Nocardioides Antarcticus Sp. Nov., Isolated from Marine Sediment Sangsang Deng, Xulu Chang, Yumin Zhang, Lvzhi Ren, Fan Jiang, Zhihao Qu and Fang Peng

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 2615–2621 DOI 10.1099/ijs.0.000309

Nocardioides antarcticus sp. nov., isolated from marine sediment Sangsang Deng, Xulu Chang, Yumin Zhang, Lvzhi Ren, Fan Jiang, Zhihao Qu and Fang Peng

Correspondence China Center for Type Culture Collection (CCTCC), College of Life Sciences, Wuhan University, Fang Peng Wuhan 430072, PR China [email protected] Strain M-SA3-94T, an aerobic, Gram-stain-positive, ovoid- to rod-shaped, non-motile bacterium, was isolated from the marine sediment of Ardley cove, King George Island, Antarctica. Strain M-SA3-94T grew optimally at pH 5.0–6.0, 20 8C and in the presence of 1.0 % (w/v) NaCl. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain M-SA3-94T belonged to the genus Nocardioides in the family Nocardioidaceae, clustering with Nocardioides plantarum NCIMB 12834T, Nocardioides ginsengagri BX5-10T, Nocardioides marinquilinus CL-GY44T and Nocardioides lianchengensis D94-1T (with 96.1 %, 95.9 %, 94.5 % and 94.7 % 16S rRNA gene sequence similarity, respectively). The chemotaxonomic properties of strain M-SA3-94T were similar to those of members of the genus Nocardioides

with validly published names. The major fatty acid was iso-C16 : 0. The polar lipid pattern contained diphosphatidylglycerol, phosphatidylglycerol and three unknown phospholipids. The diagnostic diamino acid in the cell-wall peptidoglycan was LL-2, 6-diaminopimelic acid. MK-8(H4) was the predominant menaquinone and the DNA G+C content of this strain was 66.7 mol%. On the basis of these phenotypic, phylogenetic and chemotaxonomic data, strain M-SA3-94T represents a novel species of the genus Nocardioides, for which the name Nocardioides antarcticus sp. nov. is proposed. The type strain is M-SA3-94T (5CCTCC AB2014053T5LMG 28254T).

The genus Nocardioides, belonging to the family marine (Dastager et al., 2009), soil (Dastager et al., 2008), Nocardioidaceae within the suborder Propionibacterineae meadow (Collins et al., 1994), halophyte (Song et al., (Stackebrandt et al., 1997), was originally created by 2011), the crustacean Daphnia cucullata (To´th et al., Prauser (1976) who designated Nocardioides albus KCTC 2008), a fresh water spring (Chou et al., 2008), alpine gla- 9186T as the type species. Species of the genus Nocardioides cier cryoconite (Zhang et al., 2012a) and even polluted are Gram-stain positive, aerobic, motile or non-motile and environments (Schippers et al., 2005). rod-shaped, and characterized chemotaxonomically by Strain M-SA3-94T was isolated from a sediment sample having MK-8(H4) as the principal menaquinone, LL-2, 6 collected from Ardley cove, King George Island, Antarctica diaminopimelic acid in the peptidoglycan, iso-C16 : 0 as (628 12.3999 S 0588 55.0499 W). Isolation was carried out the main fatty acid (Collins et al., 1983; Prauser, 1976) using the standard dilution plating method on marine + and a high DNA G C content. Since the polyphasic taxo- broth (MB) 2216 agar (BD) and incubated at 4 8C for nomic classification of bacteria has improved in recent one month. The purified strain was routinely cultured on years, the number of novel species of the genus Nocar- MB agar and stored by lyophilization. dioides has increased rapidly. At the time of writing, the genus Nocardioides comprises at least 73 species with The extraction and purification of genomic DNA were carried validly published names. The species of the genus Nocar- out as described previously (Hopwood et al., 1985). The univer- 9 9 dioides have been isolated from diverse habitats, such as sal primer pair, 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (59-GGTTACCTTGTTACGACTT-39), were used to amplify the 16S rRNA gene sequences and the PCR Abbreviations: AL, aminolipid; DPG, diphosphatidylglycerol; L, lipid; PG, products were purified with an ENZA gel extraction kit phosphatidylglycerol; PI, phosphatidylinositol; PL, phospholipid; PIM, (Omega Bio-Tek), ligated into pM 18-T vectors (TakaRa) The GenBank/EMBL/DDBJ accession number of the 16S rRNA gene and sequenced by Honourtech (Wuhan). The 16S rRNA T sequence of strain M-SA3-94 is KM347967. gene sequences of related taxa were obtained from the GenBank Two supplementary figures and one supplementary table are available and EzTaxon-e server (Kim et al., 2012). Multiple alignments with the online Supplementary Material. were performed by using the CLUSTAL X 1.8 program

