| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Environmental Engineering for Symbiosis, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan
2 SEAWELL Co. Ltd, 8-12-6 Ginza, Chuo-ku, Tokyo 104-0061, Japan
Correspondence
Norio Kurosawa
kurosawa{at}t.soka.ac.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KWC4T is AB254031.
A table showing the cellular fatty acid contents of strain KWC4T, T. xylanilyticus and some representatives of the genus Paenibacillus and a phylogenetic tree based on 16S rRNA gene sequences of strain KWC4T and related species constructed using the maximum-likelihood method are available as supplementary material with the online version of this paper.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Haruta et al., 2002; Narihiro et al., 2004). Therefore, composting reactors may contain many novel species of bacteria. Recently, some novel species have been isolated from FBC reactors. For example, Paenibacillus motobuensis was derived from a composting machine utilizing soil (Iida et al., 2005) and Cerasibacillus quisquiliarum was isolated from a semi-continuous decomposing system for kitchen refuse (Nakamura et al., 2004).
During a screen for novel species from an FBC reactor, a moderately thermophilic bacterium, strain KWC4T, was isolated. 16S rRNA gene sequence analysis indicated that strain KWC4T represented a novel species of the genus Thermobacillus. The genus Thermobacillus was proposed by Touzel et al. (2000) to contain Gram-negative, spore-forming, aerobic, non-motile, rod-shaped thermophiles. To date, only a single species, Thermobacillus xylanilyticus, has been described (Touzel et al., 2000). This paper reports the isolation, characterization and taxonomic classification of strain KWC4T.
An FBC reactor for household use, the ‘Namagomi-eater’ (TK401-T, Matsushita Electric Works), was used for the composting reaction. The working volume of the reactor was 15 l and the temperature was usually maintained above 40 °C and reached about 60 °C during active degradation of organic matter. The biomass carrier comprised 5 l (1.2 kg wet wt) wood chips (about 0.5–2.0 mm). The artificial organic waste [500 g dog food containing 80 % (w/w) water] was loaded daily into the reactor. The contents were gently mixed twice per minute by automated paddles. The sample from which the micro-organism was isolated was taken from the optimal conditioned reactor in which the rate of decomposition of organic matter was about 17 g l–1 day–1. The temperature and pH of the sample were 47 °C and 8.6, respectively. The diluted sample was plated onto modified Brock's basal salts (MBS) (Kurosawa et al., 1998) supplemented with 0.2 % (w/v) yeast extract, pH 7.5, solidified by 0.7 % (w/v) Gelrite and incubated at 50 °C for 24 h. Colonies were isolated and purified by repeating single-colony isolation. One of the isolates, strain KWC4T, was assumed to be a member of a novel species by preliminary analysis of 16S rRNA gene sequences.
To characterize strain KWC4T phenotypically, standard tests were performed, including Gram staining, cell morphology, motility, catalase and oxidase production and acid or gas production from glucose (Barrow & Feltham, 1993). Cell morphology and motility were examined by using a phase-contrast microscope (Axioskop 40; Carl Zeiss) and a scanning electron microscope (SEM) (JSM-5600LV; JEOL). For SEM observation of the cells, samples of exponential-phase cultures were applied onto carbon-coated SEMporeB filters (JEOL Datum) and then washed with distilled water. After freeze-drying, samples were metal-shadowed with platinum and examined in high-vacuum mode at 10 kV.
The potential for growth at various initial pH values was determined using MBS supplemented with 0.5 % (v/v) yeast extract at 50 °C. The growth temperature and optimal NaCl concentrations were determined using the same medium at pH 9.0. Anaerobic growth was examined by using an AnaeroPack jar (Mitsubishi Gas Chemical). Enzymic activities were analysed by the API ZYM kit (bioMérieux) according to the manufacturer's instructions except for the incubation temperature, which was adjusted to 50 °C. Utilization of D-cellobiose, dextrin, erythritol, D-fructose, D-galactose, glucose, lactose, maltose, D-mannose, melibiose, D-sorbitol, sucrose, trehalose and D-xylose were tested. Hydrolysis of casein, starch and xylan were also examined. Cells for fatty acid analysis were grown on MBS plates at 50 °C for 24 h. Fatty acid methyl esters were prepared and identified following the Sherlock Microbial Identification system instructions (MIDI). The resultant esters were separated using a GC (HP6890; Hewlett Packard). The G+C content of DNA of strain KWC4T was determined by HPLC (LC-10; Shimadzu) using genomic DNA digested with nuclease P1 (DNA-GC kit; SEIKAGAKU) (Katayama-Fujimura et al., 1984). DNA–DNA hybridization experiments were carried out three times independently as described by Ezaki et al. (1989). Thermobacillus xylanilyticus strain XET was kindly provided by J. P. Touzel and was used for comparison. Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v) and were analysed by using fast-atom bombardment-MS (EI/FAB mate BU25; JEOL) with diethanolamine as a matrix for the negative MS.
