S0483T;2,A. humatus DSM 16389T;3,A. ramosus T anteiso-C15 : 0 (36.8 %), anteiso-C17 : 0 (34.5 %) and DSM 43045 . All data were obtained during this study under identical iso-C (20.3 %), which was consistent with those + 2 16 : 0 growth conditions. , Positive; , negative. described for strains of the genus Agromyces, but their Characteristic 1 2 3 proportions were different from the reference strains determined in this study. Detailed fatty acid compositions Temperature range for growth (8C) 10–37 20–37 10–37 are shown in Table S1. The polar lipids of strain CFH T pH range for growth 7.0–9.0 6.0–9.0 6.0–9.0 S0483 comprised diphosphatidylglycerol, phosphatidylgly- NaCl tolerance (%, w/v) 0–2 0–2 0–4 cerol, two glycolipids and one lipid (Fig. S4). The genomic Utilization of: DNA G+C content of strain CFH S0483T was 71.6 mol%. Amygdalin 22+ DNA–DNA relatedness values between strain CFH S0483T Aesculin ++2 and its closest relatives A. humatus DSM 16389T and A. L-Fucose 22+ ramosus DSM 43045T were 35.4¡3.1 % and 29.1¡1.5 %, Fructose ++2 respectively, which were below the threshold value for deli- Galactose ++2 neation of genomic species (Wayne et al., 1987). Mannose ++2 Maltose + 2 + Phylogenetic and phenotypic analysis including chemo- T Melibiose + 2 + taxonomy indicated that strain CFH S0483 was a D-Raffinose 2 ++ member of the genus Agromyces. Although strain CFH Salicin + 22 S0483T had many properties that overlapped on most Trehalose + 22 species of the genus Agromyces, the isolate also displayed Enzyme activities a few physiological and biochemical properties that distin- b-Galactosidase ++2 guished it from the closest relatives A. humatus DSM b-Glucuronidase 2 + 2 16389T and A. ramosus DSM 43045T (see Table 1 and the b-Glucosidase + 22 species description). Therefore, based on the phenotypic, Trypsin + 22 phylogenetic and chemotaxonomic results, strain CFH b ++2 N-Acetyl- -glucosaminidase S0483T is considered to represent a novel species of the 2 ++ Presence of rhamnose in whole-cell genus Agromyces, for which the name Agromyces insulae hydrolysates sp. nov. is proposed.

Description of Agromyces insulae sp. nov. was identified using the described procedures (Collins & Jones, 1980; Minnikin et al., 1979). The genomic DNA Agromyces insulae (in9su.lae. L. gen. fem. n. insulae of an G+C content of strain CFH S0483T was determined by island). HPLC (Mesbah et al., 1989), with Escherichia coli JM-109 Cells are Gram-reaction-positive, aerobic, non-motile, 0.2– as the reference strain. 0.3 mm in diameter and 0.5–2.7 mm in length, and produce short, branched vegetative hyphae, but aerial hyphae are Quantitative microplate DNA–DNA hybridizations absent. Colonies are circular, cream–white and smooth. between strain CFH S0483T and the closest relatives, Growth occurs at 10–37 uC, at pH 7.0–9.0 and in the pre- A. humatus DSM 16389T and A. ramosus DSM 43045T, sence of 0–2.0 % (w/v) NaCl. Utilizes aesculin, cellobiose, were carried out according to the fluorometric micro- dextrin, L-fucose, D-fructose, D-galactose, gentiobiose, D- well method (Ezaki et al., 1989). The hybridizations were glucose, glycerol, maltose, D-mannitol, D-mannose, salicin, performed by using DNA probes labelled with photobiotin sorbitol, sucrose, D-turanose and trehalose as sole carbon (A1935; Sigma) and 96-well microdilution plates (Greiner sources, but not amygdalin, L-arabinose, lactose, melibiose, BioOne). Hybridizations were performed using eight repli- myo-inositol, raffinose, L-rhamnose, D-ribose, succinic