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(Thompson et al., 1997). Phylogenetic trees were reconstructed physiological properties were determined by using API using the neighbour-joining (Saitou & Nei, 1987), maximum- ZYM, API 20 NE, API 20 E, API 32 GN and API 50 CH likelihood (Felsenstein, 1981) and minimum-evolution test kits (bioMe´rieux) and Gram-positive MicroPlates (Rzhetsky & Nei, 1992) methods with Kimura 2-state par- (Biolog GP 2), according to the manufacturers’ instructions. ameter model analyses implemented in the program MEGA Susceptibility to antibiotics was tested on MB agar plates version 5 (Tamura et al., 2011). Tree topologies were evalu- using the disc-diffusion process (Bauer et al., 1966) with ated by bootstrap analysis with 1000 replications (Felsenstein, the following antibiotics (mg per disc unless otherwise speci- 1985). fied): cycline (30), neomycin (10), novobiocin (5), cefoperazone (75), ciprofloxacin (5), ceftriaxone (30), erythromycin The 16S rRNA gene sequence of strain M-SA3-94T com- (15), carbenicillin (10), kanamycin (30), norfloxacin (10), prised 1500 bp nucleotides, and highest similarity with cephazolin (30), streptomycin (10), ofloxacin (5), rifampicin Nocardioides plantarum NCIMB 12834T was revealed, (5), chloramphenicol (30), cephradine (30), gentamicin (10), with 96.1 % 16S rRNA sequence similarity. Sequence simi- polymyxin B (300 U), clindamycin (2), lincomycin (2), larities between strain M-SA3-94T and other members of furazolidone (15), metronidazole (5), vancomycin (30), the genus Nocardioides with validly published names were trimethoprim (25), cefotaxime sodium (30), doxycycline all lower than 96.0 %. The neighbour-joining phylogenetic (30), carbenicillin (100). The experimental results were tree (Fig. 1) indicated that strain M-SA3-94T belonged to recorded after 5 days’ incubation of all test plates at 20 8C. the genus Nocardioides and clustered with N. plantarum NCIMB 12834T, Nocardioides ginsengagri BX5-10T, Nocar- For cellular fatty acid analysis, strain M-SA3-94T and the dioides marinquilinus CL-GY44T and Nocardioides lian- five reference type strains were grown on MB agar plates chengensis D94-1T (with 96.1 %, 95.9 %, 94.5 % and at 20 8C. The fatty acids were extracted from cell biomass 94.7 % 16S rRNA gene sequence similarities, respectively. of the late-exponential phase, according to MIDI instruc- This relationship was also found in the tree generated tor, by the standard four-area-streaking plate method. with the maximum-likelihood and minimum-evolution When a colony was just appearing in the fourth area, the algorithm (Fig. S1, available in the online Supplementary cells of third area were approximately in the late exponen- Material). Therefore N. lianchengensis CGMCC 4.6858T, tial phase. Fatty acid methyl esters were prepared for anal- N. marinquilinus JCM 18459T, N. plantarum LMG ysis by gas chromatography (6890N, Agilent) according to 16210T, N. ginsengagri DSM 21362T and the type species the standard protocol of the Sherlock Microbial Identifi- Nocardioides albus DSM 43109T (with 93.9 % 16S rRNA cation System (MIDI Sherlock version 6.0, MIDI