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1 Referat Geomikrobiologie, Bundesanstalt für Geowissenschaften und Rohstoffe, Stilleweg 2, D-30655 Hannover, Germany
2 Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Axel Schippers
A.Schippers{at}bgr.de
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; Bosecker et al., 1991; Rueter et al., 1994; Zengler et al., 1999; Rosenberg, 2000; Van Hamme et al., 2003). Here we describe the classification of two crude-oil-degrading, Gram-positive bacteria that showed characteristics of members of the genus Microbacterium. The genus Microbacterium comprises more than 30 physiologically versatile species isolated from various environments (Yokota et al., 1993; Takeuchi & Hatano, 1998a, b; Collins & Bradbury, 1999; Schumann et al., 1999; Behrendt et al., 2001; Zlamala et al., 2002; Laffineur et al., 2003).
The two strains were isolated from different oil-containing environments. Strain BAS69T was isolated from oil storage cavern 126 near Etzel, Germany (Bock et al., 1994). Strain BNP48T was isolated from an oil-contaminated soil in Germany. For enrichment, medium supplemented with 1–5 % crude oil as carbon source in Erlenmeyer flasks was inoculated and incubated on a rotary shaker at 30 °C in the dark for several weeks. For growth of strain BAS69T, we prepared an artificial sea water medium (Fedorak & Westlake, 1981) consisting of (l–1) 23·4 g NaCl, 0·75 g KCl, 7·0 g MgSO4.7H2O, 1·0 g NH4NO3, 0·7 g K2HPO4 and 0·3 g KH2PO4 (pH 7·3), and for strain BNP48T we used a salt-poor medium (BNP; modified from Jobson et al., 1972) consisting of (l–1) 2·0 g Na2SO4, 0·2 g MgSO4.7H2O, 2·0 g KNO3, 1·0 g NH4Cl, 0·5 g K2HPO4 and traces of FeSO4 (pH 7·3). For isolation via subculturing on agar plates different media without oil were used. Strain BAS69T was isolated on a basal medium (BAS) consisting of (l–1) 23·4 g NaCl, 0·75 g KCl, 7·0 g MgSO4.7H2O, 0·5 g peptone from meat, 0·5 g peptone from casein, 1·0 g yeast extract and 18 g agar (pH 7·3). Strain BNP48T was isolated on a modified BNP medium consisting of (l–1) 2·0 g Na2SO4, 0·2 g MgSO4.7H2O, 2·0 g KNO3, 1·0 g NH4Cl, 0·5 g K2HPO4, traces of FeSO4, 1·5 g sodium lactate, 1·0 g yeast extract and 18 g agar (pH 7·3).
Cell morphology, cell motility and the occurrence of spores were examined by phase-contrast light microscopy. Gram-staining and catalase testing was performed according to Burghardt (1992) and Gerhardt et al. (1994). Anaerobic growth was investigated by incubation in the presence and absence of oxygen using the Anaerocult system (Merck). Further physiological tests were carried out as described by Kämpfer et al. (1991). Briefly, use of various carbon sources as the only substrate and hydrolysis of various compounds were studied using a complex medium containing trace elements and vitamins in microplates. To confirm oil degradation, 40 ml medium (modified from Kämpfer et al., 1991) in 100 ml Erlenmeyer flasks was supplemented with 1 ml crude oil as the only carbon source, inoculated and incubated on a rotary shaker (120 r.p.m.) at 25 °C. Cell growth was checked after 3 weeks. The modified medium (pH 7·0) comprised (l–1): 1·0 g NaCl, 0·1 g MgSO4.7H2O, 1·0 g (NH4)2SO4, 3·2 g K2HPO4, 6·18 g Na2HPO4.2H2O, 0·17 g CaSO4.2H2O, 0·001 g H3BO3, 0·002 g CuSO4.5H2O, 0·003 g ZnI2.8H2O, 0·2 g FeSO4.7H2O, 0·002 g NiCl2.6H2O, 0·004 g CoCl2.6H2O, 0·01 g MnCl2.5H2O, 0·003 g Na2MoO4.2H2O, 0·5 g EDTA dihydrate and vitamins according to Kämpfer et al. (1991).
The occurrence of diaminopimelic acid in the cell wall and the peptidoglycan type were determined as described by Schleifer (1985) and Schleifer & Kandler (1972), using plates of cellulose for TLC. Menaquinones were extracted as described by Collins et al. (1977) and were analysed by HPLC according to the method of Groth et al. (1996). The acyl type of the peptidoglycan was determined as described by Uchida et al. (1999).
