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Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, Ghent University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
Correspondence
Kim Heylen
Kim.Heylen{at}UGent.be
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A figure showing the grouping of normalized digitized SDS-PAGE patterns for members of the genus Acidovorax is available as supplementary material with the online version of this paper.
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) and Acidovorax defluvii (Schulze et al., 1999), and the phytopathogenic species Acidovorax avenae and its three subspecies, Acidovorax konjaci (Willems et al., 1992), Acidovorax anthurii (Gardan et al., 2000) and Acidovorax valerianellae (Gardan et al., 2003). This separation based on occurrence and habitat is reflected in the 16S rRNA gene sequence phylogeny of these organisms, although separate phylogenetic clustering of the genus within the Comamonadaceae has been confirmed (Willems & Gillis, 2005).
A previous cultivation-dependent study on activated sludge from an aerobic–anaerobic wastewater treatment plant used different defined growth media for specific isolation of denitrifying bacteria (Heylen et al., 2006). Nineteen denitrifiers were assigned to the genus Acidovorax based on partial 16S rRNA gene sequence analysis. The role of members of the Comamonadaceae in the removal of nitrogen in wastewater treatment plants has been recognized and described (Etchebehere et al., 2001; Gumaelius et al., 2001; Mechichi et al., 2003; Hoshino et al., 2005). Four Acidovorax-like isolates, R-24607, R-24608T, R-24613 and R-24614, were retrieved from G1M1, a mineral medium containing 15 mM sodium succinate, 3 mM potassium nitrite and different vitamins. Based on partial 16S rRNA gene sequence similarity data, these new isolates could possibly represent a novel species within the environmental cluster of Acidovorax species, and were analysed further in a polyphasic study. The type strain and a second representative strain of each recognized environmental species of the genus Acidovorax, i.e. A. facilis, A. delafieldii, A. defluvii and A. temperans, were re-examined for phenotyping, chemotaxonomy and biochemical analysis to guarantee comparable results.
To avoid studying duplicate isolates of the same strain, genotyping by random amplified polymorphism DNA PCR analysis (Coenye et al., 2002) and repetitive sequence-based PCR analysis with REP and BOX primers (Heyrman et al., 2005) were carried out. The three fingerprint methods generated different patterns for isolates R-24607, 24608T, R-24613 and R-24614, indicating genetic differences between all four isolates (data not shown). The average DNA G+C content of the four isolates, determined singly by HPLC (Mesbah et al., 1989), was 64.3±0.8 mol%. The nearly complete 16S rRNA gene sequences of R-24607, R-24608T, R-24613 and R-24614 were determined as described by Vanparys et al. (2005). Phylogenetic analysis was performed by using TREECON (Van de Peer & De Wachter, 1994) and BioNumerics software version 4.6 after multiple alignment with CLUSTAL_X (Thompson et al., 1997). Cluster analysis according to the neighbour-joining algorithm, with or without corrections for evolutionary distances as described by Jukes & Cantor (1969) and Kimura (1980), was in agreement with those based on the maximum-parsimony and maximum-likelihood methods. Strains R-24607, R-24608T, R-24613 and R-24614 clustered together with A. temperans LMG 7169T, A. delafieldii LMG 5943T, A. defluvii DSM 12644T and A. facilis LMG 2193T, but clearly formed a separate group, supported by high bootstrap values (Fig. 1). Therefore, DNA–DNA hybridization experiments were performed within this cluster, by using a modification of the microplate method of Ezaki et al. (1989) as described by Willems et al. (2001). A hybridization temperature of 45 °C (calculated with correction for 50 % formamide) was used. First, strains R-24607, R-24608T, R-24613 and R-24614 were hybridized among themselves to substantiate the hypothesis of their relatedness at the species level. Levels of DNA–DNA relatedness among the four new isolates ranged between 78.5 and 88.5 % (±1.3–10.9 %), suggesting that they represented a single species, but also confirming the genetic diversity within the strains. Strain R-24608T was further hybridized with A. temperans LMG 7169T (DNA–DNA relatedness of 26.7±5.6 %), A. delafieldii LMG 5943T (23.9±5.9 %), A. defluvii DSM 12644T (26.0±0.6 %) and A. facilis LMG 2193T (18.2±4.8 %). These results confirmed that R-24607, R-24608T, R-24613 and R-24614 belong to a novel genospecies.
