SGM Journals
Bacteroidetes

Mucilaginibacter daejeonensis sp. nov., isolated from dried rice straw

Dong-Shan An, Cheng-Ri Yin, Sung-Taik Lee, Chun-Hwi Cho

  • 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
  • 2Key Laboratory of Organism Functional Factors of the Changbai Mountain, Ministry of Education, Yanbian University, Yanji 133-002, PR China
  • 3Korea Agriculture Fertilizer Corporation Biochemistry Research Institute, ChungBuk 373-831, Republic of Korea
  • 4Department of Bio-Environmental Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea
  • Correspondence
    Chun-Hwi Cho
    kafco1002{at}hanmail.net

    • International Journal of Systematic and Evolutionary Microbiology 2009; 59(5):1122–1125 · https://doi.org/10.1099/ijs.0.003384-0

    View at publisher PubMed

    Abstract

    A novel strain, designated Jip 10T, isolated from dried rice straw, was characterized by a polyphasic approach to clarify its taxonomic position. The isolate was Gram-negative, facultatively aerobic, heterotrophic, non-motile, non-spore-forming and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate clustered with the genus Mucilaginibacter in the phylum Bacteroidetes. 16S rRNA gene sequence similarities between strain Jip 10T and the type strains of Mucilaginibacter gracilis and Mucilaginibacter paludis were 93.7 and 93.6 %, respectively. The G+C content of the genomic DNA was 48.1 mol%. Chemotaxonomic data [major menaquinone MK-7 and major fatty acids iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c)] supported the affiliation of strain Jip 10T to the genus Mucilaginibacter. However, the results of physiological and biochemical tests allowed phenotypic differentiation of strain Jip 10T from other Mucilaginibacter species with validly published names. Therefore, strain Jip 10T (=KCTC 12639T =LMG 23488T) was classified in the genus Mucilaginibacter as the type strain of a novel species, for which the name Mucilaginibacter daejeonensis sp. nov. is proposed.

    • ↵†These authors contributed equally to this work.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Jip 10T is AB267717.

    The genus Mucilaginibacter was proposed by Pankratov et al. (2007) to accommodate species characterized by Gram-negative, non-motile rods that are facultatively aerobic, produce large amounts of extracellular polymeric substances (EPS), are capable of fermenting glucose and sucrose, hydrolyse pectin, xylan, laminarin, chondroitin 6-sulfate, gellan gum, pullulan and starch, are negative for H2S production from thiosulfate or indole from tryptophan and contain iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c) as major fatty acids and MK-7 as major menaquinone. At the time of writing, it comprises two recognized species, Mucilaginibacter paludis and Mucilaginibacter gracilis (Pankratov et al., 2007). In this study, we have characterized the novel Mucilaginibacter-like strain, Jip 10T, which was isolated from dried rice straw in a field in Daejeon city in South Korea.

    Strain Jip 10T was isolated from dried rice straw via direct plating onto R2A agar (Difco). Single colonies on these plates were purified by transferring them onto new plates and subjecting them to additional incubation for 3 days at 30 °C. The purified colonies were tentatively identified by using partial 16S rRNA gene sequences and maintained as a glycerol suspension (20 %, w/v) at −70 °C.

    The Gram reaction was performed by the non-staining method as described by Buck (1982). Cell morphology was observed at ×1000 magnification with a light microscope (Nikon) using cells grown for 3 days at 30 °C on R2A agar. Catalase activity was determined by bubble production in 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethyl p-phenylenediamine. Carbon-source utilization and enzyme activities were tested by using the API 20NE, ID 32 GN, API 50 CH and API ZYM test kits (bioMérieux). The ability to use nitrate as an alternative terminal electron acceptor was tested in serum bottles by adding sodium thioglycolate (1 g l−1) to R2A broth and replacing the upper air layer with nitrogen gas. The anaerobic nitrate reduction test to determine the final electron acceptor was performed in serum bottles by adding sodium thioglycolate (1 g l−1) and 10 mM KNO3 to R2A broth, under nitrogen gas. Growth at different temperatures (4, 15, 20, 25, 30, 37 and 42 °C) and at various pH values (pH 5.0–10.0 at intervals of 0.5 pH units) was assessed after 5 days of incubation. Salt tolerance was tested on R2A agar supplemented with 1–10 % (w/v) NaCl after 5 days of incubation. Growth on nutrient agar, trypticase soy agar (TSA) and MacConkey agar was also evaluated at 30 °C.

