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1 Ecological and Toxicological Laboratory, Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University (United Graduate School of Agriculture, Gifu University), 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan
2 UltraStructure Research Laboratories Ltd, Tochiku, Yokohama 224-0007, Japan
Correspondence
Ryozo Iriye
kn53278{at}gipmc.shinshu-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Published online ahead of print on 18 October 2002 as DOI 10.1099/ijs.0.02158-0.
The DDBJ accession number for the 16S rRNA gene sequence of Serratia marcescens subsp. sakuensis KREDT is AB061685.
Present address: Fujiclean Engineering Company Ltd, Chigusa, Nagoya 464, Japan. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, the DNA G+C content, production of red pigment and biochemical characteristics of the bacterium were not in agreement with those of the genus Bacillus, but instead resembled the characteristics of the genus Serratia. The genus Serratia belongs to the Enterobacteriaceae, and some members of this genus produce pigments identified as prodigiosin (Hearn et al., 1970; Gerber, 1975). Some members of the genus Serratia also have clinical importance (Grimont & Grimont, 1992; Brenner, 1984). Production of spores among Serratia spp. has never been observed, but the strain (KREDT) described in this study produced spores. Therefore, the spore-forming strain isolated from the wastewater treatment tank posed a difficult question regarding its taxonomic position. Polyphasic identification of the novel bacterium, including 16S rDNA sequence phylogeny and genetic relatedness to confirm the identity of the isolate, and evidence for spore formation by the bacterium are presented in this work. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) (actual residence time 24 h; typical quality of treated water: biological oxygen demand, 4·6 mg l-1; suspended solids, 4 mg l-1; total nitrogen, 7·5 mg l-1; total phosphate, 2·5 mg l-1). The gross yield coefficient of the sludge was below 8 %, and the concentration of mixed-liquor suspended solids (MLSS) in the treatment tank was high (5500 mg l-1). To elucidate the bacterial flora of the activated sludge, bacteria in the suspended liquor in the first aerobic tank were analysed by the dilution heterotrophic plate-count method. The suspended liquor was diluted with 0·5 % NaCl (1x104 dilution) and plated onto culture plates (NG agar, described below); the cultures were incubated for 24 h at 32 °C and then at room temperature for 6 days. Culture medium (NG broth) was composed of 8·0 g nutrient broth (CM-1; Oxoid), 8·0 g glucose, 5·0 g NaCl and 0·5 g yeast extract (Difco) in 1 l distilled water. Agar (15 g) was added to NG broth for plate culture (NG agar).
Cell morphology and characterization.
Tests for the utilization of various carbon compounds by the novel bacterium and other biochemical tests were carried out following standard methods (Smibert & Krieg, 1994). Cooked-meat hydrolysis was tested by the decrease in the amount of cooked meat (200 mg; Oxoid) present after incubation in 0·5 % NaCl (6 ml) at 32 °C for 10 days.
Heat-resistance test.
Seven-day- and 3-month-old cultures of strain KREDT on NG agar were subjected to a heat-resistance test. Each culture was suspended in 0·5 % NaCl (1 ml) or NG broth (5 ml) (OD660 0·05) and heated at 60, 62, 65, 70 and 75 °C for up to 20 min. Each culture heated in 0·5 % NaCl was shaken at 32 °C in NG broth (5 ml) or incubated on an NG agar slant at 32 °C. Each culture heated in NG broth was incubated at 32 °C with shaking or incubated on an NG agar slant at 32 °C. Serratia marcescens JCM 1239T (=ATCC 13880T) and Bacillus subtilis IFO 13719T (=ATCC 6051T) were similarly treated and used as controls.
Analysis of dipicolinic acid (DPA).
