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Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National University, Tokiwadai 79-5, Hodogaya, Yokohama 240-8501, Japan
Correspondence
Minoru Takeda
mtake{at}ynu.ac.jp
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Published online ahead of print on 22 October 2004 as DOI 10.1099/ijs.0.63429-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains SGT and MH are AB179866 and AB179867, respectively.
A graph showing growth of strain SGT and EPS degradation, a figure showing the effect of SGT on S. natans sedimentation and a micrograph demonstrating the degradation of the sheath-associated EPS of S. natans are available as supplementary material in IJSEM Online.
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, 2003, 2004a, b). On the other hand, it is also known to be a troublesome bacterium that causes the problem of poor settling (filamentous bulking) of activated sludge (Eikelboom, 1975; Caravelli et al., 2003, 2004; Contreras et al., 2002; Gaval & Pernelle, 2003; van Veen et al., 1978). S. natans forms a sheath (Eikelboom, 1975; Takeda et al., 1998, 2003; van Veen et al., 1978) and a slimy extracellular polysaccharide (EPS) (Gaudy & Wolfe, 1962). The EPS is a straight-chain acidic polysaccharide (Takeda et al., 2002c) and is tightly associated with the sheath (Gaudy & Wolfe, 1962; Takeda et al., 2002b). The bulking effects of S. natans may due to the production of the sheath and the EPS. Biological degradation of the sheath of S. natans was revealed to be achieved by the sheath-degrading bacterium Paenibacillus koleovorans (Takeda et al., 2000, 2002a). However, degradation of the EPS has not been attempted so far. In the present study, we isolated an EPS-degrading bacterium, and the taxonomic characterization of the isolate was carried out. The results demonstrated that the isolate is a close relative of P. koleovorans and should be classified within the genus Paenibacillus as Paenibacillus hodogayensis sp. nov.
The EPS of S. natans was produced and purified as previously reported (Takeda et al., 2002b). The medium (EPS medium) used for screening for EPS-degrading bacteria was composed of the following (l–1 distilled water): 0·1 g EPS, 2 g (NH4)2SO4, 1 g K2HPO4, 0·5 g MgSO4.7H2O, 0·5 g NaCl, 0·5 g CaCO3 and 0·01 g FeSO4.7H2O. Samples of damp humus (about 0·1 g) from 50 sites in the city of Yokohama and neighbouring areas in Japan were added to test tubes containing 2 ml screening medium and were incubated statically at 30 °C for 10 days. EPS-dependent growth of the micro-organisms was detected in four cultures. After subcultivation four times, the cultures were individually spread on solidified (1·5 % agar) CaCO3-free EPS medium (EPS agar) and incubated at 30 °C for 3 months. Large (about 5 mm), yellow, convex colonies were obtained from one (a sample from the campus of Yokohama National University, Hodogaya, Yokohama, Japan) of the four cultures. However, two morphologically different rod-shaped bacteria were observed in the yellow colony that could not be grown individually on EPS agar. When the colony was transferred to TYN agar (containing, l–1 distilled water, 5 g tryptone, 2·5 g yeast extract, 2·5 g NaCl and 15 g agar), they were successfully isolated as two different colonies (yellow and white). The isolate that formed the yellow colony was designated strain MH and its cell size was about 0·3x0·7 µm. Another isolate (designated strain SGT) formed the white colony; its cells were larger than those of strain MH. Further experiments revealed that only strain SGT had the ability to degrade EPS (results are available as Supplementary Fig. A in IJSEM Online). The strain was found to be able to accelerate the sedimentation of S. natans (Supplementary Fig. B) by removing the sheath-associated EPS without having any effect on the sheath (Supplementary Fig. C).
Strain SGT was cultured on TYN agar at 30 °C for 3 days, and characterized taxonomically. Gram-staining was done using a Favour-GS kit (Nussui Pharmaceutical). Motility (18–24-h-old cells in TYN medium) and morphological properties were determined by phase-contrast microscopy (Axioskop; Zeiss). Anaerobic growth was tested using a BBL GasPak pouch (Becton Dickinson). The bacterium was inoculated into TYN medium and shaken at various temperatures (15–50 °C). Growth was monitored by measuring the optical density at 600 nm at intervals of 1 h; the specific growth rate was then calculated. To determine the effect of pH on growth, the bacterium was cultured in TYN medium at various pH values (pH 3·5–11·0, adjusted by adding HCl or NaOH) at 30 °C for 14 h, and the optical density was then measured. Growth in the presence of 5 % NaCl was tested in TYN medium supplemented with 5 % (w/v) NaCl. Strain SGT was motile and obligately aerobic. Cells were Gram-variable but Gram-negative in old cultures. Colonies on TYN agar were white, convex and opaque. Cells were straight and rod-shaped (1·3–1·7 µm in width, 2·3–2·8 µm in length), as shown in Fig. 1(a). Flagella and elliptical endospores were shown by scanning probe microscope observation (Fig. 1b). Most sporangia were not swollen, but slightly swollen sporangia were occasionally observed with phase-contrast microscopy. Growth of strain SGT occurred at pH 6·2–9·2, with an optimum at pH 8. The growth temperature range was 20–40 °C, with an optimum at 30 °C. No growth was observed at 50 °C or in the presence of 5 % NaCl. The following characteristics were tested for as described by Takeda et al. (2002a): catalase and oxidase activities were positive; urease, gelatinase, arginine dihydrolase, ornithine decarboxylase and lysine decarboxylase tests were negative; nitrate was not reduced; indole and hydrogen sulfate were not produced; citrate was not utilized; and the Voges–Proskauer reaction was negative. Acid production from various carbon sources was tested at 30 °C with API 50 CH (bioMérieux): the results are given in the species description and Table 1.