cations; the highest and lowest values in each experiment acid, D-xylitol or D-xylose. Utilizes L-glutamate, L-isoleu- were excluded, the means were calculated and expressed cine and L-proline as sole nitrogen sources, but not as DNA–DNA hybridization values. L-alanine, L-asparagine, glycine, L-histidine, L-threonine, T Whole-cell sugars found in strain CFH S0483 were man- L-tyrosine or L-valine. Catalase-, oxidase- and urease-nega- nose, galactose, glucose and ribose. Rhamnose was absent tive. Negative for degradation of Tweens 20, 40, 60, 80, in the whole-cell sugars of strain CFH S0483T, but was hydrolysis of casein, CM-cellulose, chitin, starch, tyrosine detected in the two reference strains under the identical and xylan, and nitrate reduction. Aesculin and gelatin are T conditions. Isolate CFH S0483 contained L-2,4-diamino- hydrolysed. Galactosidase is positive, but arginine dihydro- butyric acid, D-alanine, D-glutamic acid and glycine in lase, indole production from tryptophan and glucose the cell-wall peptidoglycan. The predominant menaqui- fermentation are negative. Activities of N-acetyl-b-glucosa- none of strain CFH S0483T was MK-12 (85 %), and minidase, alkaline phosphatase, acid phosphatase, cystine

Downloaded from www.microbiologyresearch.org by http://ijs.microbiologyresearch.org 2005 IP: 165.246.155.25 On: Wed, 11 May 2016 11:28:59 J.-R. Huang and others arylamidase, esterase (C4), esterase lipase(C8), b-galactosi- Felsenstein, J. (1985). Confidence limits on phylogenies: an approach dase, a-glucosidase, b-glucosidase, leucine arylamidase, using the bootstrap. Evolution 39, 783–791. naphthol-AS-BI-phosphohydrolase, trypsin and valine ary- Fitch, W. M. (1971). Toward defining the course of evolution: lamidase are present; activities of a-chymotrypsin, a-fuco- minimum change for a specific tree topology. Syst Zool 20, 406–416. sidase, a-galactosidase, b-glucuronidase, lipase (C14) and Gledhill, W. E. & Casida, L. E. (1969). Predominant catalase-negative a-mannosidase are absent. The whole-cell sugars are man- soil bacteria. III. Agromyces, gen. n., microorganisms intermediary to 18 nose, galactose, glucose and ribose, and the peptidoglycan Actinomyces and Nocardia. Appl Microbiol , 340–349. amino acids are L-2,4-diaminobutyric acid, D-alanine, Hamada, M., Shibata, C., Ishida, Y., Tamura, T., Yamamura, H., Hayakawa, M. & Suzuki, K. (2014). Agromyces iriomotensis sp. nov. D-glutamic acid and glycine. The predominant menaqui- and Agromyces subtropicus sp. nov., isolated from soil. Int J Syst none is MK-12. The major fatty acids are anteiso-C15 : 0, Evol Microbiol 64, 833–838. anteiso-C and iso-C . The polar lipid profile con- 17 : 0 16 : 0 Hudson, J. A., Morgan, H. W. & Daniel, R. M. (1986). A numerical sists of diphosphatidylglycerol, phosphatidylglycerol, two classification of some Thermus isolates. J Gen Microbiol 132, 531–540. unknown glycolipids and one lipid. Jung, S.-Y., Lee, S.-Y., Oh, T.-K. & Yoon, J.