database gene sequence similarity) were selected as reference strains TSBA6) (Sasser, 1990). To measure the DNA G+C con- for subsequent experiments. tent, genomic DNA was extracted from strain M-SA3- 94T, purified, enzymatically, degraded into nucleosides Growth on various media [R2A agar, tryptic soy agar and analysed as described by Mesbah & Whitman (1989) (TSA), nutrient agar (NA), MB agar, MacConkey agar; all using HPLC. Respiratory quinones of strain M-SA3-94T BD] was assessed. Growth at different temperatures (4, and N. albus DSM 43109T were extracted and identified 10, 15, 20, 25, 28, 30, 37, 42 8C) was monitored daily for by HPLC (UltiMate 3000, Dionex), as described by Xie & one week on MB agar. Growth at diverse pH values Yokota (2003). Polar lipids were extracted using the pro- (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) was determined in cedures of Minnikin et al. (1984) and identified by two- MB 2216 after 5 days at 20 8C. Salt tolerance was tested dimensional TLC (Tindall, 1990). The presence of diami- in R2A broth supplemented with 0–25.0 % (w/v) NaCl nopimelic acid isomers in the cell-wall peptidoglycan was (in increments of 1.0 %), with incubation for 5 days at determined by TLC after hydrolysis with 6 M HCl at 20 8C. Growth of strain M-SA3-94T under anaerobic con- 100 8C for 18 h (Komagata & Suzuki, 1987). ditions was examined by using MB agar with potassium nitrate (0.1 %, w/v) added to cultures, which were incu- Strain M-SA3-94T grew well on MB 2216 agar, NA, TSA bated for one month in a GasPak (OXOID) jar at 20 8C. and R2A agar, but did not grow on MacConkey agar. The Gram-reaction was tested by the Gram-staining MB agar was the best medium for the growth of strain method described by Doetsch (1981). Gliding motility M-SA3-94T. The temperature range for growth of strain was investigated as described by Bowman (2000). Cell mor- M-SA3-94T on MB agar was 4–37 8C (optimum 20 8C) phology was examined by phase-contrast (Olympus; BX51) and there was no growth at 42 8C. The pH range for and transmission electron microscopy (H-8100; Hitachi) growth was between pH 5.0 and 9.0 and the optimal pH using cells grown for 3 days at 20 8C on MB agar. Catalase was between 5.0 and 6.0. Growth occurred in the presence activity was determined by bubble production in 3 % (v/v) of 0–3.0 % (w/v) NaCl (optimum 1.0 %, w/v). After growth H2O2. Cytochrome oxidase activity was evaluated by using for 5 days on an MB agar plate at 20 8C, a colony of strain 1 % (w/v) N, N, N’, N’-tetramethyl-p-phenylenediamine M-SA3-94T formed circular, smooth, creamy, opaque and (Cappuccino & Sherman, 2002). The degradation of convex colonies (1.0–2.0 mm in diameter). Strain M- starch, DNA, casein, Tweens-20, -40, -60 and -80, xanthine, SA3-94T was Gram-stain positive, aerobic, non-motile hypoxanthine and guanine were investigated on MB agar and ovoid- to rod-shaped. No substrate mycelium or pri- plates after two weeks at 20 8C. In addition to enzyme activi- mary mycelium were observed. Strain M-SA3-94T was ties, substrate oxidation, carbon source utilization and other positive for catalase and negative for oxidase; it could

