Laboratory of Ecological and Toxicological Chemistry, Faculty of Agriculture, Shinshu University (United Graduate School of Gifu University), 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan1
Laboratory of Microbial Engineering and Technology, Faculty of Agriculture, Gifu University (United Graduate School of Gifu University), 1-1, Yanagido, Gifu 501-1193, Japan2
Author for correspondence: Ryozo Iriye. Tel: +81 265 77 1604. Fax: +81 265 77 1629. e-mail: kn53278{at}gipmc.shinshu-u.ac.jp
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ABSTRACT |
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Keywords: filaments, gonidia, cell-binding
Abbreviations: DAP, diaminopimelic acid; DPA, dipicolinic acid; SLRC, spore-like resting cell
The GenBank/EMBL/DDBJ accession number for the 16S rRNA sequence reported in this paper is AB049195.
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INTRODUCTION |
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In recent years a number of filamentous bacteria from activated sludge have been well characterized and classified on the basis of 16S rRNA sequence similarity (Wagner et al., 1994 ; Seviour & Blackall, 1999
). Apart from these common filamentous bacteria there are also reports on the occurrence of filamentous Bacillus spp. from wastewater treatment tanks (Farquhar & Boyle, 1971
; Storm & Jenkins, 1984
). Trick et al. (1984)
were the first to isolate filamentous Bacillus species from bulking sludge. However, to the best of our knowledge, there are no reports on the phylogenetic characterization of filamentous Bacillus from activated sludge. Iriye isolated a filamentous bacterium from Fukushima, Japan, while examining the bacterial flora of suspended water in a surplus sludge treatment tank that contained humus soil for decreasing malodour (Iriye, 1999
). This bacterium exhibited some unusual morphological characteristics and produced endospores and another unique structure which we termed a spore-like resting cell (SLRC). SLRCs grew by budding. Characterization, life cycle studies and phylogenetic analysis of this novel species are presented in this paper.
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METHODS |
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Isolation of the bacterium.
A surplus sludge treatment system to suppress malodour for a domestic wastewater treatment plant has been developed, and the examination of bacterial flora in the sludge treatment process at Nakajima, Nishishirakawa, Fukushima, Japan, has been described previously (Iriye, 1999 ). The sludge treatment process is composed of a sludge-receiving tank, sludge-circulating tank and a sludge-contacting tank. The surplus sludge settled in the sequencing batch reactors is sent to a sludge-receiving tank and then to a sludge-circulating tank. A part of the suspended water in the sludge-circulating tank is sent to a sludge-contacting tank in which the suspended water is in aerobic contact with pellets of humus soil and volcanic rocks (residence time 12 h). The suspended water (100 ml) from the sludge-circulating tank was diluted to 1x104 times with 0·5% sodium chloride solution and plated on NGS agar plates. The plates were incubated at 32 °C for 1 d and then at 20 °C for 1 week.
Characterization of bacteria.
Morphological features were studied using a microscope with phase-contrast apparatus (LABOPHOT-2; Nikon).
Biochemical tests were carried out as described by Smibert & Krieg (1994) and staining as described by Murray et al. (1994)
. For the VogesProskauer test, bacteria were incubated in NG broth with shaking at 15 r.p.m. and 32 °C.
Optimization of temperature and pH were done by measuring OD660 during growth in synthetic medium shaken at 60 r.p.m. using a bioplotter (Toyosokki). To disperse the filamentous bacteria, glass beads were added to the incubation tubes.
Heat resistance of SLRCs.
Cells grown on nutrient agar plates bearing endospores or SLRCs were suspended in minimal medium (5 ml) or 0·5% NaCl solution (0·5 ml) in test tubes (OD660=0·5). The suspension was heated at a constant temperature in an oil bath for various durations. After heat treatment, cells in the minimal medium were incubated in a slow shaking incubator at 15 r.p.m. at 30 °C. Treated cells in 0·5% NaCl were plated on NG agar or incubated in NG broth at 30 °C.
Detection of dipicolinic acid (DPA).
DPA was extracted by standard methods (Janssen et al., 1958 ; Powell, 1953
) and measured with a Hitachi M1200AP LC-mass spectrometer in APCI (atmospheric pressure chemical ionization) positive mode (Hitachiseisakusho, Hitachi) with an ODS column (AQ 312; 6x150 mm; YMC), UV-detector (275 nm) and solvent (10 mM ammonium acetate, 1 ml min-1). Authentic DPA was detected at a retention time (tR) of 3·45 min: m/z 168 (M++H), 124 (M++H-CO2), 80 (M++H-2CO2). The tR values of isomers of pyridine dicarboxylic acid were as follows (min): 2,3-isomer, 3·08; 2,4-isomer, 3·33; 2,5-isomer, 2·92; 3,4-isomer, 2·00; 3,5-isomer, 3·04. DPA could not be separated from the 2,3- and 2,4-isomers in this solvent system, but could be separated from the 2,4-isosmer (tR 3·4 min) by using 1 mM ammonium acetate in 9% methanol (DPA tR 2·7 min).
