SGM Journals
Proteobacteria

Acidovorax soli sp. nov., isolated from landfill soil

Jung-Hye Choi, Min-Soo Kim, Seong Woon Roh, Jin-Woo Bae

  • Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University, Seoul 130-701, Republic of Korea
  • Correspondence
    Jin-Woo Bae
    baejw{at}khu.ac.kr

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2715–2718 · https://doi.org/10.1099/ijs.0.019661-0

    View at publisher PubMed

    Abstract

    A Gram-negative, aerobic, rod-shaped, non-motile strain, BL21T, was isolated from landfill soil in Pohang, Korea. Strain BL21T grew optimally at pH 7.0, 30 °C and 0 % NaCl (w/v). Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain BL21T belonged to the class Betaproteobacteria and was related to the genus Acidovorax. The 16S rRNA gene sequence of strain BL21T was less than 98.30 % similar to those of other species in the genus Acidovorax. DNA–DNA hybridization values with phylogenetically related species of the genus Acidovorax were only 11.7–28.4 %. The major fatty acid components included summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), C16 : 0, C18 : 1ω7c and C10 : 0 3-OH. The DNA G+C content was 60.9 mol%. For these reasons, strain BL21T (=KCTC 22399T =JCM 15909T) is proposed as a novel species in the genus Acidovorax, with the name Acidovorax soli sp. nov.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BL21T is FJ599672.

    • Transmission electron micrographs of cells of strain BL21T are available with the online version of this paper.

    The genus Acidovorax was proposed by reclassification of Pseudomonas species by Willems et al. (1990). At the time of writing, the genus Acidovorax comprises 12 recognized species (Gardan et al., 2000, 2003; Heylen et al., 2008; Schaad et al., 2008; Schulze et al., 1999; Willems et al., 1990, 1992). The species of the genus Acidovorax can be separated into two groups by occurrence and habitat. Acidovorax defluvii, A. facilis, A. delafieldii, A. temperans and A. caeni are in the group of environmental species which are found mainly in soil and water habitats. Acidovorax citrulli, A. cattleyae, A. avenae, A. oryzae, A. anthurii, A. valerianellae and A. konjaci are the phytopathogenic species which infect corn, oats, rice and many other plants. In this paper, we describe the morphological, biochemical and phylogenetic characteristics of a novel environmental species, Acidovorax-like strain BL21T, isolated from landfill soil.

    Strain BL21T was isolated from landfill soil in Pohang, Korea, by using the standard dilution plating method on Luria–Bertani (LB, BBL) agar at 30 °C for 3 days. The culture conditions of strain BL21T were determined by growth at a variety of temperatures, pH and NaCl concentrations for up to 5 days. Growth temperatures were monitored on LB agar at 4, 10, 15, 20, 25, 30, 37, 40, 42 and 45 °C. Optimum pH was measured in LB adjusted to pH 5.0–10.0 in increments of 0.5 units. NaCl tolerance was tested in LB containing NaCl at various concentrations (0, 1, 2, 3, 4, 5, 6, 8, 10, 12 and 14 %, w/v). Strain BL21T grew at temperatures in the range 10–42 °C, with 0–1 % NaCl and at pH 6.0–9.0. Optimum conditions for growth of strain BL21T were 30 °C, 0 % NaCl and pH 7.0. For the investigation of morphological and physiological characteristics, strain BL21T was routinely cultivated on LB agar under optimum culture conditions. Cell morphology of strain BL21T was observed by light microscopy (ECLIPSE 80i, Nikon) and transmission electron microscopy. Motility was determined using semisolid agar (Motility test medium, BBL) and observed by the spreading out of cells from the line of inoculation in the tube. Gram reaction was performed using the non-staining method described by Buck (1982). Catalase and oxidase activities of the strain were determined by bubble production in 3 % (v/v) hydrogen peroxide solution and by colour development in 1 % (w/v) p-tetramethyl phenylenediamine (bioMérieux), respectively. Enzyme activities and utilization of carbon sources were tested with commercial API 20NE and API ZYM kits (bioMérieux) and the Biolog GN2 MicroPlate assay, according to the manufacturers' protocols. Hydrolysis of DNA and casein were tested using DNase test agar (BBL) and skimmed milk (BBL), respectively, as described by Atlas (1993). Hydrolysis of cellulose was tested using the method of Gerhardt et al. (1994). The morphological, physiological and biochemical traits of strain BL21T and closely related species of the genus Acidovorax are listed in Table 1⇓ and the species description (see also Supplementary Fig. S1 available in IJSEM Online). Physiological and biochemical characteristics of BL21T and the related strains A. delafieldii DSM 64T, A. defluvii DSM 12644T, A. caeni DSM 19327T, A. temperans DSM 7270T and A. facilis DSM 649T were examined under the exact same experimental conditions.