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53 Nocardioides conyzicola HWE 2-02T (KC878445) 0.01 95 Nocardioides hankookensis DS-30T (EF555584)

92 Nocardioides endophyticusMWE 3-5T (KC878444)

Nocardioides pyridinolyticusOS4T (U61298)

95 Nocardioides aquiterrae GW-9T (AF529063)

Nocardioides alpinusCr7-14T (GU784866) DS-17T (EF363712)T 72 Nocardioides terrigena 83 Nocardioides sediminis MSL-01T (EF466110)

Nocardioides panacisoli GSoil 346T (FJ666101)

Nocardioides tritolerans MSL-14T (EF466107)

Nocardioides caeni MN8T (FJ423551)

Nocardioides aestuarii JC2056T (AY423719)

Nocardioides marinus CL-DD14T (DQ401093)

VA15T (FJ423762) 93 Nocardioides terrae 68 Nocardioides panacihumi Gsoil 616T(AB271053) D287T (AM398438) 88 Nocardioides daphniae Marmoricola aurantiacus BC 361T (Y18629)

Nocardioides lianchengensis D94-1T (HQ657322)

55 Nocardioidesa ntarcticus M-SA3-94T (KM347967) 53 Nocardioides plantarum NCIMB 12834T (AF005008) 88 Nocardioides ginsengagri BX5-10T (GQ339904)

82 Nocardioides marinquilinus CL-GY44T (JX164255)

Nocardioides kribbensisKSL-2T (AY835924) RHLT -1T (JF750425) 75 Nocardioides psychrotolerans 2 RHLT -17T (JF750424) 60 Nocardioides szechwanensis 1 Nocardioides dokdonensis FR1436T (EF633986)

Nocardioides bigeumensis MSL-19T (EF466114)

95 Nocardioides marinisabuli SBS-12T (AM422448) J112T (EU143365) 99 Nocardioides basaltis 91 Nocardioides salarius CL-Z59T (DQ401092) Actinopolymorpha singaporensis IM 7744T (AF237815)

Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences, showing the relationships between strain M-SA3-94T and representative members of the genus Nocardioides. Bootstrap values (expressed as percentages of 1000 replications) are shown as percentages at each node (only if they are 50 % or greater). Actinopolymorpha singaporensis IM7744T was used as the outgroup. Bar, 0.01 substitutions per nucleotide position. hydrolyse casein, Tween-20, Tween-40, Tween-60 and shown in Table S1. The cellular fatty acids of strain M- T Tween-80, but not starch, xanthine, hypoxanthine and gua- SA3-94 were dominated by iso-C16 : 0 (comprising nine. The physiological and biochemical characteristics of 50.5 %), which is typically detected as a predominant T strain M-SA3-94 are summarized in the species descrip- fatty acid of most members of the genus Nocardioides tion and a comparison of selected characteristics with (Yoon et al., 1999); however, the proportion in strain M- those of the ﬁve reference type strains is presented in SA3-94T was obviously higher than in the reference strains. Table 1. The percentage of iso-C16 : 0 (16.9 %) in N. lianchengensis The percentage composition of the cellular fatty acids CGMCC 4.6858T was lower than the 29.2 % previously of strain M-SA3-94T and the ﬁve reference type strains is reported (Zhang et al., 2012b). The major cellular fatty

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Table 1. Differential characteristics of strain M-SA3-94T and ﬁve reference strains

Strains: 1, M-SA3-94T;2,N. albus DSM 43109T;3,N. lianchengensis CGMCC 4.6858T;4,N. marinquilinus JCM 18459T;5,N. plantarum LMG 16210T;6,N. ginsengagri DSM 21362T. All data are from this study unless indicated otherwise. All strains were cultivated at 20 8C on MB agar. +, Positive; W, weakly positive; 2, negative; AL, aminolipid; DPG diphosphatidylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PL, phospholipid; phosphatidylinositol mannoside.