-H. (2007). Agromyces allii The type strain, CFH S0483T (5KCTC 39117T 5CCTCC sp. nov., isolated from the rhizosphere of Allium victorialis var. AB 2014301T), was isolated from Catba island in Halong platyphyllum. Int J Syst Evol Microbiol 57, 588–593. Bay, Vietnam. The DNA G+C content of the type strain Jurado, V., Groth, I., Gonzalez, J. M., Laiz, L., Schuetze, B. & Saiz- is 71.6 mol%. Jimenez, C. (2005). Agromyces italicus sp. nov. Agromyces humatus sp. nov. and Agromyces lapidis sp. nov., isolated from Roman catacombs. Int J Syst Evol Microbiol 55, 871–875. Acknowledgements Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: The authors are grateful to Professor Dr Hans-Peter Klenk (Newcastle a prokaryotic 16S rRNA gene sequence database with phylotypes University, UK) for kindly providing the reference strains. This that represent uncultured species. Int J Syst Evol Microbiol 62, research was supported by Natural Science Foundation of China 716–721. (no. 3150004), Program for Innovative Research Team (in Science Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in and Technology) in University of Henan Province (14IRTSTHN013), 19 Plan for Scientific Innovation Talent of Henan Province bacterial systematics. Methods Microbiol , 161–207. (154100510009), and Scientific Research Fund of Xinxiang Medical Kovacs, N. (1956). Identification of Pseudomonas pyocyanea by the University (2013QN126). W.-J. L. was also supported by the Guang- oxidase reaction. Nature 178, 703. dong Province Higher Vocational Colleges & Schools Pearl River Kroppenstedt, R. M. (1982). Separation of bacterial menaquinones by Scholar Funded Scheme (2014). HPLC using reverse phase (RP18) and a silver loaded ion exchanger as stationary phases. J Liq Chromatogr 5, 2359–2367. Leifson, E. (1960). Atlas of Bacterial Flagellation, London: Academic References Press. Buck, J. D. (1982). Nonstaining (KOH) method for determination Li, W. J., Zhang, L.-P., Xu, P., Cui, X.-L., Xu, L.-H., Zhang, Z., of gram reactions of marine bacteria. Appl Environ Microbiol 44, Schumann, P., Stackebrandt, E. & Jiang, C.-L. (2003). Agromyces 992–993. aurantiacus sp. nov., isolated from a Chinese primeval forest. Int J Syst Evol Microbiol 53, 303–307. Chen, J., Chen, H.-M., Zhang, Y.-Q., Wei, Y.-Z., Li, Q.-P., Liu, H.-Y., Su, J., Zhang, Y.-Q. & Yu, L.-Y. (2011). Agromyces flavus sp. nov., an Li, W. J., Xu, P., Schumann, P., Zhang, Y. Q., Pukall, R., Xu, L. H., actinomycete isolated from soil. Int J Syst Evol Microbiol 61, Stackebrandt, E. & Jiang, C. L. (2007). Georgenia ruanii sp. nov., a 1705–1709. novel actinobacterium isolated from forest soil in Yunnan (China), Chung, A. P., Rainey, F. A., Valente, M., Nobre, M. F. & da Costa, M. S. and emended description of the genus Georgenia. Int J Syst Evol 57 (2000). Thermus igniterrae sp. nov. and Thermus antranikianii sp. nov., Microbiol , 1424–1428. two new species from Iceland. Int J Syst Evol Microbiol 50, 209–217. Manaia, C. M. & da Costa, M. S. (1991). Characterization of Collins, M. D. & Jones, D. (1980). halotolerant Thermus isolates from shallow marine hot springs on Lipids in the classification and 137 identification of coryneform bacteria containing peptidoglycans S. Miquel, Azores. J Gen Microbiol , 2643–2648. based on 2,4-diaminobutyric acid. J Appl Bacteriol 48, 459–470. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise + Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). measurement of the G C content of deoxyribonucleic acid by 39 Distribution of menaquinones in actinomycetes and corynebacteria. high-performance liquid chromatography. Int J Syst Bacteriol , J Gen Microbiol 100, 221–230. 159–167. Dastager, S. G., Qiang, Z.-L., Damare, S., Tang, S.-K. & Li, W.-J. Ming, H., Yin, Y.-R., Li, S., Nie, G.-X., Yu, T.-T., Zhou, E.-M., Liu, L., (2012). Agromyces indicus sp. nov., isolated from mangroves Dong, L. & Li, W.-J. (2014). Thermus caliditerrae sp. nov., a novel sediment in Chorao Island, Goa, India. Antonie van Leeuwenhoek thermophilic species isolated from a geothermal area. Int J Syst Evol 64 102, 345–352. Microbiol , 650–656. