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1 Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China
2 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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7c, C16 : 0, C18 : 1 isomers and C10 : 0 3-OH. The G+C content of the genomic DNA was 65.6 mol%. The results of DNA–DNA hybridization (15.6 %) together with phenotypic determination showed that strain Slu-5T could be distinguished from A. fungiphilus. Moreover, some phenotypic properties concerning enzyme activity, the substrates utilized as carbon sources and growth conditions distinguish strain Slu-5T from A. fungiphilus. On the basis of the results obtained in this study, Slu-05T (=DSM 17719T=KCTC 12530T=CCBAU 10199T) is the type strain of a novel species of Azonexus, for which the name Azonexus caeni sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of strain Slu-05T and the partial nifH gene sequences of strain Slu-05T and Azonexus fungiphilus LMG 19178T are AB166882, DQ029203 and DQ029204, respectively.
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. The only species in the genus is Azonexus fungiphilus, formerly Azoarcus sp. group E. This species was isolated from the resting stages of a plant-associated basidiomycete in rhizosphere soil of rice growing in Pakistan (Hurek et al., 1997). The genus Azonexus accommodates Gram-negative, non-spore-forming, highly motile, slightly curved rods. In the course of screening micro-organisms from sludge of the main aerobic treatment tanks of municipal sewage treatment plants located in Daejeon, South Korea, a Gram-negative and denitrifying bacterial strain (Slu-05T) was isolated. On the basis of 16S rRNA gene sequence analysis, strain Slu-05T was considered to be an Azonexus-like strain. To determine its exact taxonomic position, strain Slu-05T was subjected to a detailed polyphasic taxonomic investigation, including genotypic, chemotaxonomic and classical phenotypic analyses. These results indicate that strain Slu-05T should be placed in a novel species in the genus Azonexus.
Strain Slu-05T was isolated from sludge from the main aerobic treatment tanks of municipal sewage treatment plants located in Daejeon, South Korea. The suspension was spread on R2A agar plates (Difco) after being serially diluted with 50 mM phosphate buffer (pH 7.0). The plates were incubated at 30 °C for 2 weeks. Single colonies on the plates were purified by transferring them onto new plates and incubating them once again under the same conditions. The isolate was routinely cultured on R2A agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C. Azonexus fungiphilus LMG 19178T, obtained from BCCM/LMG (the Belgian Co-ordinated Collections of Microorganisms/Laboratorium voor Microbiologie, Ghent University, Ghent, Belgium), was used as a reference strain and its phenotypic properties were determined along with those of strain Slu-05T.
Extraction of the genomic DNA was done using a commercial genomic DNA-extraction kit (Core Biosystem). PCR-mediated amplification of the 16S rRNA gene and sequencing of purified PCR products were carried out according to Kim et al. (2005). The full 16S rRNA gene sequence was compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database. Multiple alignments were performed by the CLUSTAL_X program (Thompson et al., 1997). Gaps were edited using the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). Phylogenetic trees were constructed by using the neighbour-joining method (Saitou & Nei, 1987) and the maximum-parsimony method (Fitch, 1971) using the MEGA 3 program (Kumar et al., 2004) with bootstrap values based on 1000 replications (Felsenstein, 1985).
A nearly complete 16S rRNA gene sequence of strain Slu-05T (1449 bp) was obtained. Preliminary sequence comparison against 16S rRNA gene sequences deposited in the GenBank database indicated that strain Slu-05T belonged to the family Rhodocyclaceae of the Betaproteobacteria. On the basis of 16S rRNA gene sequence similarity, the closest cultured relative was A. fungiphilus LMG 19178T (96.4 %), and the phylogenetic distances from other species of the Rhodocyclaceae with validly published names were greater than 5 % (i.e. less than 95 % similarity). This relationship between strain Slu-05T and other members of the family Rhodocyclaceae was also evident in the phylogenetic tree (Fig. 1). Strain Slu-05T and A. fungiphilus LMG 19178T formed a monophyletic clade with a high bootstrap value (97 %) supported by the two separate tree-making methods employed in this study.