Characteristic 1 2 3 4 5 6

I Temperature range 4–37(20) (28) 10–40(30)* 10–35(30) 5–35(30)D 10–37(30)§ (optimum)(8C) pH range (optimum) 5.0–9.0(5.0–6.0) ND 6.0–9.0(7.0)* 6.0–9.5(7.0)D 5.5–9.5(6.5–7.0)D 5.5–9.5(7.0)§ Salt tolerance (optimum) 0–3.0(1.0) ND 0–4(0–2)* 0–5.5(0)D 0–5(0–0.5)D 0–1(0)D [% NaCl, (w/v)] Catalase +++ + + W Hydrolysis of: Starch 2 + 22 2 2 Casein +++ + + 2 Hypoxanthine 2 ++ + 22 Xanthine 2 + 22 2 2 Enzyme activity (API ZYM) Alkaline phosphatase +++ + W 2 Esterase (C4) W ++ + + W Esterase lipase (C8) W ++ + + + Lipase (C14) 222 2 WW Valine arylamidase W 2 + W ++ Cystine arylamidase 222 W ++ Trypsin 2 ++ + 22 a-Chymotrypsin 222 + 22 b-Galactosidase 2 + 22 2 2 b-Glucosidase 2 ++ 2 + 2 N-Acetyl-b-glucosaminidase W 22 2 2 2 Enzyme activity (API 20 NE) Nitrate reductase + 22 2 2 2 Urease 222 + 22 Gelatinase 2 + 2 + 22 b-Galactosidase 2 ++ 22 2 Assimilation of (API 20 NE): D-Glucose 2 ++ + 22 L-Arabinose 2 ++ 22 2 Mannose 2 + 22 2 2 D-Mannitol 2 ++ + 22 Maltose 222 + 22 Gluconate 2 + 2 ++ 2 Malic acid 2 ++ 2 + 2 Enzyme activity and oxidation of carbon source (API 20 E) O-Nitrophenyl-b-D-Galactoside 2 W 22 2 2 Arginine dihydrolase 222 2 + 2 Sodium citrate 2 W 22 2 2 Urea 222 + 22 Pyruvate W ++ + W Gelatin 2 ++ + 22 Glucose 22+ 22 2 Mannitol 22+ 22 2 Rhamnose 2 W + 22 2 Sucrose 22W 22 2 Amygdalin 22W 22 2

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Table 1. cont.

Characteristic 1 2 3 4 5 6

Arabinose W W + 2 W 2 Sole carbon source (API 32 GN) Rhamnose 2 ++ 2 + 2 D-Ribose 2 ++ 2 W 2 Sucrose + 2 ++ + 2 Maltose + 22 + 22 Itaconic acid + 22 2 2 2 Suberate ++22 W 2 Malonate 2 ++ 22 2 Acetate 2 ++ 22 2 DL-Lactate 22+ 22 2 L-Alanine ++22 2 2 Glycogen ++2 + 22 L-Serine 2 + 22 2 2 Mannitol 2 ++ + 22 D-Glucose 2 ++ + 22 Salicin + 22 w 22 Melibiose + 22 2 2 2 L-Fucose + 22 2 2 2 D-Sorbitol + 22 2 W 2 L-Arabinose +++ 22 2 Propionate 2 ++ 22 2 Valerate 2 ++ 22 2 Histidine 22+ 22 2 3-Hydroxy-butyrate +++ + + 2 L-Proline 2 ++ + 22 DNA G+C content (mol%) 66.7 67I 71.8D 71.6d 69.0§ 70.3§ Polar lipids PG,DPG,PL1, ND PG, DPG,PI, PG, PL1–3, PG, PL1–2,L3D PG, PL1–3,L2.AL§ PL2, PL3 PIM, AL* L1-2D

*Data from Zhang et al. (2012b). DData from Cho et al. (2013). dData from Collins et al. (1994). §Data from Lee et al. (2012). IData from Yoon et al. (2005).