    Table 1.

    Physiological and biochemical characteristics of strain BL21T and the type strains of phylogenetically closely related species

    Strains: 1, Acidovorax soli sp. nov. BL21T; 2, A. delafieldii DSM 64T; 3, A. temperans DSM 7270T; 4, A. defluvii DSM 12644T; 5, A. facilis DSM 649T; 6, A. caeni DSM 19327T. All strains were positive for reduction of nitrate to nitrite and activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase and naphthol-AS BI-phosphohydrolase. All strains were negative for glucose fermentation, utilization of capric acid and phenylacetic acid, and activities of oxidase, l-arginine hydrolase, valine arylamidase, cysteine arylamidase, trypsin, α-chymotrypsin, acid phospatase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. −, Negative; +, positive; w, weakly positive. Data were taken from this study.

    The 16S rRNA gene of strain BL21T was amplified by the colony PCR method with two universal primers as described by Baker et al. (2003). PCR products were purified using a PCR purification kit (LaboPass) according to the instructions of the manufacturer. Sequencing and phylogenetic analysis were performed as previously described (Choi et al., 2010). The 16S rRNA gene sequence of strain BL21T was compared with known 16S rRNA sequences in the GenBank database (NCBI) using blast. The sequence of strain BL21T was aligned with those of recognized species in the genus Acidovorax by the multiple sequence alignment program clustal x v.1.83 (Thompson et al., 1997). The phylogenetic relationships between strain BL21T and the representative species of the genus Acidovorax were defined by mega 4 (Tamura et al., 2007). A phylogenetic consensus tree was reconstructed using the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Felsenstein, 1981) and maximum-likelihood (Kluge & Farris, 1969) algorithms. The 16S rRNA gene sequence of strain BL21T shared less than 98.30 % similarity with those of other members of the genus Acidovorax and showed high similarities to those of the type strains A. delafieldii ATCC 17505T (98.26 %), A. temperans CCUG 11779T (98.19 %), A. avenae ATCC 19860T (97.98 %), A. citrulli ATCC 29625T (97.98 %), A. facilis CCUG 2113T (97.98 %), A. defluvii BSB411T (97.98 %), A. oryzae FC-143T (97.97 %), A. cattleyae NCPPB 961T (97.91 %), A. caeni R-24608T (97.70 %) and A. valerianellae CFBP 4730T (97.49 %). As a result of the phylogenetic analysis, strain BL21T was classified within the genus Acidovorax and, in particular, as highly related to environmental species (Fig. 1⇓). DNA–DNA relatedness studies were carried out between strain BL21T and phylogenetically closely related species. Genomic DNA was extracted according to Sambrook et al. (1989). DNA–DNA hybridization was performed by using a modification of the methods of Ezaki et al. (1989) and Hirayama et al. (1996), as previously described (Choi, et al., 2010). DNA–DNA hybridization values between strain BL21T and other Acidovorax strains were as follows: 28.4 % with A. delafieldii DSM 64T, 26.0 % with A. defluvii DSM 12644T, 20.7 % with A. caeni DSM 19327T, 17.7 % with A. temperans DSM 7270T and 11.7 % with A. facilis DSM 649T.

    Figure image not available in archive
    Fig. 1.

