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State Key Laboratory of Microbial Resources and 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 T3-B9T is AY500142.
The polar lipid profile of strain T3-B9T and a phylogenetic tree for Novosphingobium species constructed using the maximum-parsimony method are available as supplementary material in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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to accommodate species of the genus Sphingomonas (Yabuuchi et al., 1990) that clustered phylogenetically with Novosphingobium capsulatum [cluster III of Takeuchi et al. (2001)], have 2-OH 14 : 0 as the major 2-hydroxy fatty acid and spermidine as the major polyamine and are able to reduce nitrate (Takeuchi et al., 2001). Although Yabuuchi et al. (2002) argued that the creation of the genus Novosphingobium required more phenotypic support, many sphingomonad taxonomists (Kämpfer et al., 2002; Fujii et al., 2003; Sohn et al., 2004; Tiirola et al., 2005) adhere to the proposal of Takeuchi et al. (2001) because of phylogenetic and also chemotaxonomic studies that clearly separate Novosphingobium from the genus Sphingomonas sensu stricto. Currently, the genus Novosphingobium includes the following species with validly published names: Novosphingobium aromaticivorans (Balkwill et al., 1997), Novosphingobium capsulatum (Leifson, 1962; Yabuuchi et al., 1990), Novosphingobium hassiacum (Kämpfer et al., 2002), Novosphingobium pentaromativorans (Sohn et al., 2004), Novosphingobium rosa (Takeuchi et al., 1995), Novosphingobium stygium (Balkwill et al., 1997), Novosphingobium subarcticum (Nohynek et al., 1996), Novosphingobium subterraneum (Balkwill et al., 1997), Novosphingobium tardaugens (Fujii et al., 2003) and Novosphingobium lentum (Tiirola et al., 2005). The organisms of this genus are inhabitants of soil, sediments and other subsurfaces and are able to degrade various xenobiotic aromatic compounds (Stevens et al., 1993; Fredrickson et al., 1995; Karlson et al., 1995; Kim et al., 1996; Wildes et al., 1996; Balkwill et al., 1997). Thus, they are potentially important in the biogeochemical cycles of carbon, nitrogen or chlorine, elements of xenobiotic compounds in their surrounding environments. They have also potential biotechnological applications, for example, the bioremediation of aromatic-contaminated environments. During a systematic investigation of the microbial ecology and elemental biogeochemistry of Taihu Lake, located in south-east China, a yellow-pigmented bacterial strain, designated strain T3-B9T, was isolated from a lake sediment sample (GPS location of sampling site is 120° 02' 16·8'' E 31° 27' 10·7'' N). Sediment samples were obtained at 10 cm beneath the sediment surface. Strain T3-B9T was isolated by plating 0·1 ml of 10-fold dilutions of the sample onto diluted LB agar (0·1 % peptone, 0·05 % yeast extract, 0·1 % NaCl, 1·5 % agar). Routine cultivation was conducted aerobically with LB agar or LB broth at 30 °C.
Gram staining was conducted according to the method described by Gerhardt et al. (1994). Oxidase activity, indole production and hydrolysis of starch etc. were tested according to the methods described by Dong & Cai (2001). Spore formation was determined by malachite green staining of cells grown on LB agar. Flagellation was examined by using a transmission electron microscope (H-600; Hitachi) at 100 kV after negative staining with 1 % (w/v) phosphotungstic acid. Morphological observation was carried out using a scanning electron microscope (FEI Quanta 2000). For assaying assimilation of aromatic compounds, each compound was added at a concentration of 30 mg l–1 to mineral medium broth (Liu et al., 2002). Growth was examined after incubation at 30 °C for 1 week. Denitrification was determined by the method of Stanier et al. (1966). Catalase activity was judged by formation of bubbles after a 3 % H2O2 solution was dropped onto a fresh colony. Aerobic and anaerobic production of acids (OF reaction) from carbohydrates was determined in OF basal medium (Hugh & Leifson, 1953). Carbohydrate solutions, sterilized by filtration, were added at a final concentration of 1 % (w/v) and acid production from carbohydrates was recorded after 7 and 14 days of incubation. Cellular fatty acids were extracted from cells grown in LB broth at 30 °C and subsequently purified and analysed as described previously (Hu et al., 2004 and papers cited therein). For the analysis of polar lipids and quinones, strain T3-B9T was grown at 22 °C in R2A broth; cells were harvested at the late-exponential phase, centrifuged and lyophilized. Polar lipids were examined by two-dimensional TLC and characterized with spreading reagents specific for 
-glycols (periodate-Schiff), sugars (-naphthol-H2SO4, anisaldehyde-H2SO4) and phosphate (Zindzadze) (Ventosa et al., 1993). Quinones were determined according to Collins (1985) and Wu et al. (1989). DNA base compositions were determined by thermal denaturation (Marmur & Doty, 1962) and DNA from Escherichia coli DH-5 was used as standard for the calibration of the Tm value. The 16S rRNA gene was amplified and sequenced as described previously (Zhang et al., 2003). 16S rRNA gene sequence alignments were performed using the CLUSTAL X program (Thompson et al., 1997). Phylogenetic trees were constructed with the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony methods. DNA–DNA hybridization was performed by the thermal denaturation and renaturation method of De Ley et al. (1970) and modified by Huß et al. (1983).