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) and phase-contrast microscopy (at a magnification of x1000), respectively, for cells grown on tryptone soy agar (TSA; Oxoid) for 48 h at 28 °C. Cells were Gram stained and examined via light microscopy, and catalase and oxidase activity was determined. Utilization of carbon sources and enzyme production were tested with the API 20NE (48 h, 28 °C), API ZYM (4 h, 28 °C) (bioMérieux) and Biolog (24 h, 28 °C) systems according to the manufacturers' recommendations. The temperature range (at 4, 15, 28, 37, 45 and 52 °C), pH range (pH 4.5–10.5 at 28 °C) and salinity range (0.5–5 % NaCl, w/v, at 28 °C) for growth were recorded after incubation for 48 h in tryptone soy broth (TSB; Oxoid). The ability to denitrify was tested, as described by Smibert & Krieg (1994), after growth for 1 week in TSB supplemented with 10 mM potassium nitrate at 37 °C and in liquid isolation medium G1M1 at 37 °C, and was confirmed with N2O measurements, as described by Heylen et al. (2006). Lipolytic activity was determined after 72 h based on hydrolysis of Tween 80, as described by Sierra (1957). Differential biochemical characteristics among the test strains are given in Table 1.
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). The dominant fatty acids for strains R-24607, R-24608T, R-24613 and R-24614 were summed feature 3 (38–41 %), C18 : 1
7c (22–32 %) and C16 : 0 (25–26.5 %). Unfortunately, the Sherlock MIS software could not clearly resolve summed feature 3, referring to the peaks of C16 : 17c and/or iso-C15 : 0 2-OH. However, Sherlock lists the closest to the observed ECL first, which was C16 : 17c. In addition, comparison of the fatty acid data in the literature for the type strains of recognized species of the genus Acidovorax (Willems et al., 1990; Schulze et al., 1999) and our data on the same strains suggests C16 : 17c as the major fatty acid for this peak. No characteristic fatty acids for the novel genospecies were detected (Table 2).
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). Pearson's correlation similarity coefficients were clustered with UPGMA and analysed with the co-phenetic correlation method in BioNumerics version 4.6 (see Supplementary Fig. S1 in IJSEM Online). The different strains of each species grouped together, supported with high co-phenetic correlation values. Although strains R-24607, R-24608T, R-24613 and R-24614 demonstrated significant variation in whole-cell protein profiles, they formed a distinct group, separate from recognized species of the genus Acidovorax. Based on the polyphasic data presented here, strains R-24607, R-24608T, R-24613 and R-24614 are considered to represent a novel species of the genus Acidovorax, for which the name Acidovorax caeni sp. nov. is proposed.
Description of Acidovorax caeni sp. nov.
Acidovorax caeni (cae'ni. L. gen. neut. n. caeni of sludge).
After 48 h, colonies are round, smooth and yellow–brown. Cells are motile, non-spore-forming rods (0.9x1.8 µm). Gram-negative and catalase- and oxidase-positive. Growth is observed at 15–37 °C but not at 4 or 45–52 °C, at pH 5.5–10.5, but not at pH 4.5–5, and at salt concentrations of 0.5–2 % NaCl (w/v), but not at 3–5 % NaCl. Anaerobic respiration and growth are possible through denitrification. Positive for the following enzyme activities: hydrolysis of Tweens 40 and 80, esterase lipase, trypsin, naphthol-AS-BI-phosphohydrolase and production of indole. Malate, glycogen, formic acid, 
-hydroxybutyric acid, β-hydroxybutyric acid, -ketobutyric acid, -ketoglutaric acid, propionic acid, succinic acid, bromosuccinic acid, D-alanine, L-alanine, L-alanyl glycine, L-aspartic acid, L-glutamic acid, L-leucine, L-proline, L-pyroglutamic acid, L-serine, L-threonine, methyl pyruvate, monomethyl succinate, β-hydroxybutyric acid, -ketovaleric acid, DL-lactic acid and glycerol can be used as carbon sources. Can be differentiated from the type strains of its closest phylogenetic neighbours, A. defluvii, A. delafieldii, A. facilis and A. temperans, through SDS-PAGE analysis of whole-cell proteins, by the ability to produce indole and by the presence of trypsin and naphthol-AS-BI-phosphohydrolase.