    Cells of strain Jip 10T were facultatively aerobic, Gram-negative, heterotrophic, non-motile, non-spore-forming rods. The strain did not grow on nutrient agar or MacConkey agar. The ability to reduce nitrate was absent. Other physiological characteristics of strain Jip 10T are summarized in the species description and a comparison of selective characteristics with closely related species is shown in Table 1⇓.

    Table 1.

    Biochemical characteristics of strain Jip 10T and the type strains of Mucilaginibacter species

    Strains: 1, strain Jip 10T; 2, M. gracilis TPT18T; 3, M. paludis TPT56T. Data for reference strains were taken from Pankratov et al. (2007). All three strains are positive for copious EPS production, acid production from d-fructose, d-galactose, d-glucose and maltose and catalase and oxidase activities. All strains are negative for indole production. All three strains assimilate d-glucose, sucrose and maltose, but not acetate, citrate, malate, mannitol, melibiose, propionate or valerate.

    For the measurement of G+C content of the chromosomal DNA, genomic DNA of the novel strain was extracted and purified as described by Moore & Dowhan (1995), degraded enzymically into nucleosides and analysed as described by Mesbah et al. (1989) using reversed-phase HPLC. Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and re-extracted in n-hexane/water (1 : 1, v/v). The crude n-hexane–quinone solution was purified using Sep-Pak Vac silica cartridges (Waters) and subsequently analysed by HPLC as described by Hiraishi et al. (1996). Cellular fatty acid profiles were determined from biomass harvested from one-tenth-strength TSA (Difco) after 3 days of incubation. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System. The fatty acids were analysed by a gas chromatograph (Hewlett Packard 6890) and identified by the Microbial Identification software package (Sasser, 1990).

    The G+C content of genomic DNA of strain Jip 10T was 48.1 mol%. MK-7 was the major respiratory quinone and MK-6 was a minor component. As shown in Table 2⇓, the major fatty acids of this strain were iso-C15 : 0 (26.5 %), C16 : 0 (9.3 %), iso-C17 : 0 3-OH (7.1 %) and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c) (39.1 %). These results supported the affiliation of strain Jip 10T to the genus Mucilaginibacter.

    Table 2.

    Fatty acid profiles of strain Jip 10T and the type strains of Mucilaginibacter species

    Strains: 1, strain Jip 10T; 2, M. gracilis TPT18T; 3, M. paludis TPT56T. Data for reference strains were taken from Pankratov et al. (2007). −, <1.0 % of the total/not reported.

    Extraction of genomic DNA was performed with a commercial genomic DNA extraction kit (Solgent) and PCR amplification of the 16S rRNA gene and sequencing of the purified PCR product were performed according to Kim et al. (2005). The full sequence of the 16S rRNA gene was compiled using dnastar. The 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed by using the clustal x program (Thompson et al., 1997). Gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using Kimura's two-parameter model (Kimura, 1983) and phylogenetic trees were constructed by using the neighbour-joining method (Saitou & Nei, 1987) using the mega 3 program (Kumar et al., 2004) with bootstrap values based on 1000 replications (Felsenstein, 1985).

    The nearly complete 16S rRNA gene sequence (1469 bp) of strain Jip 10T was determined and subjected to comparative analysis. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate clustered with Mucilaginibacter in the phylum Bacteroidetes (Fig. 1⇓). 16S rRNA gene sequence similarities between strain Jip 10T and the type strains of M. gracilis and M. paludis were 93.7 and 93.6 %, respectively. These values (<97 %) were low enough to place strain Jip 10T in a novel species in the genus Mucilaginibacter according to Stackebrandt & Goebel (1994).

    Figure image not available in archive
    Fig. 1.

    Rooted phylogenetic tree based on the 16S rRNA gene sequences of strain Jip 10T and related bacteria in the phylum Bacteroidetes, made using the neighbour-joining method (Saitou & Nei, 1987) with a distance matrix generated using the two-parameter model of Kimura (1983) and pairwise deletion. Bootstrap values (expressed as percentages of 1000 replications) greater than 70 % are shown at branch points. Bar, 2 substitutions per 100 nucleotides.

    On the basis of the data and observations described above, strain Jip 10T should be assigned to the genus Mucilaginibacter as the type strain of a novel species, for which the name Mucilaginibacter daejeonensis sp. nov. is proposed.

    Description of Mucilaginibacter daejeonensis sp. nov.

    Mucilaginibacter daejeonensis (dae.jeon.en′sis. N.L. masc. adj. daejeonensis referring to Daejeon, Korea, the geographical origin of the type strain).