    Phylogenetic consensus tree based on 16S rRNA gene sequences showing the relationships between strain BL21T and type strains of species of the genus Acidovorax. The tree was reconstructed based on the neighbour-joining, maximum-parsimony and maximum-likelihood methods and numbers at nodes represent bootstrap values (based on 1000, 1000 and 300 resamplings, respectively). Only bootstrap values above 50 % are shown. Filled circles indicate generic branches that were present in phylogenetic trees generated by the neighbour-joining, maximum-parsimony and maximum-likelihood algorithms; open circles and diamonds indicate generic branches that were present in both neighbour-joining and maximum-parsimony, and both neighbour-joining and maximum-likelihood trees, respectively. The 16S rRNA gene sequence of Comamonas testosteroni ATCC 11996T was used as an outgroup. Bar, 0.005 substitutions per nucleotide position.

    The DNA G+C content was determined using the fluorimetric method proposed by Gonzalez & Saiz-Jimenez (2002) using SYBR Green and a real-time PCR thermocycler. For fatty-acid analysis, cell biomass of strain BL21T and related species was collected from LB agar plates after incubation for 2 days at 30 °C. Cellular fatty acids were extracted according to the protocol of Sasser (1990). Cellular fatty-acid compositions were determined by gas chromatography (Hewlett Packard 6890) and the Microbial Identification System. The G+C content of the genomic DNA of strain BL21T was 60.9 mol%. The fatty acids (>1.0 %) detected in strain BL21T were the saturated fatty acids C16 : 0 (32.1 %), C14 : 0 (3.7 %) and C12 : 0 (3.2 %), the 3-hydroxyoctanoic fatty acid C10 : 0 3-OH (3.3 %), the unsaturated fatty acid C18 : 1ω7c (11.7 %) and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c; 43.1 %). The fatty-acid profiles of strain BL21T and the 5 reference strains were very similar. The detailed fatty-acid compositions are shown in Table 2⇓.

    Table 2.

    Fatty acid composition of strain BL21T and the type strains of phylogenetically closely related species

    Strains: 1, Acidovorax soli sp. nov. BL21T; 2, A. delafieldii DSM 64T; 3, A. temperans DSM 7270T; 4, A. defluvii DSM 12644T; 5, A. facilis DSM 649T; 6, A. caeni DSM 19327T. All data were obtained in the current study. Values shown are percentages of total fatty acids. nd, Not detected; tr, trace component (less than 1 %).

    On the basis of the phenotypic, genetic and phylogenetic characteristics, strain BL21T is considered to represent a novel species of the genus Acidovorax, for which the name Acidovorax soli sp. nov. is proposed.

    Description of Acidovorax soli sp. nov.

    Acidovorax soli (so′li. L. gen. n. soli of soil, the isolation source of the type strain).

    Cells are Gram-negative, non-motile short rods (0.5×1.3 μm). Colonies are circular, convex, entire, bright yellow in colour and 2.0 mm in diameter after cultivation for 2 days on LB agar at 30 °C. Grows at 10–42 °C (optimum 30 °C), 0–1 % NaCl (optimum 0 %) and pH 6.0–9.0 (optimum pH 7.0) in LB medium. Catalase- and oxidase-negative. Nitrate is reduced to nitrite. Indole is not produced. Urea and DNA are hydrolysed, but cellulose, aesculin, gelatin and casein are not. Adipic acid is utilized as carbon source. Positive for activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase and naphthol-AS-BI-phosphohydrolase (API 20NE and ZYM). The following substrates can be utilized as sole carbon and energy sources: Tween 40, Tween 80, α-d-glucose, pyruvic acid methyl ester, succinic acid monomethyl ester, cis-aconitic acid, formic acid, β-hydroxybutyric acid, α-ketoglutaric acid, dl-lactic acid, succinamic acid, glucuronamide, l-alanine, l-asparagine, l-aspartic acid, l-glutamic acid, l-serine, l-threonine, γ-aminobutyric acid, dl-α-glycerol phosphate and α-d-glucose 1-phosphate (Biolog GN2). The following compounds are not used as carbon sources: α-cyclodextrin, dextrin, glycogen, N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, adonitol, l-arabinose, d-arabitol, cellobiose, i-erythritol, d-fructose, l-fucose, d-galactose, gentiobiose, myo-inositol, lactose, lactulose, maltose, d-mannitol, d-mannose, melibiose, methyl β-d-glucoside, d-psicose, raffinose, l-rhamnose, d-sorbitol, sucrose, trehalose, turanose, xylitol, acetic acid, citric acid, d-galactonic acid lactone, d-galacturonic acid, d-gluconic acid, d-glucosaminic acid, d-glucuronic acid, α-hydroxybutyric acid, γ-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, α-ketobutyric acid, α-ketovaleric acid, malonic acid, propionic acid, quinic acid, d-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, l-alaninamide, d-alanine, l-alanyl glycine, glycyl l-aspartic acid, glycyl l-glutamic acid, l-histidine, l-hydroxyproline, l-leucine, l-ornithine, l-phenylalanine, l-proline, l-pyroglutamic acid, d-serine, dl-carnitine, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol and d-glucose 6-phosphate. Major cellular fatty acids are the unsaturated fatty acid C18 : 1ω7c, the saturated fatty acid C16 : 0 and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c). The DNA G+C content of the type strain is 60.9 mol%.