DPA was extracted from strain KREDT according to published methods (Janssen et al., 1958; Powell, 1953). Cells of strain KREDT (0·2 g wet weight) that had been cultivated for 1 week on NG agar plates and washed with water were heated in 2 ml water at 100 °C for 20 min. The suspension was cooled and centrifuged; the resulting supernatant was condensed to dryness. The residue was dissolved in 0·1 ml water and then filtered through a 0·45 µm filter disc. The filtrate (0·05 ml) was analysed using an LC-MS spectrometer [column AQ-312 (ODS 6x150 mm), YMC, Kyoto, Japan; solvent 10 mM CH3COONH4; flow rate 1 ml min-1]. Authentic DPA could be detected at retention time (tR) 3·45 min (m/z values of 168, 124 and 80) [LC-MS spectra were measured with a Hitachi M-1200AP LC-MS spectrometer; flow rate 1 ml min-1; atmospheric pressure chemical ionization (APCI) positive mode].
Scanning electron microscopy.
Electron micrographs of strain KREDT incubated on NG agar for 7 days at 20 °C (cells) and on NG agar for 18 days at 20 °C (spores) were taken at UltraStructure Research Laboratories; the cells were pre-fixed in 2 % glutaraldehyde and fixed with 1 % osmic acid. A Hitachi S-4500 scanning electron microscope was used.
Transmission electron microscopy.
A study of the ultrastructure of the endospores of strain KREDT was carried out at Ultrastructure Research Laboratories. Strain KREDT was cultivated on NG agar at 32 °C for 20 h and successively for 40 h at 20 °C (liberation of spores was observed with a phase-contrast microscope). Cells of strain KREDT were pre-fixed with 2 % glutaraldehyde in phosphate buffer and fixed with 1 % osmic acid (Fig. 1c) and successively stained with 4 % uranyl acetate (Fig. 1d). Epoxy resin was used for embedding the cells. Sections (800 Å) of the cells were prepared with an LKB U5 ultramicrotome (Amersham Pharmacia) and examined with a JEOL JEM 800a electron microscope.
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). An evolutionary distance tree and sequence similarities were calculated using the programs TREEVIEW and CLUSTAL W, respectively (Saitou & Nei, 1987; Thompson et al., 1994).
DNA–DNA homology.
DNA was extracted from bacteria by the method of Marmur (1961). DNA–DNA homology was determined by fluorometric hybridization in microdilution wells [Black Cliniplate; Labsystems, catalogue no. 95029120 (enhanced binding type)] according to the method of Ezaki et al. (1989), using biotinylated DNA. DNA–DNA hybridization was performed at 50 °C for 2 h in 2xSSC containing 45 % formamide. Fluorescence intensity was measured with a FP 3000 Fluorolite microplate reader (Shinseirika, Tokyo) at 360 nm for excitation and 450 nm for emission.
Assay for antibacterial sensitivity.
Antibacterial sensitivity profile of strain KREDT was determined on NG agar plates using antibacterial discs [Showa Disc (Showa Yakuhin Kako) supplied by Nissuiseiyaku, Tokyo].
Cellular fatty acid analysis.
Cells of strain KREDT that had been cultured on NG agar (4 days) were washed with distilled water. Wet cells (200 mg) were refluxed in 10 % KOH/65 % ethanol (5 ml) for 1 h under an N2 atmosphere. The reaction mixture was acidified with 1 M H2SO4 and fatty acids were extracted with ethyl acetate. The organic layer was washed with aqueous NaCl and then dried over MgSO4. After evaporation of solvents, the resulting fatty acid mixture in ether was methylated with diazomethane and analysed using a GC-MS spectrometer by EI positive mode. The tR values of the fatty acid methyl esters (FAMEs) were compared with those of reference bacterial FAMEs purchased from Supelco. The molecular formula of each peak was determined using high-resolution GC-MS analysis (Pizzimenti et al., 1999). Low- and high-resolution GC-MS spectra were measured by using a JEOL JMS-700 spectrometer with the JEOL data processing system and a Hewlett Packard 5890 gas chromatograph [column DB-1 (HP, non-polar); temperature 140–220 °C (6 °C min-1) and kept at 220 °C for 20 min; injection temperature 240 °C; flow rate 7·2 ml min-1 (He gas)].
