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State Key Laboratory of Microbial Resources at the Institute of Microbiology, Chinese Academy of Sciences, ZhongGuanCun, Haidian, Beijing 100080, PR China
Correspondence
Shuang-Jiang Liu
shuangjiang{at}hotmail.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain H9T is AY500143.
A figure showing an electron micrograph of strain H9T and tables showing ammonia oxidation of nitrite and nitrate and cellular fatty acid composition of strain H9T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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and its description has since been subjected to repeated emendations by Warwick et al. (1994), McVeigh et al. (1994), Reichert et al. (1998) and Huang et al. (2002). The current membership of the genus Pseudonocardia includes 16 species with validly published names that form a phylogenetically coherent cluster and exhibit physiological versatility: psychrotolerant (Prabahar et al., 2004), thermophilic (Henssen, 1957) and autotrophic and heterotrophic (Takamiya & Tubaki, 1956). The physiological versatility of the species of Pseudonocardia renders them environmentally important in the removal of hazardous compounds and in biogeochemical cycles of elements. Pseudonocardia benzenivorans (Kämpfer & Kroppenstedt, 2004) and Pseudonocardia chloroethenivorans (Lee et al., 2004) were enriched for degradation of 1,2,3,5-tetrachlorobenzene and trichloroethene, respectively. Pseudonocardia asaccharolytica and Pseudonocardia sufidoxydans were isolated from biofilters and they were reported to use dimethyl disulfide as an energy source (Reichert et al., 1998). In this paper, we describe the newly isolated actinomycete strain H9T.
Strain H9T was isolated from coastal sediment (20 °C and 3·3 % salinity of the overlying sea water, GPS location of sampling site was 120° 14' 12'' E 35° 58' 48'' N) from Jiao-Dong peninsula near Tsingdao city, Shandong province, China, with a modified nitrifying medium [MNM; 2·0 g (NH4)2SO4, 0·25 g NaH2PO4, 0·75 g K2HPO4, 0·01 g MnSO4.4H2O, 0·03 g MgSO4.7H2O, 5·0 g CaCO3 and 33 g NaCl in 1000 ml deionized water (pH 8·0)]. For preparation of silica plates, silica (GF 254; Tsingdao marine chemical factory) was used as the solid matrix and was washed with 5 vols deionized water. After being washed twice with deionized water and autoclaved, 30 ml silica suspension [50 % (w/v) in deionized water] was poured into each plate (9 cm in diameter) and dried at 50 °C for 5–7 days, and 4 ml fivefold MNM was then added to the top of each plate and evaporated at 50 °C. The plates were inoculated with a loopful of sediment sample (0·1 g ml–1, suspended in saline) and were incubated at 30 °C for 1 month. One or two colonies were observed on each plate. Strain H9T was purified after several transfers and streaking onto MNM silica plates. For the growth assay of strain H9T in MNM, 20 ml of the cultural liquid was sampled and centrifuged, and the supernatant was used for analysis of ammonium (Slawyk & Maclsaac, 1972). The cell pellet was resuspended in 2 ml sterile deionized water and cells were broken by sonication. Nitrite and nitrate were determined according to van't Riet et al. (1968) and Rand et al. (1975), respectively. Results indicated that strain H9T grew autotrophically (uses CaCO3 as carbon source) and heterotrophically, and oxidized ammonia to nitrate in both MNM (nitrification) and blends of MNM and Luria–Bertani (LB) media (dissimilatory, heterotrophic ammonium oxidation; Supplementary Table S1 available in IJSEM Online). Strain H9T showed typical morphology of the genus Pseudonocardia. Chains of spores formed by acropetal budding from branched substrate mycelium. Both substrate and aerial mycelia fragmented into rod-shaped elements on trypticase soy broth (TSB; BBL) agar. The diameter of the aerial mycelium was about 0·5 µm (Supplementary Fig. S1 available in IJSEM Online).
Physiological and biochemical tests were carried out by following the procedures of Gordon et al. (1974) and Reichert et al. (1998), respectively, using Biolog GP2 plates (MicroStation), with reference strains in parallel. Nitrate reduction test was carried out according to Dong & Cai (2001). Strain H9T grew at NaCl concentrations ranging from 0 to 8 % with an optimum NaCl concentration of 3·5 %. Detailed physiological and biochemical properties of strain H9T are provided in the species description. Some characteristic and differential properties from phylogenetically closely related Pseudonocardia species are given in Table 1.