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. Cell morphology was examined by using light microscopy (Nikon) and transmission electron microscopy (EM912
; Leo Zeiss) after negative staining with 2 % (w/v) uranyl acetate. Catalase activity was determined by bubble production in 3 % (v/v) H2O2; oxidase activity was determined using 1 % (w/v) tetramethyl p-phenylenediamine. The utilization of different substrates as sole carbon sources, along with the determination of some physiological characteristics, was assessed using API 32GN and API 20NE galleries according to the instructions of the manufacturer (bioMérieux). Tests for anaerobic growth were performed in a serum bottle containing R2A broth supplemented with thioglycolate (1 g l–1) under N2. Nitrate- and nitrite-reduction tests were performed in serum bottles containing R2A broth supplemented with KNO3 (10 mM) and NaNO2 (10 mM), respectively, and the reduction of nitrate and nitrite was monitored by an ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and an anion-exchange column (Metrosep Anion Supp 4; Metrohm). The degradation of DNA (using DNA agar from Difco, supplemented with 0.01 % toluidine blue from Merck), chitin, CM-cellulose, starch (Atlas, 1993), lipid (Kouker & Jaeger, 1987) and xylan (Ten et al., 2004) was also investigated; reactions were read after 5 days. Growth at different temperatures was assessed after 5 days incubation. Nitrogen-fixing ability was determined using growth in 50 ml nitrogen-free medium (DSMZ medium no. 3) and nitrogen-free SM medium (Reinhold et al., 1986), including a carbon source which can be used for growth, contained in a 500 ml Erlenmeyer flask. Primer system PolF–PolR (Poly et al., 2001) was used to amplify the nifH gene, as described by Im et al. (2004).
Cells of strain Slu-05T were Gram-negative, non-spore-forming, slightly curved, short rods that were highly motile by means of single polar flagella. The rods were 0.7–1.1 µm in diameter and 1.6–2.3 µm in length (Fig. 2). Strain Slu-05T grew well at 25–30 °C and also grew at 40 °C. Strain Slu-05T has oxidase, catalase, urease and arginine dihydrolase activities but a negative result was obtained for indole production and for glucosidase, protease and galactosidase activities. Strain Slu-05T assimilated malate, propionate, valerate, 3-hydroxybutyrate, L-proline, acetate, DL-lactate and malonate but not most other carbon sources (Table 1). Strain Slu-05T cannot acidify glucose and cannot assimilate most polymers, such as DNA, chitin, lipid, CM-cellulose and xylan. Strain Slu-05T reduced nitrate to N2 in R2A broth, unlike A. fungiphilus LMG 19178T. Strain Slu-05T did not grow on nitrogen-free medium (DSMZ medium no. 3 or nitrogen-free SM medium), but it has a nitrogenase gene, and the nifH gene sequence of strain Slu-05T (DQ029203) showed 94 % similarity to that of A. fungiphilus LMG 19178T (DQ029204).
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). Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), purified via TLC and subsequently analysed by HPLC, as described previously (Collins & Jones, 1981; Shin et al., 1996). For the determination of G+C content, DNA was degraded enzymically into nucleosides and analysed by reversed-phase HPLC as described by Mesbah et al. (1989). Escherichia coli DNA (Sigma) was used as the calibration reference. DNA–DNA hybridization was performed fluorometrically, according to the method developed by Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. Hybridization was conducted with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the DNA relatedness values quoted are the means of the remaining three values.
The cellular fatty acid profile of strain Slu-05T was characterized by the predominance of C16 : 17c, C16 : 0, C18 : 1 isomers and C10 : 0 3-OH, and was similar to that of A. fungiphilus LMG 19178T (Table 2). Both strain Slu-05T and A. fungiphilus LMG 19178T contained Q-8 as the major respiratory lipoquinone. The G+C content of the genomic DNA of strain Slu-05T was 65.6 mol%. Strain Slu-05T exhibited a relatively low level of DNA–DNA relatedness with respect to A. fungiphilus LMG 19178T (15.6 %).
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). In addition, the DNA–DNA relatedness value between strain Slu-05T and A. fungiphilus LMG 19178T (15.6 %) was far below the threshold value suggested for species delineation (Wayne et al., 1987). Therefore, on the basis of the phenotypic, phylogenetic and genomic evidence, strain Slu-05T should be placed in the genus Azonexus as a novel species, for which we propose the name Azonexus caeni sp. nov.
Description of Azonexus caeni sp. nov.
Azonexus caeni (ca.e'ni. L. gen. neut. n. caeni of sludge).
Aerobic, Gram-negative, non-spore-forming, slightly curved, short rods (0.7–1.1x1.6–2.3 µm), highly motile by means of single polar flagella. Can reduce nitrate to N2 in R2A broth. Cells grow at 20–40 °C, with an optimum at 25–30 °C. Positive for urease and arginine dihydrolase activities, but negative for indole production and for glucosidase, protease and galactosidase activities. Carbon sources used include malate, propionate, valerate, 3-hydroxybutyrate, L-proline, acetate, DL-lactate and malonate; most other carbon sources are not utilized. The predominant respiratory lipoquinone is Q-8. The major fatty acids are C16 : 17c, C16 : 0, C18 : 1 isomers and C10 : 0 3-OH. The G+C content of the genomic DNA is 65.6 mol%.
The type strain, Slu-05T (=DSM 17719T=KCTC 12530T=CCBAU 10199T), was isolated from sludge from a wastewater treatment plant in Daejeon, South Korea.
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ACKNOWLEDGEMENTS |
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