    Cells are Gram-negative, facultatively aerobic, non-motile, non-spore-forming and rod-shaped, 0.6–0.8 μm in diameter and 1.3–1.6 μm long after 2 days on R2A agar. Colonies are opaque, circular, convex and orange in colour after 2 days on R2A agar. On R2A agar, grows at 15–37 °C, but not at 4 or 42 °C. Growth occurs at pH 4.5–8.5 and with up to 3 % NaCl. Optimal growth occurs at 30 °C, pH 7.0 and without NaCl. Substrate utilization, enzyme production and other physiological characteristics are indicated in Table 1⇑. In addition to the properties listed in Table 1⇑, l-arabinose and rhamnose are utilized as sole carbon sources. Adipate, l-alanine, 4-hydroxybenzoate, caprate, l-fucose, glycogen, histidine, 3-hydroxybenzoate, 3-hydroxybutyrate, inositol, itaconate, 2-ketogluconate, 5-ketogluconate, lactate, malonate, phenylacetate, l-proline, d-ribose, l-serine and suberate are not utilized as sole carbon sources. In API 50 CH tests, acid is produced from N-acetylglucosamine, aesculin ferric citrate, amygdalin, dl-arabinose, cellobiose, gentiobiose, lactose, melibiose, methyl α-glucopyranoside, starch, sucrose and turanose, but acid is not produced from adonitol, dl-arabitol, arbutin, dulcitol, erythritol, dl-fucose, glycerol, glycogen, inositol, inulin, d-lyxose, d-mannitol, melezitose, methyl α-d-mannopyranoside, methyl α-d-xyloside, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate, raffinose, d-ribose, d-sorbitol, l-sorbose, salicin, d-tagatose, xylitol or l-xylose. According to the API ZYM gallery, positive for N-acetyl-α-glucosaminidase, acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase (C4), esterase lipase (C8), α-galactosidase, α-galactosidase, leucine arylamidase and valine arylamidase activities, weakly positive for α-glucosidase and negative for chymotrypsin, α-fucosidase, α-glucosidase, α-glucuronidase, lipase (C14), α-mannosidase, naphthol-AS-BI-phosphohydrolase and trypsin. According to the API 20NE gallery, positive for arginine dihydrolase, urease, α-glucosidase, protease, α-galactosidase and glucose fermentation, but negative for nitrate reduction. The predominant respiratory quinone is MK-7. The major fatty acids are iso-C15 : 0, C16 : 0, iso-C17 : 0 3-OH and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c). The DNA G+C content of the type strain is 40.4 mol%.

    The type strain, strain Jip 10T (=KCTC 12639T =LMG 23488T), was isolated from dried rice straw in a field in Daejeon city in South Korea.

    Acknowledgments

    This work was supported by the Key Project of Chinese Ministry of Education (no. 206039). We thank Jean Euzéby for his help with the etymology of the species epithet.

    References

    1. Buck, J. D. (1982). Nonstaining (KOH) method for determination of gram reactions of marine bacteria. Appl Environ Microbiol 44, 992–993.
    2. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.
    3. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
    4. Hiraishi, A., Ueda, Y., Ishihara, J. & Mori, T. (1996). Comparative lipoquinone analysis of influent sewage and activated sludge by high-performance liquid chromatography and photodiode array detection. J Gen Appl Microbiol 42, 457–469.
    5. Kim, M. K., Im, W.-T., Ohta, H., Lee, M. & Lee, S.-T. (2005). Sphingopyxis granuli sp. nov., a β-glucosidase producing bacterium in the family Sphingomonadaceae in α-4 subclass of the Proteobacteria. J Microbiol 43, 152–157.
    6. Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.
    7. Kumar, S., Tamura, K. & Nei, M. (2004). mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
    8. 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.
    9. Moore, D. D. & Dowhan, D. (1995). Preparation and analysis of DNA. In Current Protocols in Molecular Biology, chapter 2, pp. 2–11. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Wiley.
    10. Pankratov, T. A., Tindall, B. J., Liesack, W & Dedysh, S. N. (2007). Mucilaginibacter paludis gen. nov., sp. nov. and Mucilaginibacter gracilis sp. nov., pectin-, xylan- and laminarin-degrading members of the family Sphingobacteriaceae from acidic Sphagnum peat bog. Int J Syst Evol Microbiol 57, 2349–2354.
    11. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
    12. Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
    13. Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
    14. 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.