Cells of strain T3-B9T were Gram-negative, aerobic, non-spore-forming, non-motile rods, about 0·45x1·8
4·0 µm. The strain produced small (about 2 mm), yellow colonies on 10-fold-diluted LB agar, LB agar or R2A agar after 3 days cultivation. The optimal temperature for growth of strain T3-B9T was 25 °C; growth was observed at 10–37 °C, but not at 4 or 40 °C. The optimal pH for growth was 6·0; growth occurred at pH 4·5–8·5, but not at pH 4·0 or 9·0. It assimilated the following aromatic compounds for growth: phenol, 3-aminophenol, aniline, nitrobenzene, 4-chloronitrobenzene, 3-hydroxybenzoate and phenanthrene, but not 3- or 4-chloroaniline, chlorobenzene, 3,5-dihydroxytoluene, anthracene or pyrene.
Strain T3-B9T reduced nitrate, but did not produce N2, consistent with the properties of the genus Novosphingobium (Takeuchi et al., 2001). H2S was not produced. Catalase activity was positive. Additional features and some properties that differentiate strain T3-B9T from other members of the genus Novosphingobium are given in Table 1. Strain T3-B9T contained 18 : 1 as the dominant fatty acid and 2-OH 14 : 0 as the major hydroxylated fatty acid. The cellular fatty acid profile of strain T3-B9T is outlined in the species description. Strain T3-B9T contained sphingoglycolipid, phosphatidylethanolamine and diphosphatidylglycerol as major identified polar lipids (see Supplementary Fig. A in IJSEM Online). The unidentified phospholipid PLx1 was most likely identical to the corresponding unidentified phospholipids of N. lentum (PLx1; Tiirola et al., 2005) and of N. hassiacum (PL; Kämpfer et al., 2002). The unidentified polar lipids Lx1 and PLx2 of strain T3-B9T were most likely identical to the corresponding polar lipids Lx1 and PLx2 of N. lentum (Tiirola et al., 2005). Strain T3-B9T showed more unidentified polar lipids, Lx2 and Lx3, when compared to N. lentum (Tiirola et al., 2005), and phosphatidylmonomethylethanolamine (PME) was not detected. The combined presence of unknown polar lipids and the absence of PME clearly distinguishes T3-B9T from other species of Novosphingobium, including N. aromaticivorans, N. capsulatum, N. stygium, N. subterraneum, N. subarcticum, N. rosa (Busse et al., 1999), N. hassiacum (Kämpfer et al., 2002) and N. lentum (Tiirola et al., 2005). The predominant isoprenoid quinone of strain T3-B9T was Q-10. The G+C content of strain T3-B9T was 63·3 mol% (Tm), which is in accordance with the range described for the genus Novosphingobium (62–67 mol%; Takeuchi et al., 2001). All these data indicate that strain T3-B9T is a member of the genus Novosphingobium.
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). Strain T3-B9T showed levels of 16S rDNA gene sequence (1362 base pairs) similarity ranging from 91·6 to 97·5 % to members of the genus Novosphingobium, with the highest to N. subterraneum DSM 12447T (97·5 %) and N. aromaticivorans ATCC 700278T (97·2 %). A neighbour-joining tree (Fig. 1) showed that strain T3-B9T formed coherent clades with other members of the genus Novosphingobium and it clustered closely with N. stygium ATCC 700280T (75 % bootstrap support). A tree derived by the maximum-parsimony method is available as Supplementary Fig. B in IJSEM Online. The DNA–DNA relatedness of strain T3-B9T to N. subterraneum DSM 12447T, N. aromaticivorans ATCC 700278T and N. stygium ATCC 700280T was 31, 33 and 14 %, respectively.
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Description of Novosphingobium taihuense sp. nov.
Novosphingobium taihuense (tai.hu.en'se. N.L. neut. adj. taihuense pertaining to Taihu Lake, the source of the sediment from which the type strain was isolated).
Cells are Gram-negative, aerobic, non-spore-forming, non-motile rods. Produces small (about 2 mm), yellow colonies on 10-fold dilution of LB agar, LB agar or R2A agar, after incubation at 30 °C for 3 days. The colonies are smooth, shiny, flat, non-transparent, elevated and rubbery. Growth occurs at 10–37 °C, pH 4·5–8·5 and 0–2·0 % NaCl; optimal growth temperature, pH and salinity are 25 °C, pH 6·0 and 0·5 % NaCl. Positive for catalase activity and nitrate reduction. Activity of oxidase, urease and arginine dihydrolase, production of indole, growth on citrate, hydrolysis of starch and gelatin and glucose fermentation are all negative. Other characteristics are listed in Table 1
. The dominant fatty acid is 18 : 1 (36·5 %) and the major hydroxylated fatty acid is 2-OH 14 : 0 (12·8 %). The fatty acid profile also contains 15 : 0 (2·4 %), 16 : 0 (9·9 %), 16 : 1 (11·9 %), 17 : 0 (1·9 %), 17 : 1 (17·3 %), 18 : 0 (0·7 %), 13 : 0 2-OH (0·9 %), 2-OH 15 : 0 (4·6 %) and 2-OH 16 : 0 (0·6 %). The predominant isoprenoid quinone is Q-10. Cells contain phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, sphingoglycolipid and an unknown polar lipid as major polar lipids; it also contains phosphatidylcholine, unknown phosphoglycolipids and other unknown polar lipids or phospholipids. DNA G+C content is 63·3 mol% (Tm).
The type strain is T3-B9T (=AS 1.3432T=JCM 12465T), isolated from sediment of Taihu Lake, Jiangsu Province, China.
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ACKNOWLEDGEMENTS |
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-alanine, citrate, D-sorbitol, L-phenylalanine, phenylacetate and D-ribose.