T acids of N. ginsengagri DSM 21362 were C18 : 1v9c (37.2 %), Schumann 1999; Holt et al., 1994). Diphosphatidylglycerol C16 : 0 (16.8 %) and iso-C16 : 0 (15.7 %), which was also (DPG), phosphatidylglycerol (PG) and three unknown somewhat different from the data [C18 : 1v9c (33.2 %), phospholipids were detected in the polar lipid proﬁle C16 : 0 (17.7 %) and C17 : 1v8c (8.7 %)] reported by Lee (two-dimensional TLC, Fig. S2). DPG and PG are particu- et al. (2012). These differences were caused by the different larly common among the polar lipids of species of the culture conditions used, such as the medium and tempera- genus Nocardioides. The presence of LL-2,6-diaminopimelic ture. Other major fatty acids (.5.0 %) of strain M-SA3-94T acid in the cell-wall peptidoglycan also supported the pla- T comprised C17 : 1v8c (6.6 %), C17 : 1v6c (6.3 %), iso-H- cement of strain M-SA3-94 in the genus Nocardioides. C (6.1 %) and C v9c (5.8 %). 16 : 1 18 : 1 The phylogenetic trees, chemotaxonomic properties and The DNA G+C content of strain M-SA3-94T (66.7 mol%) physiological characteristics of strain M-SA3-94T corre- was within the range for members of the genus Nocar- sponded with descriptions of species of the genus Nocar- dioides. Strain M-SA3-94T contained MK-8(H4) as the dioides. However, the 16S rRNA gene sequence, cellular major respiratory quinone because of the same retention fatty acid proﬁle and a number of differential phenotypic time as N. albus DSM 43109T. Menaquinone MK-8(H4) characteristics distinguished strain M-SA3-94T from is the principal menaquinone of members of the genus species of the genus Nocardioides with validly published Nocardioides with validly published names (Busse & names. Therefore, strain M-SA3-94T represents a novel