The 16S rRNA gene of strain KWC4T was amplified by PCR using the bacterial universal primers B27F (forward; 5'-AGAGTTTGATCMTGGCTCAG, positions 8–27 based on Escherichia coli numbering) and U1492RM (reverse; 5'-GGYTACCTTGTTACGACTT, positions 1510–1492 based on E. coli numbering). The following thermal cycle was used for 25 cycles: 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1.5 min. DNA sequencing was carried out by the dideoxynucleotide chain-termination method with Texas-red-labelled primers using the ThermoSequenase Primer Cycle Sequencing kit (GE Healthcare Biosciences) and an automated DNA sequencer (SQ5500E; Hitachi). The 16S rRNA gene sequence of strain KWC4T was compared with available 16S rRNA gene sequences in the NCBI nucleotide sequence database using BLAST (http://www.ncbi.nlm.nih.gov/blast/) (Altschul et al., 1990). Twenty-nine 16S rRNA gene sequences of related species were aligned using CLUSTAL W (Thompson et al., 1994) and all sites with gaps in any sequences and regions of the PCR primers were removed from the alignment. Phylogenetic trees were reconstructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981) methods and algorithms were integrated in the PHYLIP package (Felsenstein, 1993). The stability of relationships was assessed by performing bootstrap analyses of the neighbour-joining data based on 1000 resamplings.
Colonies of strain KWC4T on MBS plates were 0.5–1.0 mm in diameter, light-cream in colour, round and half-opaque, but sometimes exhibited an irregular, flat morphology and had rather undulate margins. Cells of strain KWC4T were 2.0–5.0 µm long and 0.5–0.7 µm in diameter (Fig. 1). Endospores were formed at the middle of the cell (data not shown). Cells of strain KWC4T were aerobic, Gram-negative, spore-forming, occurred singly and occasionally in pairs or chains, and were non-motile and catalase- and oxidase-positive. Strain KWC4T utilized D-cellobiose, D-fructose, D-galactose, D-xylose, D-mannose, lactose, melibiose, starch, trehalose and xylan; T. xylanilyticus, the type species of the genus, also utilized these substrates. Strain KWC4T also utilized D-glucose, maltose, sucrose and dextrin. Alkaline phosphatase, esterase, leucine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase and -fucosidase activities were detected; no lipase, valine arylamidase, chymotrypsin or -mannosidase activities were found. Acid or gas production from glucose was negative.
|
). The major fatty acids of strain KWC4T were iso-16 : 0 (39.0 %) and anteiso-15 : 0 (33.3 %). This fatty acid composition also distinguished strain KWC4T from T. xylanilyticus and species of the genus Paenibacillus (see Supplementary Table S1 available in IJSEM Online). The major isoprenoid quinone of strain KWC4T was MK-6(H8), in contrast to T. xylanilyticus, which had MK-7 as the major isoprenoid quinone (Touzel et al., 2000).
|
). This cluster was also distinct from other genera, i.e. Cohnella and Paenibacillus, in the maximum-likelihood tree (see Supplementary Fig. S1 available in IJSEM Online). Interestingly, strain KWC4T has a 38 bp insertion sequence near the 3' end of the 16S rRNA gene compared with the sequence of T. xylanilyticus. The 16S rRNA gene sequences of strain KWC4T and T. xylanilyticus showed a similarity value of 98.3 % when the insertion sequence was removed from the alignment. This unusual insertion sequence was also found in the 16S rRNA gene sequence of Paenibacillus nematophilus strain NEM3 (Enright et al., 2003). In addition, when compared with other bacteria, strain KWC4T and T. xylanilyticus have a 36 bp insertion sequence located near the 5' end of the 16S rRNA gene. This insertion sequence may be unique for the genus Thermobacillus. DNA–DNA hybridization experiments were also performed between strain KWC4T and T. xylanilyticus strain XET. The DNA–DNA reassociation value was 66 % (SD 5.4 %). This value is relatively high for members of different species, but phenotypic signatures suggest that strain KWC4T should be defined independently from T. xylanilyticus. Based on phylogenetic evidence, together with phenotypic and chemotaxonomic data, strain KWC4T is proposed as a representative of a novel species of the genus Thermobacillus, Thermobacillus composti sp. nov.
