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Asticcacaulis taihuensis sp. nov., a novel stalked bacterium isolated from Taihu Lake, China

State Key Laboratory of Microbial Resource at the Institute of Microbiology, Chinese Academy of Sciences, ZhongGuanCun, Haidian, Beijing 100080, People's Republic of China

Correspondence
Shuang-Jiang Liu
shuangjiang{at}hotmail.com

	ABSTRACT

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A novel stalked bacterium, designated strain T3-B7^T, was isolated from sediment of Taihu Lake, Jiangsu Province, China, and its taxonomy was studied by using a polyphasic approach. Cell morphology, physiological and biochemical properties, and polar lipids indicated that strain T3-B7^T represented a member of the genus Asticcacaulis. Based on 16S rRNA gene sequence similarity analysis, strain T3-B7^T was found to be phylogenetically related to Asticcacaulis biprosthecium DSM 4723^T (98·5 %) and Asticcacaulis excentricus DSM 4724^T (95·0 %), but could be differentiated from these two species on the basis of the number and position of prosthecae, assimilation of sugars, nitrate reduction and tolerance to NaCl. Levels of DNA–DNA relatedness of strain T3-B7^T to A. biprosthecium DSM 4723^T and A. excentricus DSM 4724^T were 37·1 and 18·0 %, respectively. The G+C content of strain T3-B7^T was 59 mol% (T_m). It is concluded that strain T3-B7^T represents a novel species of the genus Asticcacaulis, for which the name of Asticcacaulis taihuensis sp. nov. is proposed. The type strain is T3-B7^T (=AS 1.3431^T=JCM 12463^T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain T3-B7^T is AY500141.

Polar lipid profiles of A. taihuensis T3-B7^T and A. biprosthecium DSM 4723^T are shown in a supplementary figure available in IJSEM Online.

	MAIN TEXT

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The genus Asticcacaulis was established by Poindexter (1964) to accommodate isolates that were regarded as representing Caulobacter species but with prosthecae that were not adhesive. Subsequent studies on 16S rRNA gene sequence similarities and chemotaxonomic features indicated that members of the genus Asticcacaulis were distantly related to members of the genera Caulobacter and Brevundimonas (Abraham et al., 2001; Sly et al., 1999). Asticcacaulis comprises only two species (Poindexter, 1989), Asticcacaulis excentricus Poindexter 1964 and Asticcacaulis biprosthecium Pate et al. 1973. All strains representative of this genus have been isolated from freshwater environments.

During ecological surveys on the freshwater Taihu Lake (31° 27' 10·7'' N 120° 02' 16·8'' E), which is located in Jiangsu Province, China, strain T3-B7^T was isolated from lake sediment by plating 10-fold dilutions of samples on diluted LB agar (0·5 g yeast extract, 1 g peptone, 1 g NaCl, 15 g Difco agar, 1 litre distilled water). Sediment samples were obtained from 10 cm beneath the sediment surface. Routine cultivation was conducted with LB agar or LB broth at 30 °C.

Gram reactions were determined by staining cells grown on LB agar at 30 °C for 24 h according to the method described by Gerhardt et al. (1994). Endospore formation was determined after malachite green staining of cells grown on LB agar. Flagellation and stalk generation were examined by transmission electron microscopy (H-600; Hitachi) at 100 kV after negative staining with 1 % (w/v) phosphotungstic acid. Denitrification capacity was determined by the method of Stanier et al. (1966). For assimilation of carbon sources, the standard mineral base of Stanier et al. (1966) was used. After the mineral base was autoclaved, each compound was added at a concentration of 0·2 % (w/v). Growth was examined after incubation at 30 °C for 1, 3, 7 and 14 days. Aerobic and anaerobic production of acids (OF reaction) from carbohydrates was determined in OF basal medium (Hugh & Leifson, 1953). Carbohydrate solution sterilized by filtration was added at a concentration of 1 % (w/v), and acid production was recorded after 7 and 14 days of incubation. Cellular fatty acids were extracted and analysed by using the Sherlock system (MIDI Inc.) following the recommendations of the manufacturer. Polar lipids were separated with one- and two-dimensional TLC and were characterized with spreading reagents specific for -glycols (periodate-Schiff), sugars (-naphthol-H₂SO₄, anisaldehyde-H₂SO₄), phosphate (Zindzadze), free amino groups (ninhydrin) and quaternary nitrogen compounds (Dragendorff) (Ventosa et al., 1993). The developed spots were identified by comparison with those of A. biprosthecium DSM 4723^T.

DNA base composition was determined by thermal denaturation (Marmur & Doty, 1962) using DNA from Escherichia coli DH-5 as a control. DNA–DNA hybridizations were carried out according to the method of De Ley et al. (1970). Renaturation rates and levels of relatedness were calculated as described by Jahnke (1992). 16S rRNA genes were amplified and sequenced as described by Zhang et al. (2003), and 16S rRNA gene sequence alignments were performed with the CLUSTAL X program (version 1.64b; Thompson et al., 1997). A phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, 1987) with Kimura's two-parameter calculation model in TREECON W version 1.3b.