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and purification of PCR products were carried out as described by Rainey et al. (1996). Purified PCR products were sequenced with Taq Dyedeoxy terminator cycle sequencing kits (Applied Biosystems) according to the manufacturer's protocol. An Applied Biosystems 373A DNA sequencer was used for electrophoresis of the sequence reaction products. The ae2 editor (Maidak et al., 1999) was used to align the 16S rRNA gene sequences determined here against those of representatives of the main bacterial lineages available from the public databases. Pairwise evolutionary distances were computed using the correction of Jukes & Cantor (1969). The least-squares distance method of De Soete (1983) contained in the PHYLIP package (Felsenstein, 1993) was used in the construction of the phylogenetic dendrogram from distance matrices.
For DNA–DNA reassociation experiments, DNA was isolated using a French pressure cell (Thermo Spectronic) followed by purification by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA hybridization was carried out by the method described by De Ley et al. (1970), with the modifications described by Huß et al. (1983) and Escara & Hutton (1980), using a model 2600 spectrophotometer equipped with a model 2527-R thermoprogrammer and plotter (Gilford). Renaturation rates were computed with the TRANSFER.BAS program (Jahnke, 1992).
The almost complete 16S rRNA gene sequences of strains BAS69T and BNP48T, consisting of 1490 and 1495 nt, respectively, were compared to sequences of members of the genus Microbacterium, which were their closest phylogenetic neighbours (Fig. 1).
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For strain BNP48T, maximum pairwise similarity values of 99·0 % were obtained for M. luteolum DSM 20143T, 98·9 % for M. foliorum DSM 12966T and 98·8 % for M. oxydans DSM 20578T, M. maritypicum ATCC 19260T, M. phyllosphaerae DSM 13468T and M. saperdae DSM 20169T. Pairwise similarity values above 98 % were also found for M. liquefaciens DSM 20638T and M. paraoxydans DSM 15019T (98·6 % each), strain BAS69T (98·2 %), M. schleiferi DSM 20489T (98·2 %), M. testaceum DSM 20166T and M. keratanolyticum DSM 8606T (98·1 % each).
Based on these high pairwise 16S rRNA gene sequence similarity values, DNA–DNA relatedness of strains BAS69T and BNP48T to several closest phylogenetic neighbours was determined. Levels of DNA–DNA relatedness of strain BAS69T to M. saperdae, M. foliorum and M. phyllosphaerae were 18, 21 and 19 %, respectively. Those of strain BNP48T to M. maritypicum, M. oxydans, M. luteolum, M. foliorum and M. phyllosphaerae were 23, 16, 38, 36 and 11 %, respectively. DNA–DNA relatedness between the two novel strains was 41 %. These low values demonstrate that strains BAS69T and BNP48T each represent members of single novel species of the genus Microbacterium.
Morphological, physiological and chemotaxonomic characteristics of the two strains are different from those of phylogenetically closely related Microbacterium species (Table 1). The two strains were obligately aerobic, Gram-positive, irregular rods without spores. Cells of strain BAS69T were immotile, whereas those of strain BNP48T were motile. Cells were small, varying in size from 0·3 to 1·1 µm for strain BAS69T and from 0·3 to 1·5 µm for strain BNP48T. Colonies of both strains were circular, smooth, translucent and pigmented. Maximum colony diameters were 3 and 5 mm after 2 weeks of growth for strains BAS69T and BNP48T, respectively. Neither strain contained meso-diaminopimelic acid in the cell wall. The peptidoglycan type of strain BAS69T was B1
, [L-glu] D-glu(hyg)–gly1–2–L-Lys and that of strain BNP48T was B2
, [L-hsr] D-glu(hyg)–gly–D-Orn (Schleifer & Kandler, 1972). The major menaquinones were MK-11 and MK-12, with minor amounts of MK-9, MK-10 and MK-13 for both strains.