|
Cells are non-motile, spore-forming rods (approx. 2.0–5.0 µm long). Aerobic and Gram-negative. Catalase- and oxidase-positive. Colonies are light-cream in colour, round and half-opaque, but sometimes exhibit an irregular, flat morphology. Grows at 32–61 °C and pH 5.6–10.1, with optimal growth at 50 °C and around pH 9.0. Optimal NaCl concentration is almost 0 %, but is able to grow at NaCl concentrations up to 4.4 % (w/v). The following compounds are utilized: D-cellobiose, dextrin, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, melibiose, starch, sucrose, trehalose, D-xylose and xylan. Erythritol, sorbitol, and casein are not utilized. The major fatty acids are iso-16 : 0 (39.0 %) and anteiso-15 : 0 (33.3 %). The major isoprenoid quinone is MK-6(H8). DNA–DNA relatedness between strain KWC4T and Thermobacillus xylanilyticus strain XET is 66 %. The DNA G+C content of the type strain is 60.0 mol%.
The type strain, KWC4T (=DSM 18247T=JCM 13945T), was isolated from a fed-batch composting reactor.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Barrow, G. I. & Feltham, R. K. A. (1993). Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press.
Dees, P. M. & Ghiorse, W. C. (2001). Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA. FEMS Microbiol Ecol 35, 207–216.[CrossRef][Medline]
Enright, M. R., Mclnerney, J. O. & Griffin, C. T. (2003). Characterization of endospore-forming bacteria associated with entomopathogenic nematodes, Heterorhabditis spp., and description of Paenibacillus nematophilus sp. nov. Int J Syst Evol Microbiol 53, 435–441.
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.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef][Medline]
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Department of Genome Sciences, University of Washington, Seattle, USA.
Haruta, S., Kondo, M., Nakamura, K., Aiba, H., Ueno, S., Ishii, M. & Igarashi, Y. (2002). Microbial community changes during organic solid waste treatment analyzed by double gradient-denaturing gradient gel electrophoresis and fluorescence in situ hybridization. Appl Microbiol Biotechnol 60, 224–231.[CrossRef][Medline]
Iida, K., Ueda, Y., Kawamura, Y., Ezaki, T., Takade, A., Yoshida, S. & Amako, K. (2005). Paenibacillus motobuensis sp. nov., isolated from a composting machine utilizing soil from Motobu-town, Okinawa, Japan. Int J Syst Evol Microbiol 55, 1811–1816.
Katayama-Fujimura, Y., Komatsu, Y., Kuraishi, H. & Kaneko, T. (1984). Estimation of DNA base composition by high performance liquid chromatography of its nuclease P1 hydrolysate. Agric Biol Chem 48, 3169–3172.
Kurosawa, N., Itoh, Y. H., Iwai, T., Sugai, A., Uda, I., Kimura, N., Horiuchi, T. & Itoh, T. (1998). Sulfurisphaera ohwakuensis gen. nov., sp. nov., a novel extremely thermophilic acidophile of the order Sulfolobales. Int J Syst Bacteriol 48, 451–456.
Nakamura, K., Haruta, S., Ueno, S., Ishii, M., Yokota, A. & Igarashi, Y. (2004). Cerasibacillus quisquiliarum gen. nov., sp. nov., isolated from a semi-continuous decomposing system of kitchen refuse. Int J Syst Evol Microbiol 54, 1063–1069.
Narihiro, T., Abe, T., Yamanaka, Y. & Hiraishi, A. (2004). Microbial population dynamics during fed-batch operation of commercially available garbage composters. Appl Microbiol Biotechnol 65, 488–495.[CrossRef][Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL_W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Touzel, J. P., O'Donohue, M., Debeire, P., Samain, E. & Breton, C. (2000). Thermobacillus xylanilyticus gen. nov., sp. nov., a new aerobic thermophilic xylan-degrading bacterium isolated from farm soil. Int J Syst Evol Microbiol 50, 315–320.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  R. Rivas, P. Garcia-Fraile, J. L. Zurdo-Pineiro, P. F. Mateos, E. Martinez-Molina, E. J. Bedmar, J. Sanchez-Raya, and E. Velazquez Saccharibacillus sacchari gen. nov., sp. nov., isolated from sugar cane Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1850 - 1854. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