    The type strain, BL21T (=KCTC 22399T =JCM 15909T), was isolated from a landfill site in Pohang, Republic of Korea.

    Acknowledgments

    We thank Dr J. P. Euzéby (École Nationale Vétérinaire, France) for etymological advice. This work was supported by the CAER (Center for Aquatic Ecosystem Restoration) of the Eco-STAR project, the 21C Frontier Microbial Genomics and Application Center Program, and the Eco-technopia 21 project.

    References

    1. Atlas, R. M. (1993). Handbook of Microbiological Media. Edited by Parks, L. C.. Boca Raton, FL. : CRC Press.
    2. Baker, G. C., Smith, J. J. & Cowan, D. A. (2003). Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55, 541–555.
    3. Buck, J. D. (1982). Nonstaining (KOH) method for determination of gram reactions of marine bacteria. Appl Environ Microbiol 44, 992–993.
    4. Choi, J.-H., Kim, M.-S., Roh, S. W. & Bae, J.-W. (2010). Brevundimonas basaltis sp. nov., isolated from black sand. Int J Syst Evol Microbiol 60, 1488-1492.
    5. 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.
    6. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.
    7. 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.
    8. 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.
    9. Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC. : American Society for Microbiology.
    10. Gonzalez, J. M. & Saiz-Jimenez, C. (2002). A fluorimetric method for the estimation of G+C mol% content in microorganisms by thermal denaturation temperature. Environ Microbiol 4, 770–773.
    11. Heylen, K., Lebbe, L. & De Vos, P. (2008). Acidovorax caeni sp. nov., a denitrifying species with genetically diverse isolates from activated sludge. Int J Syst Evol Microbiol 58, 73–77.
    12. Hirayama, H., Tamaoka, J. & Horikoshi, K. (1996). Improved immobilization of DNA to microwell plates for DNA-DNA hybridization. Nucleic Acids Res 24, 4098–4099.
    13. Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the evolution of anurans. Syst Zool 18, 1–32.
    14. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
    15. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    16. Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
    17. Schaad, N. W., Postnikova, E., Sechler, A., Claflin, L. E., Vidaver, A. K., Jones, J. B., Agarkova, I., Ignatov, A., Dickstein, E. & Ramundo, B. A. (2008). Reclassification of subspecies of Acidovorax avenae as A. avenae (Manns 1905) emend., A. cattleyae (Pavarino, 1911) comb. nov., A. citrulli Schaad et al., 1978) comb. nov., and proposal of A. oryzae sp. nov. Syst Appl Microbiol 31, 434–446.
    18. 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.
    19. Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). mega4: molecular evolutionary genetics analysis (mega) software version 4.0. Mol Biol Evol 24, 1596–1599.
    20. 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.
    21. 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.
    22. Willems, A., Goor, M., Thielemans, S., Gillis, M., Kersters, K. & De Ley, J. (1992). Transfer of several phytopathogenic Pseudomonas species to Acidovorax as 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.