%paper no. ije048512 charlesworth ref: ije048512& New Taxa - Actinobacteria International Journal of Systematic and Evolutionary Microbiology (2013), 63, 000–000 DOI 10.1099/ijs.0.048512-0 Actinotalea ferrariae sp. nov., isolated from an iron mine, and emended description of the genus Actinotalea Yanzhi Li, Fang Chen, Kun Dong and Gejiao Wang Correspondence State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Gejiao Wang Huazhong Agricultural University, Wuhan, Hubei 430070, PR China [email protected] or [email protected] ; A Gram-stain-positive, aerobic, non-motile, rod-shaped bacterium, designated strain CF5-4T, was isolated from iron mining powder. 16S rRNA gene sequence analysis grouped strain CF5-4T in a single cluster with Actinotalea fermentans DSM 3133T (97.6 % similarity). The major fatty acids T (.5 %) of strain CF5-4 were anteiso-C15 : 0, anteiso-C15 : 1 A, C16 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C17 : 0. The predominant respiratory quinone was MK-10(H4) and the genomic DNA G+C content was 74.7 mol%. The major polar lipids were diphosphatidylglycerol and one unidentified phosphoglycolipid. The peptidoglycan type of strain CF5-4T was A4b, containing L-Orn–D-Ser–D-Asp. The cell-wall sugars were rhamnose, fucose, mannose and galactose. The results of DNA–DNA hybridization in combination with the comparison of phenotypic and phylogenetic characteristics among strain CF5-4T and related micro-organisms revealed that the isolate represents a novel species of the genus Actinotalea, for which the name Actinotalea ferrariae sp. nov. is proposed. The type strain is CF5-4T (5KCTC 29134T5CCTCC AB2012198T). The family Cellulomonadaceae was originally defined by genus Cellulomonas and MK-10(H4) for the genus Stackebrandt & Prauser (1991) and included the genera Actinotalea (Stackebrandt et al., 2006, Yi et al., 2007; Shi Actinotalea (one species), Cellulomonas (21 species), et al., 2012)]. Oerskovia (four species), Paraoerskovia (one species) and Iron mining powder was collected from Hongshan Iron Tropheryma (one species). The genus Actinotalea was first Mine (about 100 m underground, 30u 049 38.770 N described by Yi et al. (2007) with Actinotalea fermentans 114u 579 24.070 E) of Daye City, Hubei Province, China. DSM 3133T as the type strain. This type strain was The pH and the total C, N, P, S and Fe concentrations were previously isolated from dumping ground samples described in a previous study (Chen et al., 2012). For and identified as representing Cellulomonas fermentans bacterial isolation, serially diluted samples were spread on (Bagnara et al., 1985). It was reclassified in the new genus R2A (Difco) agar plates and incubated at 28 uC for 7 days. Actinotalea based on the major respiratory quinone MK- A single colony of strain CF5-4T was picked and 10(H ) and phylogenetic analysis results (Yi et al., 2007). 4 subcultivated. Routine cultivation was performed at The characteristics of A. fermentans were Gram-stain- 28 uC for 4 days on trypticase soy agar (TSA) and the positive, non-motile, facultatively anaerobic rods, contain- strain was preserved at 280 uC in trypticase soy broth ing L-Orn–D-Asp (type A4b) in the peptidoglycan, anteiso- (TSB) supplemented with glycerol (25 %, v/v). C15 : 0,C16 : 0, iso-C16 : 0,C14 : 0 and iso-C14 : 0 as the major fatty acids (.5 %), MK-10(H4) as the major isoprenoid For analyses of morphological, physiological and biochem- quinone, diphosphatidylglycerol (DPG) and phosphatidyl- ical characteristics, strain CF5-4T and the type strains glycerol (PG) as the major polar lipids, and possessing of four close relatives, A. fermentans DSM 3133T, relatively high DNA G+C content of 75.8 mol% (Bagnara Cellulomonas carbonis KCTC 19824T, Cellulomonas bogo- et al., 1985; Yi et al., 2007; Shi et al., 2012). The main riensis DSM 16987T and Cellulomonas flavigena DSM difference between the genera Cellulomonas and Actinotalea 20109T, were cultivated on TSA agar or in TSB and [ was the major respiratory quinone MK-9(H4) for the incubated at 28 uC for 4 days unless otherwise mentioned. Gram staining was performed using the method described Abbreviations: DPG, diphosphatidylglycerol; PG, phosphatidylglycerol. by Dussault (1955) combined with the KOH lysis method The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene (Ryu, 1938). Cell morphology was observed using a sequence of Actinotalea ferrariae CF5-4T is HQ730135. scanning electron microscope (JSM-6390; JEOL) and a Three supplementary figures are available with the online version of this transmission electron microscope (H-7650; Hitachi). paper. Growth at 4, 15, 20, 28, 37, 40 and 45 uC was tested on 048512 G 2013 IUMS Printed in Great Britain 1 %paper no. ije048512 charlesworth ref: ije048512& Y. Li and others TSA for 2 weeks. Various NaCl (1–10 %, w/v) and pH The DNA G+C content was determined by HPLC as (4.0–11.0) ranges were assessed