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric Minnikin, D. E., Collins, M. D. & Goodfellow, M. (1979). Fatty acid and deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro- polar lipid composition in the classification of Cellulomonas, dilution wells as an alternative to membrane filter hybridization in Oerskovia and related taxa. J Appl Bacteriol 47, 87–95. which radioisotopes are used to determine genetic relatedness Nie, G.-X., Ming, H., Li, S., Zhou, E.-M., Cheng, J., Yu, T.-T., Zhang, J., among bacterial strains. Int J Syst Bacteriol 39, 224–229. Feng, H.-G., Tang, S.-K. & Li, W.-J. (2012). Geodermatophilus Felsenstein, J. (1981). Evolutionary trees from DNA sequences: nigrescens sp. nov., isolated from a dry-hot valley. Antonie van a maximum likelihood approach. J Mol Evol 17, 368–376. Leeuwenhoek 101, 811–817.

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Park, E.-J., Kim, M.-S., Jung, M.-J., Roh, S. W., Chang, H.-W., Shin, Tang, S. K., Wang, Y., Chen, Y., Lou, K., Cao, L. L., Xu, L. H. & Li, W. J. K.-S. & Bae, J.-W. (2010). Agromyces atrinae sp. nov., isolated from (2009). Zhihengliuella alba sp. nov., and emended description of the fermented seafood. Int J Syst Evol Microbiol 60, 1056–1059. genus Zhihengliuella. Int J Syst Evol Microbiol 59, 2025–2031. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method Thawai, C., Tanasupawat, S., Suwanborirux, K. & Kudo, T. (2011). for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Agromyces tropicus sp. nov., isolated from soil. Int J Syst Evol 61 Sasser, M. (1990). Identification of bacteria by gas chromatography Microbiol , 605–609. of cellular fatty acids. USFCC Newsl 20, 16. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477. strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882. Suzuki, K., Sasaki, J., Uramoto, M., Nakase, T. & Komagata, K. (1996). Agromyces mediolanus sp. nov., nom. rev., comb. nov., a Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., species for ‘‘Corynebacterium mediolanum’’ Mamoli 1939 and for Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., some aniline-assimilating bacteria which contain 2,4-diaminobutyric Murray, R. G. E. & other authors (1987). International Committee acid in the cell wall peptidoglycan. Int J Syst Bacteriol 46, 88–93. on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Takeuchi, M. & Hatano, K. (2001). Agromyces luteolus sp. nov., Agromyces Bacteriol 37, 463–464. rhizospherae sp. nov. and Agromyces bracchium sp. nov., from the mangrove rhizosphere. Int J Syst Evol Microbiol 51, 1529–1537. Zgurskaya, H. I., Evtushenko, L. I., Akimov, V. N., Voyevoda, H. V., Dobrovolskaya, T. G., Lysak, L. V. & Kalakoutskii, L. V. (1992). Tamaoka, J., Katayama-Fujimura, Y. & Kuraishi, H. (1983). Analysis Emended description of the genus Agromyces and description of of bacterial menaquinone mixtures by high performance liquid 54 Agromyces cerinus subsp., cerinus sp. nov., subsp. nov., Agromyces chromatography. J Appl Bacteriol , 31–36. cerinus subsp. nitratus sp. nov., subsp. nov., Agromyces fucosus Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). subsp. fucosus sp. nov., subsp. nov., and Agromyces MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol fucosus subsp. hippuratus sp. nov., subsp. nov. Int J Syst Bacteriol Evol 30, 2725–2729. 42, 635–641.

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