Isolation of an acidic compound (lactic acid).
The culture broth after 72 h incubation (NG broth, 200 ml) was eluted through a Dowex 50W (100 ml) column and then through Dowex 1 (basic, 100 ml). The latter column was washed with 6 M ammonium hydroxide and the eluate was condensed. The precipitate thus obtained was dried and analysed in D2O by a DRX-500 NMR spectrometer (Bruker; 1H 500·13 MHz), using tetramethylsilane as an external standard. The lactic acid spectral character was [ 1H (
13C) p.p.m.]: 1·02 [3H, d, J=6·9 Hz, CH3 (19·74)], 3·88 [1H, q, J=6·9 Hz, CH (67·55)], (169·12 COO).
Electron microscopic studies.
Ultrastructural studies with a transmission electron microscope were performed at the Ultrastructure Research Laboratories, Yokohama, Japan. Cells were cultured on NG agar for 1 d at room temperature (20 °C) and pre-fixed in 2·5% (w/v) glutaraldehyde plus 0·15% ruthenium red in 0·1 M cacodylate buffer (pH 7·3) for 24 h at 6 °C. Epoxy resin was used for embedding. Thin sections (800 ) were prepared with a LKB U5 ultramicrotome (Amersham Pharmacia) and examined using a JEOL JEM-800a after staining with 10% (w/v) uranyl acetate.
Antibiotic sensitivity.
Antibiotic sensitivity was carried out on NG agar plates with antibiotic discs for the estimation of the dose [Showa Disc (Showa Yakuhin Kako) supplied by Nissuiseiyaku).
Cell wall analysis.
Preparation of cell walls for the detection of diaminopimelic acid (DAP) was carried out by the method of Schleifer & Kandler (1972) . The cell wall preparation was analysed by TLC for qualitative detection of DAP (Staneck & Roberts, 1974
).
DNA base composition.
Genomic DNA was extracted by treating the bacterial colonies collected from NG agar with guanidine thiocyanate/EDTA/Sarkosyl (Rademaker & de Bruijn, 1997 ). DNA was hydrolysed with nuclease P1 using a GC-kit (Seikagaku Kogyo). G+C content was estimated by HPLC using a Shimadzu LC-9A pump and an ODS AQ-312 column. The solvent used was 10 mM H3PO4+10 mM KH2PO4 (pH 3·5) at a flow rate of 1·5 ml min-1 and a Shimadzu SPD-6A detector set at 270 nm was used to monitor eluates.
PCR amplification and sequencing of 16S rRNA gene.
The 16S rRNA gene was amplified using the primer set from the MicroSeq 16S rRNA gene kit quick card (Perkin Elmer Applied Biosystems) and also by using the following primers: forward primer 5'-TCYGKTTGATCCYGSCRGAG-3' (Escherichia coli positions, 827) and reverse primer 5'-GGTTACCTTGTTACGACTT-3' (E. coli positions, 15101492) (Alm et al., 1996 ). The reaction mixture contained (100 µl): 10 pmol each primer, 250 µmol each dNTP, PCR buffer containing 15 mM MgCl2 and 1·25 U AmpliTaq Gold (Applied Biosystems). PCR was performed as follows: 95 °C for 9 min, followed by 35 cycles consisting of 95 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min. The PCR products were sequenced with an ABI 377 automatic sequencer (Applied Biosystems). Sequences were compared by using the BLAST program from the National Center for Biotechnology Information and the Ribosomal Database Project (RDP; Maidak et al., 1996
). The evolutionary distance tree and sequence similarities were calculated with the CLUSTAL W program and the TreeView package (Saitou & Nei, 1987
; Thompson et al., 1994
).
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RESULTS |
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Morphology and life cycle
A schematic life cycle of strain NAF001 is depicted in Fig. 1.
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These two different growth forms (i.e. the trichome via endospores and SLRCs leading to budding cells) were regularly observed as a mixture when strain NAF001 was incubated on NG agar for 1 month at room temperature.
Heat resistance of SLRCs and detection of DPA
To confirm the presence of endospores in strain NAF001, heat resistance and the presence of DPA were tested. Endospores of strain NAF001 survived heat treatment at 90 °C for 20 min, confirming that they were endospores. From the extract of bacteria at the stage where endospores began to form, DPA was detected [38 µg (g bacteria)-1]. SLRCs also survived by heat treatment at 80 °C for 10 min.