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and Lechevalier & Lechevalier (1980). Strain H9T contained meso-diaminopimelic acid and arabinose and galactose, thus giving a cell wall type IV, according to Lechevalier & Lechevalier (1970, 1980). Cells of strain H9T had type PIII phospholipid (Lechevalier et al., 1977; Lechevalier & Lechevalier, 1980). No glucosamine-containing phospholipids were detected. Menaquinones were extracted and purified from freeze-dried biomass according to Collins (1985), and were determined by using an HPLC procedure (Wu et al., 1989). The major menaquinone of strain H9T was MK-8 (H4). Cellular fatty acids were determined as described previously (Hu et al., 2004). The results (Supplementary Table S2 available in IJSEM Online) indicated that the major fatty acids of strain H9T were 16 : 0 (41·1 %), iso-16 : 1 (15·7 %) and 17 : 1
8c (12·1 %).
The 16S rRNA gene of strain H9T was amplified and sequenced as described previously (Zhang et al., 2003), and the sequence was aligned by using the CLUSTAL X program (Thompson et al., 1997). The 16S rRNA gene sequence similarities of strain H9T to members of the genus Pseudonocardia ranged from 93·0 to 97·5 %. The closest relatives were Pseudonocardia kongjuensis (97·5 %), Pseudonocardia autotrophica (97·1 %) and Pseudonocardia compacta (96·8 %), according to 16S rRNA gene similarity. Phylogenetic trees were constructed with neighbour-joining and maximum-parsimony methods, all trees showed similar topology. The tree constructed with the neighbour-joining method (Saitou & Nei, 1987) using Kimura's two-parameter calculation model in TREECON W version 1.3b (Van de Peer & De Wachter, 1994) is shown in Fig. 1. Strain H9T, together with P. kongjuensis, P. autotrophica and P. compacta, formed a phyletic clade with 100 % support.
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) and genomic DNA from Escherichia coli DH5
was analysed in parallel for calibration of the Tm value. The result showed that the G+C content of strain H9T was 69·6 mol% (Tm). DNA–DNA hybridizations were carried out according to De Ley et al. (1970). Renaturation rates and relatedness values were calculated as described by Jahnke (1992). DNA–DNA relatedness values between strain H9T and P. kongjuensis DSM 44525T, P. autotrophica IMSNU 20050T and P. compacta IMSNU 20111T were 42, 13 and 11 %, respectively.
Description of Pseudonocardia ammonioxydans sp. nov.
Pseudonocardia ammonioxydans (N.L. n. ammonia ammonia; N.L. part. adj. oxydans oxidizing; N.L. part. adj. ammonioxydans oxidizing ammonia).
Aerobic, Gram-positive. Forms branched, brown substrate mycelium and white aerial mycelium on TSB and LB agar. The mycelium fragments into rod-shaped elements. Smooth spores are borne in short chains by acropetal budding from the substrate mycelium. No pigment is produced. Growth occurs at 10–40 °C. Growth occurs on MNM and ammonia is oxidized to nitrate as the sole energy source. Ammonia is also oxidized to nitrate during growth on a blend of MNM and LB media. Growth occurs at NaCl concentrations ranging from 0 to 8 % with an optimum at 3·5 % NaCl. Catalase-positive. Acid is produced from D-fructose, D-glucose, N-acetyl-D-glucosamine, D-ribose, D-arabitol, D-galacturonic acid, D-gluconic acid and glycerol, but not from N-acetyl-
-D-mannosamine, arbutin, L-fucose, gentiobiose, -D-lactose, lactulose, maltotriose, methyl -D-galactoside, methyl -D-galactoside, methyl -D-glucoside, methyl -D-mannoside, palatinose, D-psicose, D-raffinose, L-rhamnose, D-gagatose, turanose or xylitol. Uses acetic acid, -hydroxybutyric acid, -ketoglutaric acid, lactamide, D-lactic acid methyl ester, L-lactic acid, L-malic acid, pyruvic acid methyl ester, succinic acid mono-methyl ester, propionic acid, succinamic acid, succinic acid, N-acetyl-L-glutamic acid, L-alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-glutamic acid, glycyl L-glutamic acid, L-pyroglutamic acid, L-serine, putrescine, but not -hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketovaleric acid, D-malic acid or pyruvic acid. Other physiological properties are listed in Table 1
. The cell-wall chemotype is type IV. Predominant menaquinone is MK-8 (H4). It contains phosphatidylcholine, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylinositol mannosides and diphosphatidylglycerol, but does not contain glucosamine-containing phospholipids or mycolic acids. The major fatty acids are iso-hexadecanoic acid (16 : 0-iso, 41·1 %), iso-hexadecenoic acid (16 : 1-iso, 15·7 %) and heptadecenoic acid (17 : 18c, 12·1 %). The G+C content of the DNA is 69·6 mol% (Tm).
The type strain, H9T (=CGMCC 4.1877T=JCM 12462T), was isolated from coastal sediment collected from Jiao-Dong peninsula near Qingdao, Shandong province, China.
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ACKNOWLEDGEMENTS |
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