Isoprenoid quinone analysis.
Cells of strain KREDT that had been cultured on NG agar (4 days) were washed with distilled water. Isoprenoid quinones were extracted from 500 mg wet cells with 150 ml acetone (three times) by stirring for 2 h each at room temperature. After centrifugation, the supernatant was condensed to dryness. The extracts dissolved in acetone were applied to preparative silica-gel TLC, and then developed with benzene. The isoprenoid quinones were detected under an UV lamp. The bands due to menaquinones and ubiquinones were collected and extracted with acetone. Each fraction was analysed using an LC-MS spectrometer [Column Zorbax-ODS (4·5x250 mm), solvent methanol/di-isopropyl ether (4 : 1, v/v), flow rate 1 ml min-1 (Collins & Jones, 1981; Hiraishi, 1999)]. LC-MS spectra were measured using a Hitachi M-1200AP LC-MS spectrometer (flow rate 1 ml min-1; APCI positive mode).
Pigment analysis.
Cells of strain KREDT that had been cultured on NG agar (2 days) were washed with distilled water. Pigments (66 mg) were extracted from 8 g wet cells with 150 ml ethyl acetate in a separating funnel. The organic layer was washed twice with saturated NaCl solution and then dried over MgSO4. After evaporation of the solvents, the extracts (66 mg) were separated by silica-gel TLC (benzene/acetone, 5 : 1). The red-coloured band (Rf 0·35–0·6) was collected and extracted with ethyl acetate/ethanol (5 : 1). After evaporation of the solvents, the pigment (4·3 mg) was dissolved in 5 ml ethyl acetate containing a few drops of 2 M HCl and shaken. The organic layer was washed with saturated NaCl solution and then dried over MgSO4. After evaporation of the solvent, the pigment was analysed by using a Bruker DRX 500 spectrometer with tetramethylsilane as an internal standard and Bruker's Pulse programme (1H at 500·13 MHz and 13C at 125·77 MHz). The red pigment was extracted from S. marcescens JCM 1239T using the same method as described above and analysed using an NMR spectrometer.
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RESULTS |
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) were rods (0·5–0·6x1·3–2 µm) that formed granules (generally 0·4–0·6 µm, but some measured 1·5–1·8 µm; Fig. 1b) identified as endospores (Fig. 1c, d). Colonies of strain KREDT were smooth and round, and on nutrient agar the strain produced a bright-red pigment. Because of endospore formation, some cells of strain KREDT were found to be swollen when first isolated, though such swollen cells could not be observed after successive transfers and maintenance. The biochemical characteristics of strain KREDT are given in Table 1, and they were found to be very similar to those of S. marcescens. Strain KREDT differed from the type strain of S. marcescens in terms of its methyl red test [negative on sucrose (5 days), D-sorbitol (7 days) and L-arabitol, whereas S. marcescens was positive on sucrose, D-sorbitol and L-arabitol (7 days)]. Strain KREDT did not produce acid from L-arabitol, whereas S. marcescens did.
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). Cells of strain KREDT had an outer membrane, a peptidoglycan layer and a cell membrane (not visible), all characteristics of Gram-negative bacteria (Fig. 1c). Inside the cells, granules with cortex layers were observed (Fig. 1c, a white ring), indicating that endospores were present. Although a spore coat and spore membrane could not be detected in the cells under the conditions tested, spore coats could be clearly observed from some endospores in which a cortex layer was covered with the peptidoglycan layer of the cell, by staining with uranyl acetate (Fig. 1d). The spore membrane of strain KREDT endospores could not be observed, and was presumed to be very thin. The structure of the endospores of strain KREDT was similar to that of an endospore of Bacillus megaterium (Ralph et al., 1992).