Physiological and biochemical characteristics
The optimum pH and temperature for the growth of strain NAF001 were standardized using minimal medium with glucose as sole source of carbon and energy. For all subsequent experiments, these optimum conditions were used. This strain was able grow well between 25 and 35 °C, but growth was poor below 20 °C. The strain was not able to grow well when the starting pH was 5·0 or below, but could tolerate pH up to 9·0. Under optimum conditions (30 °C, pH 7·0), the generation time was found to be 40 min. NAF001 did not grow under anaerobic conditions.
Strain NAF001 grew well in synthetic medium with glucose, fructose, galactose, maltose and glycerol, and moderately (poor growth) on rhamnose, -gluconolactone and xylose. No growth was observed on lactose, raffinose, arabinose and inositol. Moderate growth was observed in ethyl, butyl, propyl and isoamyl alcohols. Glucose and fructose were found to be the best carbon sources for growth.
NAF001 was resistant to polymyxin B but sensitive to tetracycline (MIC=0·05 µg ml-1), chloramphenicol (0·39 µg ml-1), streptomycin (0·39 µg ml-1), gentamycin (0·78 µg ml-1), benzyl penicillin (0·1 µg ml-1), erythromycin (0·39 µg ml-1), carbenicillin (12 µg ml-1), kanamycin (0·39 µg ml-1), lyncomycin (12·5 µg ml-1), bacitracin (0·05 µg ml-1) and ampicillin (0·78 µg ml-1).
The strain was oxidase-negative and weakly catalase-positive. Nitrate was reduced. Casein and Tween 80 hydrolysis were both negative, but urea (weak) and starch hydrolysis were positive. The VogesProskauer test was positive after incubation for 36 h. Polyhydroxybutyrate tested positive by staining. During growth in NG broth with shaking at 60 r.p.m., the pH was found to drop to 5·25·5 and no appreciable further growth was observed. This indicated the secretion of some acidic compounds into the growth medium. The acidic compound isolated was found by NMR analysis to be lactic acid.
The cell wall of strain NAF001 contained DAP and the G+C content of the DNA was determined by HPLC to be 37·2 mol%.
Phylogenetic analysis based on 16S rDNA homology
The sequence (1532 bp) of the 16S rDNA of strain NAF001 was determined. A phylogenetic tree (Fig. 5) and 16S rDNA similarity values (data not shown) show that this isolate is phylogenetically related to members of the Bacillaceae. The highest similarity values were for Bacillus megaterium and Bacillus cohnii (95·2% each) followed by Bacillus cereus, Bacillus simplex and Bacillus flexus (95·0% each); Bacillus halmapalus (94·9%), and Bacillus mycoides and Bacillus weihenstephanensis (94·8% each). There was no close relationship to various groups of filamentous bacteria for which the 16S rRNA sequences were available (similarity <86%).
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DISCUSSION |
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Morphological features and the presence of endospores initially led us to believe strain NAF001 to be Achroonema, an endospore-forming filamentous bacterium observed by Skuja (1974) . Based on the 16S rDNA sequence, strain NAF001 should be placed in the Bacillaceae, though this does not explain its budding growth pattern. Although budding growth is reported for certain bacterial species (Staley & Fuerst, 1989
), there is no such finding among Bacillus spp. Trick et al. (1984)
had reported the isolation of a filamentous Bacillus spp. from activated sludge and identified them as B. mycoides and B. cereus strains. Emtiazi et al. (1989)
also reported the isolation of endospore-forming filamentous iron bacteria from activated sludge, but did not place them in the Bacillus group. There was also a report on asporogenous division mutant strains of B. megaterium which formed a long filamentous trichome (Lach et al., 1990
). However, strain NAF001 is filamentous in its natural habitat and at no stage did we observe a non-filamentous growth form. Strain NAF001 also differed from Thermoactinomyces (Lacey & Cross, 1989
) due to a large difference in the G+C content of the DNA (37·2 mol% for NAF001 compared to 53 mol% for Thermoactinomyces), morphology and 16S rDNA sequence.
16S rDNA phylogenetic analysis showed no close relatives at the species level. Maximum sequence similarity (95·294·8%) was with members of rRNA homology group I (Ash et al., 1991 ) and also to B. cohnii (95·2%), an alkaliphile without meso-DAP in the cell wall (Spanka & Fritze, 1993
). Although strain NAF001 can grow at pH 9·0, its optimum growth pH is around 7·0 and its cell wall contained DAP. Differential morphological and biochemical characters of strain NAF001 are compared to phylogenetically close members of the genus Bacillus in Table 1
. Considering the unique life cycle and its 16S rDNA sequence, we could not assign strain NAF001 to any species group and propose it to be a novel species of the genus Bacillus. To the best of our knowledge, this is the first report of a Bacillus sp. that exhibits budding growth.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 October 2000;
revised 20 February 2001;
accepted 14 March 2001.
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