using TSB after incubation described by Mesbah et al. (1989). The respiratory for 7 days. In the pH tests, the broth was adjusted to an quinones were extracted and identified by HPLC as initial pH with citrate/phosphate or Tris/HCl buffers described by Minnikin et al. (1984). Levels of DNA–DNA (Breznak & Costilow, 1994). Anaerobic growth was tested relatedness were determined using the thermal denatura- on TSA with incubation in an anaerobic chamber tion and renaturation method of Huss et al. (1983). (Mitsubishi Gas Chemical) at 28 uC for 2 weeks. Growth Cells of strain CF5-4T were Gram-stain-positive, aerobic, on nutrient agar, MacConkey agar, R2A agar and Luria– non-motile rods. Colonies were yellow, convex, circular, Bertani (LB) agar (all from Difco) was also investigated. Catalase activity was determined by assessing bubble smooth and non-transparent after 4 days incubation on TSA at 28 uC. The strain grew on TSA, nutrient agar, R2A production in 3 % (v/v) H2O2, and oxidase activity was determined using 1 % (w/v) tetramethyl-b-phenylenedia- agar and LB agar, but did not grow on MacConkey agar. Detailed results of the polyphasic characterization of mine (Cappuccino & Sherman, 2002). Hydrolysis of T gelatin, starch, casein, tyrosine, CM-cellulose, DNA, urea strain CF5-4 are given in the species description. A and Tween 80 was investigated according to Cowan & Steel scanning electron micrograph and a transmission electron micrograph showing the general morphologies of strain (1965). Nitrate reduction was tested by the method T described by La´nyı´ (1987). H S, indole production, methyl CF5-4 are available in IJSEM Online (Fig. S1). The main 2 differential phenotypic characteristics between strain red and Voges–Proskauer tests were carried out using the T method described by Smibert & Krieg (1994). Acid CF5-4 and the four reference strains are shown in production from various carbohydrates was determined Table 1. as described by Hugh & Leifson (1953). Antibiotic- The 1483 bp 16S rRNA gene sequence of strain CF5-4T was susceptibility tests were performed by spreading bacterial analysed to determine its phylogenetic position. Strain suspensions on TSA plates and applying filter-paper discs CF5-4T showed high sequence similarities to species of the containing different antibiotics (Hangzhou Microbial genera Cellulomonas and Actinotalea. Highest 16S rRNA Reagent), incubated at 28 uC for 7 days as described by gene sequence similarities were to C. carbonis T26T Chen et al. (2012). Other physiological and biochemical (5KCTC 19824T) (98.4 %), followed by A. fermentans characteristics and enzyme activities were determined using DSM 3133T (97.6 %) and C. bogoriensis 69B4T (5DSM API 20 NE, API ID 32 GN and API ZYM systems according 16987T) (96.6 %). The 16S rRNA gene sequence similarity to the manufacturer’s instructions (bioMe´rieux). The between strain CF5-4T and C. flavigena DSM 20109T (type results from API ZYM test strips were observed after species of the genus Cellulomonas) was 94.6 %. These four incubation at 28 uC for 6 h, and the API 20 NE and API ID type strains were therefore used as reference strains. A 32 GN test results were examined after incubation at 28 uC phylogenetic tree reconstructed using the neighbour-join- for 48 and 72 h, respectively. ing algorithm grouped strain CF5-4T in a cluster with A. T T The nearly full-length 16S rRNA gene sequence was fermentans DSM 3133 . Although strain CF5-4 showed T amplified as described by Fan et al. (2008) and compared highest similarity to C. carbonis T26 , all species of the T with the sequences available in EzTaxon-e server (http:// genus Cellulomonas, including C. carbonis T26 , fell into eztaxon-e.ezbiocloud.net/) (Kim et al., 2012) and NCBI other nearby positions (Fig. 1). The maximum-parsimony GenBank database. Multiple alignments of sequences were tree and the maximum-likelihood trees (Fig. S2) also carried out using CLUSTAL X (Thompson et al., 1997). supported the phylogenetic position obtained with the Neighbour-joining (Saitou & Nei, 1987) and maximum- neighbour-joining method. parsimony (Fitch, 1971) trees were reconstructed using The major cellular fatty acids of strain CF5-4T (.5 %) were MEGA 4.0 (Tamura et al., 2007), and a maximum-likelihood anteiso-C15 : 0 (38.4 %), anteiso-C15 : 1 A (19.2 %), C16 : 0 tree was generated using the PHYML online web server (9.5 %), iso-C16 : 0 (6.9 %), iso-C15 : 0 (7.7 %) and anteiso- (Guindon et al., 2005). To obtain the confidence level of T C17 : 0 (7.1 %) (Table 2). Strain CF5-4 exhibited similar the branches, bootstrap values were calculated based on polar lipids of DPG and PG as the four reference type 1000 replications (Felsenstein, 1985). strains. It also had one unidentified phosphoglycolipid For whole-cell fatty acid analysis, strain CF5-4T and A. (PGL), one unidentified phospholipid (PL), one uniden- fermentans DSM 3133T, C.