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1 Division of Applied Life Science, EB-NCRC, PMBBRC, Gyeongsang National University, Jinju 660-701, Republic of Korea
2 Jeju Hi-Tech industry Development Institute, 4-8 Ara-1 dong, Jeju 690-121, Republic of Korea
Correspondence
Che Ok Jeon
cojeon{at}gnu.ac.kr
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ABSTRACT |
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7c, C16 : 0 and C15 : 0. The predominant polar lipid was phosphatidylglycerol. The G+C content of the genomic DNA was 64.1 mol% and the major quinone was Q-10. Comparative 16S rRNA gene sequence analyses showed that strain EMB102T formed a distinct phyletic lineage within the genus Brevundimonas. The levels of 16S rRNA gene sequence similarity between the type strains of Brevundimonas species ranged from 95.8 to 97.5 %. DNA–DNA relatedness levels between the EMB102T and closely related Brevundimonas species were below 15.0 %. On the basis of chemotaxonomic data and molecular properties, strain EMB102T represents a novel species within the genus Brevundimonas, for which the name Brevundimonas aveniformis sp. nov. is proposed. The type strain is EMB102T (=KCTC 12609T=DSM 17977T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain EMB102T is DQ372984.
The cellular fatty acid compositions and substrate-assimilation data for strain EMB102T and some related Brevundimonas species are available in supplementary tables available with the online version of this paper.
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MAIN TEXT |
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; Jeon & Park, 2000). Insight into the bacterial community responsible for phosphorus removal is a prerequisite for an understanding of the enhanced biological phosphorus removal (EBPR) mechanism and for controlling the EBPR processes. Pseudomonas diminuta and Pseudomonas vesicularis, first described by Leifson & Hugh (1954), were reclassified into a novel genus, Brevundimonas (in the Alphaproteobacteria) with Brevundimonas diminuta by Segers et al. (1994). Later, several Caulobacter species and subspecies were transferred to the genus Brevundimonas, and novel Brevundimonas species isolated from diverse habitats, especially marine habitats, were described (Abraham et al., 1999; Fritz et al., 2005; Li et al., 2004). In this study, we describe the taxonomic characterization of a novel species, isolated from activated sludge that was able to perform EBPR, and we propose a novel species of the genus Brevundimonas.
Strain EMB102T was isolated from activated sludge performing EBPR in a laboratory-scale sequencing batch reactor. Sodium acetate was supplied as a sole carbon source and the operation of the sequencing batch reactor was as described elsewhere (Jeon et al., 2003). The sludge sample was serially diluted with 1 % (w/v) saline solution and spread on R2A agar (Difco) and incubated at 20 °C for 7 days. Subcultivation was conducted on R2A agar at 30 °C for 3 days.
Physiological characteristics of strain EMB102T and type strains of members of the genus Brevundimonas were examined; the isolate was grown on R2A medium at different temperatures and pH values. R2A media with different pHs were prepared as described previously (Gomori, 1955). Gram staining was performed using a bioMérieux Gram stain kit according to the instructions of the manufacturer. Oxidase activity was tested by assessing the oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck), and catalase activity was evaluated by observing the production of oxygen bubbles in a 3 % (v/v) aqueous hydrogen peroxide solution. Cell morphology and motility were studied using phase-contrast microscopy and transmission electron microscopy (JEM-1010; JEOL) at different growth stages, as described by Jeon et al. (2005). Hydrolysis of casein, gelatin, Tweens 80 and 20, aesculin, urea, tyrosine and starch was investigated on R2A agar after 7 days incubation, as described previously (Lanyi, 1987; Gerhardt et al., 1994). Growth at various NaCl concentrations was investigated in 10-fold-diluted trypticase soy broth (Difco). Nitrate reduction was determined according to the method of Lanyi (1987), and acid production from carbohydrates was tested as described by Leifson (1963). Oxidation of various substrates was determined by using the Biolog GN2 MicroPlate assay as recommended by the manufacturer, and additional enzyme activities and biochemical features were determined by using API kits (API ZYM and API 20E) at 30 °C as recommended by the manufacturer (bioMérieux). Growth of strain EMB102T was obtained at temperatures between 15 and 35 °C, the optimum growth temperature being 30 °C. The strain grew at pH 