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). Quinone was extracted and purified according to a previously described method (Takeda et al., 2004) and was identified by fast atom bombardment MS (JMS-AX500 apparatus; JEOL). Cellular fatty acids were identified by using previously described methods (Takeda et al., 2004). The DNA G+C content of strain SGT was 55·0 mol%. Most Paenibacillus strains exhibit a G+C content of 39–54 mol% (Shida et al., 1997), although the strains of Paenibacillus dendritiformis (Tcherpakov et al., 1999) and P. koleovorans (Takeda et al., 2002a) have a rather higher G+C content, of 54–55 mol%. The major respiratory quinone was MK-7(H2). The major cellular fatty acids were anteiso-C15 : 0 (39·7 %), C15 : 0 (19·2 %), iso-C16 : 0 (9·6 %) and C16 : 0 (7·6 %). Paenibacillus strains are reported to have MK-7 as the major respiratory quinone and anteiso-C15 : 0 as the major fatty acid (Shida et al., 1997).
The 16S rRNA gene sequences of strains SGT (1488 bp) and MH (1345 bp) were amplified by PCRs with a universal primer (8F: 5'-AGAGTTTGATCATGGCTCAG-3') (Hallbeck et al., 1995) in combination with universal primer 1392R (5'-ACGGGCGGTGTGTAC-3') (Hallbeck et al., 1995) or 1522R (5'-AAGGAGGTGATCCAGCCGCA-3') (Ash et al., 1993). The PCR products were cloned into pCR-TOPO (Invitrogen) and the nucleotide sequences were subsequently determined using vector-specific primers (Invitrogen) and a series of primers designed by Hallbeck et al. (1995). Phylogenetic analysis (neighbour-joining method) was done according to previously described methods (Takeda et al., 2000a). Sequences specific for the genus Paenibacillus, the PAEN515F primer-binding site (Shida et al., 1997) and the 22-base sequence (Ash et al., 1993), were found in the sequence determined for strain SGT. The closest relative of strain MH was Chryseobacterium indoltheticum (AY468448), a species that has been inadequately characterized (Vandamme et al., 1994), which showed a high level of similarity (97·5 %). A phylogenetic analysis revealed that strain SGT could be placed in the cluster that included P. koleovorans, Paenibacillus chondroitinus, Paenibacillus alginolyticus, Paenibacillus larvae subsp. larvae, P. larvae subsp. pulvifaciens, Paenibacillus chinjuensis, Paenibacillus koreensis, Paenibacillus validus and Paenibacillus naphthalenovorans (Fig. 2). The sequence similarity values for the type strains of the genus Paenibacillus ranged from 94·2 % (P. koleovorans) to 89·9 % (P. dendritiformis). The closest relative of strain SGT, P. koleovorans, is capable of degrading the sheath of S. natans (Takeda et al., 2000, 2002a). It is interesting that two bacteria involved in the degradation of extracellular macromolecules of S. natans are closely related. Table 1
summarizes the properties of strain SGT that can be used to differentiate it from phylogenetically related species of the genus Paenibacillus. Strain SGT produced acids from several carbohydrates but P. koleovorans does not. Most strains of the genus Paenibacillus form swollen sporangia, but strain SGT formed non-swollen or only slightly swollen sporangia. A few strains of P. larvae subsp. larvae and P. larvae subsp. pulvifaciens are known to form non-swollen sporangia (Heyndrickx et al., 1996). The DNA G+C content of these subspecies of P. larvae (42–43 mol%) is much lower than that of strain SGT (55 %), allowing easy discrimination. The genetic and phenotypic features of strain SGT suggest that it represents a novel species of the genus Paenibacillus, for which the name Paenibacillus hodogayensis sp. nov. is proposed.
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Cells are rod-shaped (1·3–1·7x2·3–2·8 µm) and motile. Gram-variable or Gram-negative. Ellipsoidal spores are formed in non-swollen or slightly swollen sporangia. Growth occurs only under aerobic conditions. Colonies on TYN agar are white, convex and opaque. The optimum growth temperature is 30 °C. The optimum pH for growth is 8. No growth occurs at 50 °C or in the presence of 5 % (w/v) NaCl. The Voges–Proskauer reaction is negative. Catalase- and oxidase-positive. Nitrate is not reduced. Indole is not produced. Citrate is not utilized. Acids are produced from glycerol, methyl 
-xyloside, glucose, mannitol, aesculin, cellobiose, maltose, melibiose, sucrose, trehalose and turanose. Small amounts of acid are produced from ribose, methyl 
-mannoside, methyl -glucoside, N-acetylglucosamine, amygdalin, salicin, lactose and 5-ketogluconate. Acid is not produced from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, adonitol, galactose, fructose, mannose, sorbose, rhamnose, dulcitol, inositol, sorbitol, arbutin, inulin, melezitose, raffinose, starch, glycogen, xylitol, -gentiobiose, lyxose, tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate or 2-ketogluconate. Acidic exopolysaccharide from S. natans is degraded and utilized. The predominant cellular fatty acid is anteiso-C15 : 0. The major quinone is MK-7(H2). The DNA G+C content is 55 mol%. The PAEN515F binding site exists in the 16S rRNA gene.
The type strain is SGT (=JCM 12520T=KCTC 3919T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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