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1 Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Road, Nan-Tzu, Kaohsiung City 811, Taiwan
2 Department of Marine Biotechnology, National Kaohsiung Marine University, No. 142, Hai-Chuan Road, Nan-Tzu, Kaohsiung City 811, Taiwan
3 Department of Biotechnology, Tajen University, Pingtung, Taiwan
Correspondence
Wen-Ming Chen
p62365{at}ms28.hinet.net
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ABSTRACT |
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A table detailing the chemical composition of the Guanzingling hot spring water sample is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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and belongs to the subgroup Rubrivivax of the Betaproteobacteria. The genus Schlegelella currently comprises a single species with a validly published name, Schlegelella thermodepolymerans. The two strains of S. thermodepolymerans (K14T and DhA-71) (Elbanna et al., 2003) and an independently isolated bacterium, Caenibacterium thermophilum (N2-680T) (Manaia et al., 2003), were described simultaneously as gen. nov., sp. nov. However, after confirming their closer relationship, Lütke-Eversloh et al. (2004) reclassified C. thermophilum N2-680T as a later synonym of S. thermodepolymerans. All of the strains of S. thermodepolymerans are moderately thermophilic (optimum growth at 45–50 °C) and have been isolated from man-made habitats. The type strain of S. thermodepolymerans was isolated from activated sludge, whilst C. thermophilum N2-680T was isolated from an aerobic digester of municipal sludge. The aim of the present study was to determine the taxonomic position of the isolate wcf1T, which was isolated from a water sample (natural habitat) collected from a hot spring located in the Tainan area, southern Taiwan.
The water sample was collected in July 2005 from a hot spring in Guanzingling located in southern Taiwan. The temperature of the spring water ranged from 60 to 65 °C and the pH value was approximately 8. The chemical composition of the Guanzingling hot spring water sample is listed in Supplementary Table S1 in IJSEM Online. After diluting with sterile distilled water, the water sample was plated on 10 % Luria–Bertani (LB) agar plates (1.0 g tryptone, 0.5 g yeast extract, 1.0 g NaCl and 15 g agar in 1 l distilled water) and incubated at 55 °C. A bacterial strain, designated wcf1T, was isolated, maintained and subcultivated further on 10 % LB medium. The organism was the subject of a polyphasic taxonomic study, which showed that it represented a novel species of Schlegelella.
Cell morphology was observed under a light microscope. The motility of cells was tested by the hanging-drop method. A Gram stain Set S (Difco) kit was used for testing the Gram reaction, and poly-
-hydroxybutyrate granule accumulation was observed by light microscopy after staining the cells with Sudan black. Flagellar staining was performed using a spot test flagella stain (Difco). The pH range for growth was determined by measuring OD595 of the culture grown in 10 % LB broth that had been adjusted prior to sterilization to various pH values (pH 3–11 at intervals of 0.5 pH units) using appropriate biological buffers (Chung et al., 1995). Growth at various temperatures (15–70 °C) was measured on 10 % LB medium. Growth under anaerobic conditions was determined after incubation in an Oxoid AnaeroGen system. The performance of growth was recorded by measuring the OD of the culture with respect to time.
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described previously (Chen et al., 2001). Sequence analysis was achieved using a DNA sequencer (ABI PRISM 310; Applied Biosystems) and sequence assembly by using the Fragment Assembly System program from the Wisconsin Package 9.1 supplied by the National Health Research Institute of Taiwan. The resultant sequence was compared with available 16S rRNA gene sequences in GenBank. Multiple-sequence alignment including strain wcf1T and its closest relatives was performed using BIOEDIT software (Hall, 1999). Phylogenetic trees were inferred using the maximum-parsimony and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms. An evolutionary distance matrix was generated for the neighbour-joining algorithm using the Jukes & Cantor (1969) distance model and bootstrap analysis (1000 resamplings).
For determination of G+C content, a DNA sample was prepared and degraded enzymically into nucleosides as described by Mesbah et al. (1989). The obtained nucleoside mixture was then separated by HPLC. DNA–DNA hybridization experiments were performed with photobiotin-labelled probes as described by Ezaki et al. (1989). The degree of hybridization was determined from two replicate experiments. Further differentiation of strain wcf1T from its closest phylogenetic neighbour was determined by cellular fatty acid profiling using gas chromatography according to the instructions of the Microbial Identification System (MIDI; Microbial ID).
Strain wcf1T was examined for a broad range of phenotypic properties. Additional biochemical tests were performed at 50 °C using the Biolog GN2 (Biolog), API ZYM (bioMérieux) and API 20NE (bioMérieux) microtest systems according to the methods outlined by the manufacturers. Sensitivity to antibiotics was examined after spreading cells (0.5 McFarland) on 10 % LB medium and incubating at 50 °C. Antibiotic discs (Difco) contained the following antibiotics: ampicillin (10 µg), cefotaxime (30 µg), chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), rifampicin (5 µg), penicillin G (10 µg), streptomycin (10 µg) or tetracycline (30 µg). The effect of antibiotics on cell growth was assessed after 3 days and susceptibility was scored based on the distance from the edge of clear zone to that of the disc. If the distance was larger than 3 mm the strain was classified as susceptible, between 1 and 3 mm it was classified as moderately susceptible and if the clear zone was less than 1 mm it was considered resistant.
Strain wcf1T grew well aerobically in complex media with a reduced concentration of nutrients, such as 10 % LB medium. When higher concentrations of nutrients (such as trypticase soy, nutrient and LB medium) were provided, growth was comparatively poor. Strain wcf1T formed visible, cream or white colonies of circular and convex shape with entire edges. The colony size was approximately 1.0–1.5 mm in diameter on 10 % LB agar plates after incubation for 48 h at 55 °C. Strain wcf1T grew well at temperatures ranging from 30 to 60 °C and from pH 6 to 8. Optimal growth occurred at 50 °C and pH 7.0. Strain wcf1T did not grow after 120 h of incubation at 50 °C under anaerobic conditions.