6.0–9.0, the optimum being at pH 7.5–8.5. Cells of strain EMB102T were found to be Gram-negative and oxidase- and catalase-positive. During the early growth stage, the cells were motile by means of single polar flagella, but they gradually lost their flagella and each produced a stalk at the flagellated pole: this is the typical lifestyle of some species of the genera Caulobacter and Brevundimonas (Fig. 1). The number of stalked cells lacking motility increased with increasing cultivation time. The cylindrical body of the cell was approximately 0.3–0.4 µm in diameter and 1.0–2.0 µm in length. Anaerobic growth was not observed at 7 days cultivation on R2A agar at 30 °C, but weak growth was observed after 16 days. The strain reduced nitrate to nitrite and produced nitrogen gas. Other phenotypic features of strain EMB102T and type strains of Brevundimonas species are presented in Table 1 and in the description of the novel species. The oxidation results for various substrates tested with the Biolog GN2 MicroPlate system are shown in Supplementary Table S1 ( available with the online version of this paper). Some of these results are in accordance with the characteristics of members of the genus Brevundimonas, whereas some others allow the differentiation of strain EMB102T from closely related species of this genus (Table 1).
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and Batrakov et al. (1997), respectively. The DNA G+C content of strain EMB102T was determined using HPLC apparatus fitted with a reversed-phase column (GROM-SIL 100 ODS-2FE, GROM) according to the method of Tamaoka & Komagata (1984). The major cellular fatty acids present were C18 : 17c, C16 : 0 and C15 : 0. The main hydroxy fatty acid was iso-C17 : 0 3-OH on LMG medium no. 221, and the fatty acid profile was generally similar to those of other Brevundimonas species; however, the proportions of some fatty acids, such as C18 : 1 11-methyl 7c, C17 : 17c and C19 : 0 cyclo 8c were different (see Supplementary Table S2 available with the online version of this paper). The predominant polar lipid of strain EMB102T was phosphatidylglycerol, the major respiratory lipoquinone was Q-10 and the DNA G+C content was 64.1 mol%. The major fatty acids, the major lipoquinone, the polar lipid profile and the G+C content of strain EMB102T are in accordance with those of members of the genus Brevundimonas (Abraham et al., 1999; Li et al., 2004; Fritz et al., 2005; Yoon et al., 2006).
The sequencing and assembly of the 16S rRNA gene were carried out as described by Lane (1991). The resultant 16S rRNA gene sequence (1403 nt) of strain EMB102T was compared with those available from GenBank by using the BLAST program (www.ncbi.nlm.nih.gov/blast/) to determine an approximate phylogenetic affiliation; the gene sequences were aligned with those of closely related species by using the CLUSTAL W software program (Thompson et al., 1994). Phylogenetic trees were constructed using three different methods, i.e. the neighbour-joining, maximum-likelihood and maximum-parsimony algorithms; these methods are available in PHYLIP software, version 3.6 (Felsenstein, 2002). Values for sequence similarity between the isolate and related members of the genus Brevundimonas were computed using SIMILARITY MATRIX, version 1.1 (Ribosomal Database Project II; http://35.8.164.52/html/; Cole et al., 2003). A bootstrap analysis was performed according to the algorithm of the Kimura two-parameter model (Kimura, 1980) of the neighbour-joining method in the PHYLIP package. DNA–DNA hybridization experiments were carried out to evaluate the genomic DNA relatedness between strain EMB102T and Brevundimonas nasdae W1-2BT, Brevundimonas vesicularis LMG 2350T, Brevundimonas intermedia ATCC 15262T, Brevundimonas kwangchunensis KSL-102T and Brevundimonas aurantiaca DSM 4731T, which exhibited 16S rRNA gene sequence similarities above 97 %. The extracted genomic DNAs were fragmented with HaeIII for slot hybridization. The digested DNAs were serially diluted and loaded into slots (three replications for each sample) and their DNAs used individually as labelled DNA probes for cross-hybridization. Random-primed DNA labelling with digoxigenin-dUTP and hybridization (hybridization at 51.5 °C; washing at 68 °C) were performed using the DIG High Prime DNA labelling kit (Roche Applied Science) according to the instructions of the manufacturer and using standard procedures (Sambrook & Russell, 2001; Lim et al., 2005). The signals of the series of dilutions were quantified using Bio-Rad GelDoc scanning software. The signals produced by self-hybridization were inferred as 100 % and the homology percentages were calculated from samples analysed in triplicate.
