Glycomyces Artemisiae Sp. Nov., an Endophytic Actinomycete Isolated from the Roots of Artemisia Argyi

International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3492–3495 DOI 10.1099/ijs.0.063388-0 Glycomyces artemisiae sp. nov., an endophytic actinomycete isolated from the roots of Artemisia argyi Xiumin Zhang,1 Kai Ren,1 Jiao Du,1 Haiyan Liu2 and Liping Zhang1 Correspondence 1Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Liping Zhang College of Life Sciences, Hebei University, Baoding 071002, PR China [email protected] 2Central laboratory, No. 252 Hospital of PLA, Baoding, PR China An endophytic actinomycete strain, IXS4T, was isolated from the root of Artemisia argyi,a medicinal plant collected from Yesanpo located in Laishui county, Hebei province, China. The 16S rRNA gene sequence of strain IXST showed most similarity to Glycomyces mayteni YIM 61331T (98.23 % 16S rRNA gene sequence similarity), Glycomyces scopariae YIM 56256T (98.00 %), Glycomyces sambucus E71T (97.90 %) and Glycomyces algeriensis NRRL B-16327T (97.10 %). DNA–DNA hybridization values between strain IXS4T and the closely related type strains were well below 70 %. The strain also showed a number of physiological and biochemical characteristics that were distinct from the closely related species. The strain contained MK-10(H2) and MK-11(H0) as the detected menaquinones. The peptidoglycan was mainly meso-diaminopimelic acid and the whole-cell sugars contained galactose, glucose, mannose, xylose and ribose. The major cellular fatty acids were iso-C14 : 0, iso-C15 : 0, iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. Based on the genetic and phenotypic properties, it is proposed that strain IXS4T represents a novel species of the genus Glycomyces, with the name http://dx. doi.org/10.1601/nm.7671Glycomyces artemisiae sp. nov. The type strain is IXS4T (5HBUM178000T5CGMCC 4.7067T5NBRC 109773T). The genus Glycomyces was initially established by Labeda to a modified five-step surface-sterilization procedure et al. (1985), and at the time of writing, it holds 11 species according to Qin et al. (2008): a 3 min wash in 5 % with validly published names. While most strains have been NaOCl, followed by a 10 min wash in 2.5 % Na2S2O3, isolated from soil collected in diverse geographical regions a 3 min wash in 75 % ethanol, a 10 min wash in 10 % (Labeda & Kroppenstedt, 2004; Guan et al., 2011; Labeda NaHCO3 and a final rinse in sterile distilled water. Surface- et al., 1985; Evtushenko et al., 1991), members of the genus sterilized roots were pulverized in a ceramic mortar, Glycomyces also appear to be closely associated with plant distributed on tap water-yeast extract medium (Crawford tissues (Gu et al., 2007; Qin et al., 2008, 2009). In the et al., 1993) and incubated at 28 uC for 3 weeks. Isolates present paper, we describe the phenotypic and genotypic were purified and maintained on yeast extract-malt extract characterization of a Glycomyces-like strain (IXS4T), isolated agar [International Streptomyces Project (ISP) medium 2] from the roots of Artemisiae argyi, a medicinal plant (Shirling & Gottlieb, 1966) at 4 uC and as glycerol collected from Yesanpo in Hebei province, north China suspensions (20 %, v/v) at 220 uC. (115u 279 E39u 409 N) at an elevation of 1980 m. Extraction of genomic DNA and PCR amplification of T Healthy root samples of Artemisiae argyi were used as the 16S rRNA gene from strain IXS4 was performed as sources for isolation of endophytic actinomycetes. The described by Coombs & Franco (2003). The PCR products fresh root samples were air-dried for 48 h and then washed was purified and cloned into the pMD 18-T vector thoroughly as described by Coombs & Franco (2003). (TaKaRa) and sequenced by using an Applied Biosystems Subsequently, the root samples were excised and subjected DNA sequencer (model 3730XL) and the software provided by the manufacturer. The nearly full-length 16S rRNA gene sequence (1487 bp) was aligned manually in the GenBank Abbreviation: DAP, diaminopimelic acid. database with BLAST (Altschul et al., 1990), which showed T The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene that strain IXS4 was closely related to members of the genus T sequence of strain IXS4 is JN408755. Glycomyces. MEGA software version 5.0 (Tamura et al., 2011) Six supplementary figures and two supplementary tables are available was used for the multiple alignment and for the phylogenetic with the online version of this paper. tree reconstruction using the neighbour-joining (Saitou & 3492 063388 G 2014 IUMS Printed in Great Britain Glycomyces artemisiae sp. nov. Nei, 1987), maximum-likelihood (Felsenstein, 1981) and The DNA G+C content of strain IXS4T was determined maximum-parsimony (Kluge & Farris, 1969; Fitch, 1971) by HPLC (Mesbah et al., 1989) to be 74 mol%, which is tree-making algorithms. Evolutionary distance matrices marginally higher than other members of the genus (70– were calculated according to the neighbour-joining method 72 mol%) (Guan et al., 2011; Gu et al., 2007; Qin et al., with Kimura’s two-parameter model (Kimura, 1980). The 2008; Qin et al., 2009). robustness of the tree topology was evaluated by bootstrap Biomass for chemotaxonomic studies were obtained by analysis (Felsenstein, 1985) based on 1000 resamplings. 