Cells of strain wcf1T were Gram-negative rods, motile by means of polar flagellum, non-spore-forming, 0.4–0.5 µm in diameter and 0.8–2.0 µm in length. Poly--hydroxybutyrate granules were observed after staining with Sudan black.
A nearly complete 16S rRNA gene sequence (1419 nt) was obtained for strain wcf1T. A comparison of the sequence with those of representatives of genera classified in the Betaproteobacteria showed that the organism fell within the evolutionary radiation occupied by the genus Schlegelella (Fig. 1). According to the sequence similarity calculations, the organism was most closely related to S. thermodepolymerans strain K14T (97.8 % similarity) (Elbanna et al., 2003), S. thermodepolymerans strain N2-680 (97.9 % similarity) (Manaia et al., 2003), S. thermodepolymerans strain SA8 (97.9 % similarity), S. thermodepolymerans strain SA1 (97.9 % similarity) (Romen et al., 2004) and S. thermodepolymerans strain DhA-71 (97.8 % similarity) (Yu & Mohn, 1999) (Fig. 1). The similarity levels of strain wcf1T with other bacterial species with validly published names within the Betaproteobacteria were less than 95 %.
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The G+C content of strain wcf1T was 69.2 mol%, which is lower than that of S. thermodepolymerans (70.0 mol%).
The cellular fatty acid composition of strain wcf1T is shown in Table 1 in comparison with S. thermodepolymerans K14T. The fatty acid profile of strain wcf1T was similar to that of S. thermodepolymerans K14T, although the amounts of 16 : 0 and 17 : 0 cyclo differed. However, strain wcf1T could clearly be distinguished from S. thermodepolymerans K14T by the presence of a significant amount of summed feature 3 (19.5 %).
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-galactosidase and assimilation of arabinose, mannose, mannitol, N-acetylglucosamine, caprate, malate and citrate. In API ZYM tests, wcf1T was positive for alkaline phosphatase, C4 esterase, C8 lipase, C14 lipase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase and 
-glucosidase and negative for valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, acid phosphatase, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. The following compounds were utilized as sole carbon sources in the Biolog GN2 test system: glycogen, methyl pyruvate, monomethyl succinate, acetic acid, -hydroxybutyric acid, -hydroxybutyric acid, 
-hydroxyphenylacetic acid, -ketobutyric acid, -ketoglutaric acid, DL-lactate, propionic acid, succinic acid, succinamic acid, L-alaninamide, D-alanine, L-alanine, L-asparagine, L-proline, L-pyroglutamic acid and urocanic acid. However, strain wcf1T could not oxidize -cyclodextrin, dextrin, Tween 40, Tween 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, arabinose, arabitol, cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose, gentiobiose, myo-inositol, -D-lactose, lactulose, D-mannitol, D-mannose, melibiose, methyl -D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, cis-aconitic acid, citrate, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-glucosaminic acid, D-glucuronic acid, 
-hydroxybutyric acid, itaconic acid, -ketovaleric acid, malonic acid, quinic acid, D-saccharic acid, sebacic acid, bromosuccinic acid, glucuronamide, L-alanyl glycine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, D-serine, L-serine, L-threonine, DL-carnitine, -aminobutyric acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, DL--glycerol phosphate, D-glucose 6-phosphate and -D-glucose 1-phosphate. Strain wcf1T was susceptible to ampicillin, cefotaxime, chloramphenicol, gentamicin, kanamycin, nalidixic acid, novobiocin, penicillin G, rifampicin, streptomycin and tetracycline.
Further comparisons of physiological and biochemical characteristics between strain wcf1T and S. thermodepolymerans K14T are presented in Table 2. It is apparent from Table 2 that strain wcf1T could be distinguished from S. thermodepolymerans K14T using a combination of phenotypic attributes (nitrate reduction, assimilation of carbon substrates such as arabinose, adipate, citrate, maltose and phenyl acetate and some enzyme activities such as catalase, cystine arylamidase and -glucosidase). Despite physiological and biochemical attributes, the results of fatty acid composition and DNA–DNA hybridization clearly distinguished strain wcf1T from S. thermodepolymerans K14T. On the basis of the results of this polyphasic taxonomic study, strain wcf1T represents a novel species of the genus Schlegelella for which the name Schlegelella aquatica sp. nov. is proposed.
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Cells are Gram-negative, non-spore-forming, rod-shaped, 0.4–0.5 µm in diameter and 0.8–2.0 µm in length. Cells occur singly and are motile with a single polar flagellum. Growth is evident at temperatures of 30–60 °C and pH 6.0–8.0. Optimum growth is at 50 °C and pH 7.0. Positive for nitrate reduction, gelatin hydrolysis and aesculin hydrolysis and weakly positive for oxidase activity. Assimilates glucose, maltose, gluconate, adipate and phenyl acetate. Major fatty acids are 16 : 0, 17 : 0 cyclo and summed feature 3 (16 : 1
7c and/or 15 : 0 iso 2-OH). Additional biochemical characteristics are listed above and in Table 2
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The DNA G+C content of the type strain is 69.2 mol%. The type strain, wcf1T (=BCRC 17557T=LMG 23380T), was isolated from a water sample from a hot spring in Guanzingling located in the Tainan area of southern Taiwan.
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Chung, Y. C., Kobayashi, T., Kanai, H., Akiba, T. & Kudo, T. (1995). Purification and properties of extracellular amylase from the hyperthermophilic archeon Thermococccus profundus DT5432. Appl Environ Microbiol 61, 1502–1506.[Abstract]
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