Heat resistance
Cultures of strain KREDT that had been incubated for 7 days and 3 months, respectively, survived heat treatment at 62 °C for 15 min in 0·5 % NaCl and 75 °C for 20 min in NG broth. B. subtilis IFO 13719T incubated for 10 days survived similar heat treatment, but S. marcescens JCM 1239T did not survive heat treatment at 60 °C for 15 min. Growth from cultures of strain KREDT that had been subjected to heat treatment could be observed after 24 h, whereas growth of the controls (not subjected to heat treatment) was observed after 12 h. The heat resistance of strain KREDT could be attributed to it forming spores.
DPA
DPA could be detected [10 µg (g bacteria)-1] by LC-MS analysis [tR 3·5 min; m/z 168 (M+H)+, 124 (M+H–CO2)+ and 80 (M+H–2CO2)+].
DNA G+C content
DNA G+C content (as determined by HPLC) of strain KREDT was 58 mol%, and agreed with the published values for S. marcescens (Grimont & Grimont, 1992; Brenner, 1984).
Phylogenetic analysis and DNA–DNA homology
An almost complete sequence (1532 bp) of the 16S rDNA of strain KREDT was determined (DDJB accession no. AB061685). A phylogenetic tree of related sequences based on BLAST sequence homology (Altschul et al., 1990) was constructed using the DDBJ data analysis server (CLUSTAL W) and TREECON software (Van de Peer & De Wachter, 1997); the tree is shown in Fig. 2. The 16S rDNA sequence of strain KREDT showed 99·6 % similarity with S. marcescens, 98·2 % with Serratia entomophila, 98·1 % with Serratia ficaria, 98·0 % with Serratia odorifera, 97·8 % with Serratia rubidaea, 97·4 % with Enterobacter cloacae, 97·4 % with Klebsiella pneumoniae and 97·4 % with Citrobacter freundii.
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Fatty acids
Whole-cell fatty acids of strain KREDT were detected as saturated fatty acids C12 : 0 (1·25 %), C15 : 0 (0·58 %), C16 : 0 (33·2 %), C17 : 0 (0·66 %) and C18 : 0 (1·93 %), mono-hydroxylated fatty acids C14 : 0 (2·83 %) and C16 : 0 (0·14 %), and unsaturated fatty acids C12 : 2 (2·94 %), C14 : 1 lpar;9·81 %), C17 : 1 (28·31 %), C18 : 2 (0·77 %) and C19 : 1 (19·94 %).
The profile of strain KREDT was similar to that of S. marcescens (Grimont & Grimont, 1992; Pizzimenti et al., 1999).
Isoprenoid quinones
Ubiquinone (Q-8; tR 10·8 min) was detected as the major respiratory quinone of strain KREDT, along with very small amounts of menaquinones [MK-8, MK-8(H2), MK-8(2H2); tR 16·1–16·3 min]. The presence of Q-8 as the major quinone in the type strain of S. marcescens has been reported previously (Grimont & Grimont, 1992; Collins & Jones, 1981; Hiraishi, 1999).
Antibacterial sensitivity
Strain KREDT was resistant to tetracycline, erythromycin, polymyxin, lyncomycin and benzylpencillin, but sensitive to ampicillin (MIC 50·0 µg ml-1), chloramphenicol (MIC 6·25 µg ml-1), gentamicin (MIC 6·25 µg ml-1), kanamycin (MIC 12·5 µg ml-1), streptomycin (MIC 50·0 µg ml-1) and carbenicillin (MIC 12·5 µg ml-1).