Downloaded from www.microbiologyresearch.org by http://ijs.sgmjournals.org 2619 IP: 128.95.40.200 On: Mon, 30 Nov 2015 03:27:23 S. Deng and others species within the genus Nocardioides, for which the name The type strain, M-SA3-94T (5CCTCC AB2014053T5LMG Nocardioides antarcticus sp. nov. is proposed. 28254T), was isolated from the marine sediment of Ardley cove, King George Island, Antarctica. The genomic DNA + Description of Nocardioides antarcticus sp. nov. G C content of the type strain is 66.7 mol% Nocardioides antarcticus (ant.arc’ti.cus. L. masc. adj. antarcticus southern, by extension pertaining to the Antarctic, Acknowledgements referring to its isolation source). This work was supported by the National Basic Research Program Aerobic, Gram-stain-positive, catalase-positive, oxidase- of China (973 Program) (2011CB808800), National Infrastructure of negative and non-motile. Rods or ovoid, 0.6–1.8 mmin Natural Resources for Science and Technology Program of China (No. diameter and 1.3–3.7 mm long. After incubation for NIMR-2014-8), National Natural Science Foundation of China (grant 5 days at 20 uC on MB agar colonies are circular, smooth, No. 31270538) and State Oceanic Administration, PR. China (Project convex, opaque, creamy and approximately 1.0–2.0 mm no. 10/11YR06). in diameter. No substrate mycelium or primary mycelium are formed. Growth occurs in 0–3.0% (w/v) NaCl (opti- References mum 1.0%, w/v) at 4–37 uC (optimum 20 uC) and at pH 5.0–9.0 (optimum pH 5.0–6.0). Good growth occurs Bauer, A. W., Kirby, W. M. M., Sherris, J. C. & Turck, M. (1966). on MB, NA, TSA and R2A, but not on MacConkey agar. Antibiotic susceptibility testing by a standardized single disk method. Casein, Tween-20, Tween-40, Tween-60 and Tween-80 Am J Clin Pathol 45, 493–496. are hydrolysed, but starch, xanthine, hypoxanthine and Bowman, J. P. (2000). ). Description of Cellulophaga algicola sp. nov., guanine are not. Susceptible to the following antibiotics: isolated from the surfaces of Antarctic algae, and reclassification of penicillin G, novobiocin, cefoperazone, erythromycin, car- Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as 50 benicillin, kanamycin, cephazolin, streptomycin, rifampi- Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol , 1861–1868. cin, gentamicin, polymyxin B, clindamycin, lincomycin and vancomycin. Resistant to the following antibiotics: Busse, H.-J. & Schumann, P. (1999). Polyamine profiles within genera of the class Actinobacteria with -diaminopimelic acid in the cycline, ciprofloxacin, ceftriaxone, norfloxacin, ofloxacin, peptidoglycan. Int J Syst Bacteriol 49, 179–184. cephradine, furazolidone, metronidazole, trimethoprim, Cappuccino, J. G. & Sherman, N. (2002). Microbiology: a Laboratory cefotaxime sodium and oxycycline. According to the results Manual, 6th edn. Menlo Park, CA: Benjamin/Cummings. of API ZYM tests, alkaline phosphatase, leucine arylami- Cho, Y., Jang, G. I. & Cho, B. C. (2013). Nocardioides marinquilinus sp. dase, acid phosphatase, naphthol-AS-BI-phosphohydrolase 63 a nov., isolated from coastal seawater. Int J Syst Evol Microbiol , and -glucosidase are present; esterase (C4), esterase lipase 2594–2599. b (C8), valine arylamidase and N-acetyl- -glucosaminidase Chou, J. H., Cho, N. T., Arun, A. B., Young, C. C. & Chen, W. M. (2008). are weakly positive, but lipase (C14), cystine arylamidase, Nocardioides fonticola sp. nov., a novel actinomycete isolated from trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, spring water. Int J Syst Evol Microbiol 58, 1864–1868. b b a a -glucuronidase, -glucosidase, -mannosidase and - Collins, M. D., Keddie, R. M. & Kroppenstedt, R. M. (1983). Lipid fucosidase are absent. All results are negative in the API 20 composition of Arthrobacter simplex, Arthrobacter tumescens and NE test strips, except for nitrate reduction and aesculin possibly related taxa. Syst Appl Microbiol 4, 18–26. activity. The Voges–Proskauer reaction is the only positive Collins, M. D., Cockcroft, S. & Wallbanks, S. (1994). Phylogenetic result in API 20 E. API 50 CH strips showed that only aesculin analysis of a new LL-diaminopimelic acid-containing coryneform and 5-keto-gluconate are hydrolysed. In API ID 32 GN kits, bacterium from herbage, Nocardioides plantarum sp. nov. Int J Syst 44 sucrose, maltose, itaconic acid, suberate, L-alanine, glycogen, Bacteriol , 523–526. salicin, melibiose, L-fucose, D-sorbitol, L-arabinose and Dastager, S. G., Lee, J. C., Ju, Y. J., Park, D. J. & Kim, C. J. (2008). 3-hydroxy-butyrate can act as sole carbon sources. Utilizes Nocardioides koreensis sp. nov., Nocardioides bigeumensis sp. nov. and Nocardioides agariphilus sp. nov., isolated from soil from dextrin, Tween-40, L-arabinose, D-fructose, D-galactose, Bigeum Island, Korea. Int J Syst Evol Microbiol 58, 2292–2296. D-gluconic acid, a-D-glucose, myo-inositol, D-mannitol, Dastager, S. G., Lee, J. C., Ju, Y. J., Park, D. J. & Kim, C. J. (2009). D-mannose, 3-methyl-D-glucose, D-ribose, D-sorbitol, Nocardioides sediminis sp. nov., isolated from a sediment sample. D D -xylose, acetic acid, a-ketoglutaric acid, lactamide, -malic Int J Syst Evol Microbiol 59, 280–284. acid, succinic acid mono-methyl ester, propionic acid, pyruvic Doetsch, R. N. (1981). Determinative methods of light microscopy. In acid,succinicacid,glycerol,adenosine,inosine,thymidine and Manual of Methods for General Bacteriology, pp. 21–33. Edited by uridine as carbon and energy sources, according to Biolog P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. 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