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Cells are obligate aerobic, Gram-positive, non-spore-forming, immotile, irregular rods 0·3–1·1 µm in size. Colonies are circular, smooth, translucent and orange pigmented with a maximum colony diameter of 3 mm after 2 weeks. Growth occurs at 30 and 37 °C and at 2 and 4 % NaCl. Catalase-positive and oxidase-negative. Crude oil is used as substrate. H2S is not produced, arginine is not hydrolysed, urease is not present and the Voges–Proskauer reaction is negative. Acid is produced from sucrose and xylose. Acid is not produced from D-glucose, rhamnose, adonitol, inositol or sorbitol. The following are utilized: L-arabinose, D-cellobiose, D-fructose, D-galactose, gluconate, D-glucose, D-maltose, D-mannose, 
-D-melibiose, L-rhamnose, D-ribose, D-sucrose, salicin, D-trehalose, L-xylose, D-mannitol, sorbitol, fumarate, DL-lactate, L-malate, pyruvate, L-aspartate, L-histidine, putrescine and 4-hydroxybenzoate. The following are not utilized: N-acetyl-D-glucosamine, -D-galacturonate, glycogen, adonitol, i-inositol, acetate, propionate, trans-aconitate, adipate, citrate, DL-3-hydroxybutyrate, suberate, L-alanine, L-hydroxyproline, L-proline, L-serine, 3-hydroxybenzoate, phenylacetate, N-acetyl-D-galactosamine and L-ornithine. The following are hydrolysed: aesculin, p-nitrophenyl (pNP) N-acetyl--D-galactosaminide, pNP N-acetyl--D-glucosaminide, pNP -L-arabinopyranoside, pNP -D-cellobioside, pNP -D-galactopyranoside, pNP -D-glucopyranoside, pNP -D-glucopyranoside, pNP -D-mannoside, pNP -D-maltoside, pNP -D-xyloside, bis-pNP phosphate, benzolphosphonacid-pNP ester, L-alanine p-nitroanilide (pNA), glycine pNA, L-leucine pNA, L-lysine pna and L-proline pNA. The following are not hydrolysed: pNP -D-glucuronide, pNP -D-lactoside, pNP phosphocholine, 2-deoxythymidine-5'-pNP phosphate, -L-glutamate pNA, L-glutamate--3-carboxy pna and L-valine pNA. The type strain does not contain meso-diaminopimelic acid in its cell wall and the peptidoglycan-type is B1, [L-glu] D-glu(hyg)–gly1–2–L-Lys. The major menaquinones are MK-11 and MK-12.
The type strain, BAS69T(=DSM 16091T=NCIMB 14003T), was isolated from oil storage cavern 126 near Etzel, Germany (Bock et al., 1994).
Description of Microbacterium hydrocarbonoxydans sp. nov.
Microbacterium hydrocarbonoxydans (hy'dro.car.bon.ox'y.dans. N.L. part. adj. hydrocarbonoxydans oxidizing hydrocarbons).
Cells are obligate aerobic, Gram-positive, non-spore-forming, motile, irregular rods 0·3–1·5 µm in size. Colonies are circular, smooth, translucent and yellow pigmented with a maximum colony diameter of 5 mm after 2 weeks. Growth occurs at 30 and 37 °C and at 2 and 4 % NaCl. Catalase-positive and oxidase-negative. Crude oil is used as substrate. H2S is not produced, arginine is not hydrolysed, urease is not present and the Voges–Proskauer reaction is negative. Acid is produced from rhamnose, sucrose and xylose. Acid is not produced from D-glucose, adonitol, inositol or sorbitol. The following are utilized: N-acetyl-D-glucosamine, L-arabinose, D-cellobiose, D-fructose, D-galactose, gluconate, D-glucose, glycogen, D-maltose, D-mannose, L-rhamnose, D-ribose, D-sucrose, salicin, D-trehalose, L-xylose, D-mannitol, acetate, propionate, citrate, fumarate, DL-lactate, L-malate, pyruvate, L-alanine, L-aspartate, L-histidine, L-hydroxyproline, L-proline, L-serine, putrescine, phenylacetate and N-acetyl-D-galactosamine. The following are not utilized: -D-galacturonate, -D-melibiose, adonitol, i-inositol, sorbitol, trans-aconitate, adipate, DL-3-hydroxybutyrate, suberate, 3-hydroxybenzoate, 4-hydroxybenzoate and L-ornithine. The following are hydrolysed: pNP N-acetyl--D-galactosaminide, pNP N-acetyl--D-glucosaminide, pNP -L-arabinopyranoside, pNP -D-cellopyranoside, pNP -D-galactopyranoside, pNP -D-glucopyranoside, pNP -D-glucopyranoside, pNP -D-lactoside, pNP -D-mannoside, pNP -D-maltoside, pNP -D-xyloside, bis-pNP phosphate, benzolphosphonacid-pNP ester, pNP phosphocholine, 2-deoxythymidine-5'-pNP phosphate, L-alanine pNA, glycine pNA, L-leucine pNA, L-lysine pna and L-proline pNA. The following are not hydrolysed: pNP -D-glucuronide, -L-glutamate pNA, L-glutamate--3-carboxy pna and L-valine pNA. The type strain does not contain meso-diaminopimelic acid in its cell wall and the peptidoglycan-type is B2, [L-hsr] D-glu(hyg)–gly–D-Orn. The major menaquinones are MK-11 and MK-12.
The type strain, BNP48T (=DSM 16089T=NCIMB 14002T), was isolated from an oil-contaminated soil in Germany.
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ACKNOWLEDGEMENTS |
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