The neighbour-joining phylogenetic tree constructed on the basis of 16S rRNA gene sequences indicated that strain EMB102T formed a distinct phyletic lineage within the genus Brevundimonas (Fig. 2). The topologies of the phylogenetic trees constructed using the maximum-likelihood and maximum-parsimony algorithms also supported the notion that the isolate belongs to the genus Brevundimonas and can be differentiated from the other species of the genus (data not shown). Comparative 16S rRNA gene sequence analyses showed that the isolate was most closely related to B. nasdae W1-2BT, B. vesicularis LMG 2350T, B. intermedia ATCC 15262T, B. kwangchunensis KSL-102T and B. aurantiaca DSM 4731T, with similarities of 97.5, 97.5, 97.3, 97.2 and 97.2 %, respectively. However, the values for DNA–DNA relatedness between strain EMB102T and the related Brevundimonas species were below 15.0 %. Therefore, the physiological, biochemical and phylogenetic properties of strain EMB102T support its description as a novel species within the genus Brevundimonas, for which the name Brevundimonas aveniformis sp. nov. is proposed.
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Cells are Gram-negative, rod-like bacteria that can produce stalks; the size of the main cell body is 0.3–0.4x1.0–2.0 µm. Colonies on R2A agar are white, slightly raised and circular with entire margins. Growth occurs optimally at 30 °C and pH 7.5–8.5. The optimum NaCl concentration is 0–1 % (w/v); no growth occurs at 
3 %. Catalase- and oxidase-positive. Nitrate is reduced to nitrite and nitrogen gas is produced. Hydrolyses casein, urea, tyrosine and gelatin, but not Tween 80 and 20, aesculin or starch. Negative for indole, H2S and acetoin production and citrate utilization (API 20E). Produces acids from raffinose, myo-inositol, D-mannose and D-mannitol, but not from D-glucose, D-fructose, D-galactose, L-arabinose, melibiose, lactose, arbutin or salicin. Produces leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase and urease, but not esterase (C4), lipase (C14), arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, cystine arylamidase, 
-galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase or -fucosidase. Weak enzymic activities are observed for esterase lipase (C8), alkaline phosphatase, trypsin, tryptophan deaminase, -chymotrypsin and acid phosphatase. The predominant polar lipid is phosphatidylglycerol. Utilizes glycogen, pyruvic acid methyl ester, -hydroxybutyric acid, L-alaninamide, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, L-proline, L-serine, L-threonine and urocanic acid (Biolog GN2). Other organic substrates included in Biolog GN2 MicroPlate system are not utilized. The major isoprenoid quinone is Q-10. The major cellular fatty acids are C18 : 17c, C16 : 0 and C15 : 0, and the main hydroxy fatty acid is iso-C17 : 0 3-OH. The DNA G+C content is 64.1 mol% (HPLC).
The type strain, EMB102T (=KCTC 12609T=DSM 17977T), was isolated from activated sludge that performed EBPR in a laboratory-scale sequencing batch reactor.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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