The type strain, R-24608T (=LMG 24103T =DSM 19327T), has a DNA G+C content of 65.7 mol% and was isolated from activated sludge from an aerobic–anaerobic wastewater treatment plant (Bourgoyen-Ossemeersen) in Gent, Belgium. Due to the genetic variation within this species, R-24607, R-24613 and R-24614 were also deposited in the BCCM/LMG collection with strain numbers LMG 24104, LMG 24105 and LMG 24106, respectively.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Etchebehere, C., Errazquin, I., Barrandeguy, E., Dabert, P., Moletta, R. & Muxi, L. (2001). Evaluation of the denitrifying microbiota of anoxic reactors. FEMS Microbiol Ecol 35, 259–265.[CrossRef][Medline]
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Gardan, L., Dauga, C., Prior, P., Gillis, M. & Saddler, G. S. (2000). Acidovorax anthurii sp. nov., a new phytopathogenic bacterium which causes bacterial leaf-spot of anthurium. Int J Syst Evol Microbiol 50, 235–246.[Abstract]
Gardan, L., Stead, D. E., Dauga, C. & Gillis, M. (2003). Acidovorax valerianellae sp. nov., a novel pathogen of lamb's lettuce [Valerianella locusta (L.) Laterr.]. Int J Syst Evol Microbiol 53, 795–800.
Gumaelius, L., Magnusson, G., Pettersson, B. & Dalhammar, G. (2001). Comamonas denitrificans sp. nov., an efficient denitrifying bacterium from activated sludge. Int J Syst Evol Microbiol 51, 999–1006.[Abstract]
Heylen, K., Vanparys, B., Wittebolle, L., Verstraete, W., Boon, N. & De Vos, P. (2006). Cultivation of denitrifying bacteria: optimization of isolation conditions and diversity study. Appl Environ Microbiol 72, 2637–2643.
Heyrman, J., Verbeeren, J., Schumann, P., Swings, J. & De Vos, P. (2005). Six novel Arthrobacter species isolated from deteriorated mural paintings. Int J Syst Evol Microbiol 55, 1457–1464.
Hoshino, T., Terahara, T., Tsuneda, S., Hirata, A. & Inamori, Y. (2005). Molecular analysis of microbial population transition associated with the start of denitrification in a wastewater treatment process. J Appl Microbiol 99, 1165–1175.[CrossRef][Medline]
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Mechichi, T., Stackebrandt, E. & Fuchs, G. (2003). Alicycliphilus denitrificans gen. nov., sp. nov., a cyclohexanol-degrading, nitrate-reducing β-proteobacterium. Int J Syst Evol Microbiol 53, 147–152.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electrophoretic whole organism protein fingerprints. In Chemical Methods in Prokaryotic Systematics, pp. 493–521. Edited by M. Goodfellow & A. G. O'Donnell. Chichester: Wiley.
Schulze, R., Spring, S., Amann, R., Huber, I., Ludwig, W., Schleifer, K.-H. & Kämpfer, P. (1999). Genotypic diversity of Acidovorax strains isolated from activated sludge and description of Acidovorax defluvii sp. nov. Syst Appl Microbiol 22, 205–214.[Medline]
Sierra, G. (1957). A simple method for the detection of lipolytic activity of micro-organisms and some observations on the influence of the contact between cells and fatty substrates. Antonie van Leeuwenhoek 23, 15–22.[Medline]
Smibert, R. M. & Krieg, N. R. (1994). General characterization. In Manual of Methods for General Microbiology, pp. 409–443. Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American Society for Microbiology.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
Vanparys, B., Heylen, K., Lebbe, L. & De Vos, P. (2005). Pedobacter caeni sp. nov., a novel species isolated from a nitrifying inoculum. Int J Syst Evol Microbiol 55, 1315–1318.
Willems, A. & Gillis, M. (2005). Genus II. Acidovorax Willems, Falsen, Pot, Jantzen, Hoste, Vandamme, Gillis, Kersters and De Ley 1990, 394VP. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, part C, pp. 696–703. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer.
Willems, A., Falsen, E., Pot, B., Jantzen, E., Hoste, B., Vandamme, P., Gillis, M., Kersters, K. & De Ley, J. (1990). Acidovorax, a new genus for Pseudomonas facilis, Pseudomonas delafieldii, E. Falsen (EF) group 13, EF group 16, and several clinical isolates, with the species Acidovorax facilis comb. nov., Acidovorax delafieldii comb. nov., and Acidovorax temperans sp. nov. Int J Syst Bacteriol 40, 384–398.
Willems, A., Goor, M., Thielemans, S., Gillis, M., Kersters, K. & De Ley, J. (1992). Transfer of several phytopathogenic Pseudomonas species to Acidovorax avenae subsp. avenae subsp. nov., comb. nov., Acidovorax avenae subsp. citrulli, Acidovorax avenae subsp. cattleyae, and Acidovorax konjaci. Int J Syst Bacteriol 42, 107–119.
Willems, A., Doignon-Bourcier, F., Goris, J., Coopman, R., de Lajudie, P., De Vos, P. & Gillis, M. (2001). DNA–DNA hybridization study of Bradyrhizobium strains. Int J Syst Evol Microbiol 51, 1315–1322.[Abstract]
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