Cells of strain T3-B7^T are Gram-negative, aerobic, non-spore-forming rods. They are motile with a single polar flagellum (Fig. 1a). A single stalk was located near the cell pole and the stalk possessed no adhesive material (Fig. 1b). 16S rRNA gene sequence analysis indicated that strain T3-B7^T was phylogenetically related to members of the genus Asticcacaulis, with similarities of 95·0 % to A. excentricus DSM 4724^T and 98·5 % to A. biprosthecium DSM 4723^T. Based on these 16S rRNA gene sequence analyses, a neighbour-joining tree was constructed (Fig. 2); the results showed that strain T3-B7^T and the two recognized species of the genus Asticcacaulis were grouped together to form a cluster with 100 % bootstrap support. Investigation of polar lipids revealed that strain T3-B7^T contained phosphatidylglycerol, 1,2-diacyl-3-O--D-glucopyranosyl glycerol, 1,2-diacyl-3-O--D-glucuronopyranosyl glycerol and three unidentified phospholipids (see Supplementary Fig. A in IJSEM Online). Comparison with A. biprosthecium DSM 4723^T reported by Abraham et al. (2001) showed that both strains contained phosphatidylglycerol, 1,2-diacyl-3-O--D-glucopyranosyl glycerol and 1,2-diacyl-3-O--D-glucuronopyranosyl glycerol, but that the two strains differed in their unidentified polar lipids (compare Supplementary Figs A and B). Strain T3-B7^T also differed from A. excentricus and A. biprosthecium with regard to the number and position of its stalks, assimilation of sugars (L-arabinose, D-mannose, D-ribose and sucrose), tolerance to NaCl and nitrite reduction (Table 1). Cellular fatty acid profiles of strains T3-B7^T, A. biprosthecium DSM 4723^T and A. excentricus DSM 4724^T are given in Table 2. Levels of DNA–DNA relatedness of strain T3-B7^T to A. excentricus DSM 4724^T and A. biprosthecium DSM 4723^T were 18·0 and 37·1 %, respectively. Based on the above results we conclude that strain T3-B7^T represents a novel species of the genus Asticcacaulis, for which the name Asticcacaulis taihuensis is proposed.

View larger version (66K): [in this window] [in a new window]   Fig. 1. Transmission electron micrographs of cells of strain T3-B7T showing a single polar flagellum (a) and the typical stalk near the cell pole (b). Bars, 1 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 2. Phylogenetic tree constructed with the neighbour-joining method based on 16S rRNA gene sequence analysis of strain T3-B7T and related bacteria. Numbers at nodes indicate percentages of bootstrap support based on 1000 resampled datasets. Phenylobacterium immobile was used as the outgroup. Bar, evolutionary distance (Knuc) of 0·1.

Gram-negative, aerobic, non-spore-forming rods. Motile with a single polar flagellum. Forms a single typical stalk near its cell pole and the stalk possesses no adhesive material. Assimilates L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, D-mannose, D-maltose, D-melibiose, raffinose, L-rhamnose, lactose, sucrose, D-trehalose, D-xylose, melezitose, L-alanine and L-proline but not D-ribose, D-mannitol, D-sorbitol, -alanine, L-arginine, L-phenylalanine, L-lysine or citrate. Acids are produced oxidatively from L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose, D-maltose, L-rhamnose, lactose, D-xylose and melezitose but not from raffinose, D-ribose, sucrose, trehalose, D-mannitol or D-sorbitol. Cells grow well at NaCl concentrations below 20 g l^–1. Starch and aesculin are hydrolysed, but gelatin is not. Nitrate is reduced to nitrite. Does not denitrify. Arginine dihydrolase and urease are negative. Major cellular fatty acids are octadecanoic acid (18 : 1), hexadecanoic acid (16 : 0), 2-hydroxy-hexadecanoic acid (16 : 0 2-OH) and hexadecanoic acid (16 : 1). Contains phosphatidylglycerol, 1,2-diacyl-3-O--D-glucopyranosyl glycerol, 1,2-diacyl-3-O--D-glucuronopyranosyl glycerol and three unidentified phospholipids. The G+C content of the DNA is 59 mol% (T_m).

The type strain, T3-B7^T (=AS 1.3431^T=JCM 12463^T), was isolated from sediment of Taihu Lake, Jiangsu Province, China.

	ACKNOWLEDGEMENTS

 
This work was supported by grants from the Chinese Academy of Sciences (KJCX1-SW-12-II-02-02) and from the Natural Science Foundation of China (20177034).

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This Article Abstract Full Text (PDF) Polar lipid profiles Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Liu, Z.-P. Articles by Liu, Y.-H. Search for Related Content PubMed PubMed Citation Articles by Liu, Z.-P. Articles by Liu, Y.-H. Agricola Articles by Liu, Z.-P. Articles by Liu, Y.-H.