16S rRNA gene sequence similarities between the strains were growth in ISP 2 broth or TSB (trypticase soy broth) for calculated on the basis of pairwise alignment using the fatty acids, at 28 uC for 14 days on a rotary shaker till EzTaxon server ((http://www.ezbiocloud.net/eztaxon; Kim stationary phase, and harvested by centrifugation and et al., 2012). Strain IXS4T was closely related to Glycomyces washed with distilled water. Diaminopimelic acid (DAP) mayteni YIM 61331T (98.23 % 16S rRNA gene sequence was analysed by TLC (Bousfield et al., 1985) and whole-cell similarity), Glycomyces scopariae YIM 56256T (98.00 %), sugars were analysed by TLC (Hasegawa et al., 1983). Meso- Glycomyces sambucus E71T (97.90 %) and Glycomyces DAP and a minor amount of LL-DAP were present, and the algeriensis NRRL B-16327T (97.10 %); the similarities whole-cell sugars contained galactose, glucose, mannose, between strain IXS4T and other members of the Glycomyces xylose and a minor amount of ribose (Fig. S3), while the whole-cell sugars of the closest related type strain, G. were all below 97 %. In the phylogenetic trees, the affiliation T between strain IXS4T and its closest neighbour, G. mayteni mayteni YIM 61331 , did not contain mannose and only YIM 61331T, was supported with bootstrap values of 53 %, contained meso-DAP. The isoprenoid quinones were 67 % and 70 % in the neighbour-joining (Fig. 1), maximum- extracted with chloroform/methanol (2 : 1, v/v) and purified likelihood (Fig. S1, available in the online Supplementary by using TLC on Gel 60 F254 plates (10620 cm, 0.5 mm Material) and maximum-parsimony (Fig. S2) trees, respect- thickness) and using n-hexane/diethyl ether (85 : 15, v/v) as ively, and the node branches were identical in two different the solvent. The identities of the quinones were analysed algorithms. by HPLC with a Cosmosil 5C18column (4.66250 mm; Thermo Electron Corporation). Strain IXS4T contained The liquid reassociation rate method (De Ley et al., 1970) MK-10(H2) as the predominant menaquinone, with MK- was used for determining the percentage of DNA–DNA 11(H0) also present (Fig. S4). The menaquinone profile of hybridization, in duplicate, by using a spectrophotometer the closest related type strain, G. mayteni YIM 61331T, was (model CE9500; Cecil Instruments) equipped with a very different, it contained MK-11(H0), MK-11(H4) and programmable melting-temperature control unit. The MK-10(H ) (Qin et al., 2009). Detailed menaquinone data DNA–DNA relatedness values between strain IXS4T and 4 T T for the novel strain and related species of the genus G. mayteni YIM 61331 , G. scopariae YIM 56256 , G. Glycomyces are given in Table S1. sambucus E71T and G. algeriensis NRRL B-16327T were 59.4 %, 24.3 %, 20.2 % and 8.6 %, respectively, all of which Polar lipids were extracted and identified by two- are below the 70 % cut-off point for recognition of dimensional TLC (silica gel 60, 10610 cm) as described genomic species (Wayne et al., 1987; Stackebrandt & by Minnikin et al. (1984). Major lipids were diphospha- Goebel, 1994). tidylglycerol, phosphatidylglycerol, phosphatidylcholine, T 89 G. lechevalierae NRRL B-16149 (AY462041) 99 G. rutgersensis IMSNU 22074T (AJ293748) 0.01 66 G. algeriensis NRRL B-16327T (AY462044) 100 G. endophyticus YIM 56134T (EU200681) . NBRC 14487T (D85483) 100 G harbinensis 52 Glycomyces artemisiae IXS4T (JN408755) G. mayteni YIM 61331T (EU814511) 94 G. sambucus E71T (DQ460469) 100 G. scopariae YIM 56256T (EU200682) G. halotolerans TRM 40137T (HQ651156) 70 G. arizonensis DSM 44726T (AY462042) 91 G. tenuis ATCC 49849T (D85482) Stackebrandtia nassauensis DSM 44728T (CP001778) Fig. 1. Neighbour-joining tree showing the relationship of strain IXS4T with related members of the genus Glycomyces. All branches of the tree were also recovered by using the maximum-likelihood (Fig. S1) and maximum-parsimony (Fig. S2) algorithms. Bootstrap values based on 1000 replications are shown at branch nodes. Stackebrandtia nassauensis DSM 44728T was used as the outgroup. Bar, 0.01 substitutions per nucleotide position. http://ijs.sgmjournals.org 3493 X. Zhang and others phosphatidylinositol, phosphatidylinositol mannosides and Table 1. Differential characteristics of strain IXS4T and type an unidentified phospholipid (Fig. S5). strains of related species of the genus Glycomyces T For the analysis of whole-cell fatty acids, strain IXS4 was Strains: 1, IXS4 T;2,G. mayteni YIM 61331T;3,G. scopariae YIM grown for 14 days at 28 uC in TSB (trypticase soy broth) in 56256T;4,G. sambucus E71T;5,G. algeriensis NRRL B-16327T. All an Erlenmeyer flask at 180 r.p.m., and harvested by data were obtained in this study.

Load more