Pigment analysis
The pigment produced by strain KREDT was identified as prodigiosin by high-resolution MS (calculated for C20H25ON3, found 323.1998, measured 323.1990) and on the NMR spectra with the authentic sample extracted from the type strain of S. marcescens.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). From transmission electron microscopy analysis of strain KREDT, cells of the novel strain were found to possess an outer membrane, a peptidoglycan layer and a cell membrane (not visible), all characteristics of Gram-negative bacteria. The presence of endospores in cells of the novel strain could be confirmed due to the presence of a granule with a cortex layer and a spore coat formed inside cells of strain KREDT. The structure of the spores of strain KREDT was similar to that of spores of B. megaterium, while some endospores of strain KREDT were wrapped in the peptidoglycan layer of the cell. The spore membrane of strain KREDT was presumed to be very thin and it could not be observed under the conditions described above. The formation of endospores by strain KREDT was also confirmed by the results of the heat-resistance test and by the presence of DPA in the cells, although the temperature of heat resistance was lower than that of Bacillus spp. On the basis of the above results, cells of strain KREDT were shown to form endospores which possessed similar characteristics to spores of Bacillus spp. Strain KREDT was identified as S. marcescens on the basis of FAME analysis, quinone profiling, pigment analysis and DNA analysis. The 16S rDNA sequence of strain KREDT showed 99·6 % similarity with the 16S rDNA sequence of S. marcescens. The close relationship of the novel strain with S. marcescens was further confirmed by DNA–DNA homology analysis. Although DNA–DNA homology and 16S rDNA sequence analyses showed strain KREDT to be very similar to S. marcescens JCM 1239T, the unique spore-forming characteristic of strain KREDT supported its classification as a novel subspecies of S. marcescens, for which we propose the name Serratia marcescens subsp. sakuensis.
Endospore formation is known to be a characteristic of some Gram-positive bacteria, including those grouped in genera such as Acetonema, Bacillus, Clostridium, Desulfotomaculum, Sporomusa, Sporosarcina and Thermoactinomyces (Edward, 1997; Sneath, 1989). The work presented here is the first report of the isolation of a spore-forming bacteria belonging to the Enterobacteriaceae, and is a deviation from the general concept regarding endospore-forming bacteria. We propose that either (i) S. marcescens strains that possess the gene related to endospore formation may be present in nature but have been unreported until now or (ii) endospores were present in strain KREDT due to gene transfer. The latter explanation is more acceptable at this moment in time, since endospore-forming S. marcescens have not been found in nature and the gene related to spore formation has not been reported for S. marcescens. The similarity of the biochemical characteristics of strain KREDT to those of S. marcescens also support the hypothesis for gene transfer. The activated sludge from the wastewater treatment facility at Komaba, Japan, had abundant Bacillus spp. and high concentrations of magnesium and silicate present (22·5 mg Mg2+ l-1 and 31·9 mg silicate l-1 in pipe water, and 26·1 mg Mg2+ l-1 and 32·7 mg silicate l-1 in the supernatant of the first aerobic treatment tank). The possibility of gene transfer between Bacillus and Serratia spp. should be investigated in activated sludge containing high concentrations of Mg2+ and silicate ions.
Description of Serratia marcescens subsp. sakuensis subsp. nov.
Serratia marcescens subsp. sakuensis (sa.ku.en'sis. N.L. adj. saku referring to Saku, Nagano, Japan, where the strain was isolated).
Cells are Gram-negative, short rods (0·5x1·3 µm). Facultatively anaerobic. Motile. Forms round endospores. Colonies on nutrient agar produce the pigment prodigiosin. Catalase-positive and oxidase-negative. Hydrolyses casein, cooked meat and soybean oil, but not starch. Reduces nitrate to nitrite. Indole is not produced. Can grow in 7 % NaCl. Utilizes glucose, sucrose, D-sorbitol, L-arabitol, 4-hydroxybenzoic acid, gentisic acid and m-erythritol as sole carbon sources, but not D-tartrate, L-rhamnose, L-arabinose, D-xylose, lactose, benzoic acid, 3-hydroxybenzoic acid, trigonelline or raffinose. Produces acids from sucrose, D-glucose and D-sorbitol, but not from L-arabitol. Fermentative on glucose. Methyl red test is negative on sucrose (5 days), glucose (7 days), D-sorbitol (7 days) and L-arabitol. DNA G+C content is 58 mol%. Other characteristics of the species can be found in Table 1. The type strain of Serratia marcescens subsp. sakuensis is KREDT (JCM 11315T =CIP 107489T). Isolated from the suspended water of a domestic wastewater treatment tank in Komaba, Saku, Nagano, Japan.
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ACKNOWLEDGEMENTS |
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