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1 Department of Ecology, Ecotoxicology and Ecochemistry, Aachen University, Worringerweg 1, D-52056 Aachen, Germany
2 Unit of Applied Microbiology, Department of Microbiology and Genetics, Aachen University, Worringerweg 1, D-52056 Aachen, Germany
3 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Reiner M. Kroppenstedt
kdt{at}dsmz.de
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Published online ahead of print on 30 July 2004 as DOI 10.1099/ijs.0.03003-0.
The GenBank/EMBL/DDBJ accession number of the 16S rRNA gene sequence of Mycobacterium pyrenivorans 17A3T is AJ431371.
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), is the taxon that harbours most xenobiotic degraders among Gram-positive bacteria. Namely, species of the genera Gordonia, Rhodococcus and Mycobacterium are well known for their capacity to degrade environmentally hazardous chemicals (Bell et al., 1998; Williumsen & Karlson, 1997). In this paper, we describe a novel pyrene-degrading, rapidly growing isolate that represents a novel species of the genus Mycobacterium.
Strain 17A3T was isolated from an enrichment culture obtained from soil that was highly contaminated with polycyclic aromatic hydrocarbons (PAHs). The soil sample was collected on the site of a former coking plant at Übach-Palenberg, Germany. An enrichment culture of indigenous bacteria was extracted from soil with 0·2 % tetrasodium pyrophosphate and was cultivated in mineral medium amended with phenanthrene, anthracene, pyrene, fluoranthene and benzo[a]pyrene (100 mg l–1 each) as sole sources of carbon and energy (Schwiening, 1999). 17A3T was purified by alternately streaking dilutions of the sample on R2A agar plates and cultivating isolated colonies in mineral medium with PAHs. Purity was confirmed by plating on R2A agar and the recovered single isolate was maintained by repeated transfer to mineral medium with PAH or pyrene (50 mg l–1) and R2A agar plates.
Gram staining, acid–alcohol-fastness, colony morphology, the ability to grow at various temperatures, pigment production and photoreactivity were determined after 2 weeks of growth on R2A agar plates at 28–30 °C using the methods described by Vincent Lévy-Frébault & Portaels (1992) and Wayne et al. (1974). The catalase test was performed as described by Kubica & Pool (1960). Nitrate reductase and Tween 80 hydrolysis were detected as described by Wayne et al. (1974) and by BBL Taxo nitrite test strips. The protocols used to determine growth on xylose, trehalose, mannitol and sorbitol as sole sources of carbon were those of Silcox et al. (1981). The type strains of the closely related species Mycobacterium austroafricanum (DSM 44191T) (Tsukamura et al., 1983), Mycobacterium aurum (DSM 43999T) (Tsukamura, 1966) and Mycobacterium vaccae (DSM 43292T) (Bönike & Juhasz, 1964) were tested under identical conditions. Mycobacterium vanbaalenii DSM 7251T (Khan et al., 2002) could not be included in the tests because it was described after performing the experiments.
The capacity of strain 17A3T to degrade different PAHs in liquid batch experiments was tested over a period of 8 weeks by using mineral medium amended with phenanthrene, anthracene, fluoranthene, pyrene and benzo[a]pyrene (20 mg l–1 each). Remaining PAHs were extracted four times with ethyl acetate by vigorous mixing on a stirring plate. Extracts were analysed by GC, which was performed on a Hewlett Packard 5890 Series II gas chromatograph equipped with a flame-ionization detector. A fused silica column (HP-5, cross-linked 5 % PhMe-Silicone, 30x0·25 mm, 0·25 µm film thickness) with an uncoated deactivated precolumn (Hewlett Packard; Retention Gap, 5 mx0·32 mm) was used. GC conditions were: injector temperature 270 °C; detector temperature 285 °C; the column temperature program was initial temperature 40 °C for 4 min followed by a linear gradient of 10 °C min–1 to a final temperature of 280 °C, which was maintained for 15 min.
The strain was grown for fatty acid and mycolic acid analysis (HPLC) for 7 days at 35 °C in Petri dishes on Middlebrook 7H10 medium supplemented with glycerol and OADC (DSMZ medium 645; DSMZ, 2001). Three to four inoculation loops of cell material were scraped from the plates and used directly for the analyses. TLC analyses of mycolic acids were performed with whole-cell methanolysates from freeze-dried cells as described previously (Springer et al., 1995).
Fatty acids were analysed as methyl esters obtained from cells after saponification, methylation and extraction by GC as described previously (Sasser, 1990; Schröder et al., 1997). For mycolic acid analyses by HPLC, the cells were saponified by KOH and transferred to their bromophenacyl esters as described previously by Butler et al. (1996) and Miller (1997). Mycolic acid bromophenacyl esters were analysed by HPLC operated by the Sherlock System software (MIDI Inc.).
rRNA gene internal spacer analyses (RISA) were carried out with strain 17A3T and M. vanbaalenii DSM 7251T, M. aurum DSM 43999T and M. austroafricanum DSM 44191T as references using methods described previously (Garcia-Martinez et al., 1999; Gürtler & Stanisich, 1996; Jensen et al., 1993; Ranjard et al., 2000).
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and purification of PCR products were carried out as described previously (Rainey et al., 1996). The ae2 editor (Maidak et al., 1999) was used to align the almost complete 16S rRNA gene sequence of strain 17A3T (1451 nt) against the 16S rRNA gene sequences of type strains of the genus Mycobacterium. Phylogenetic analyses (De Soete, 1983; Felsenstein, 1993) followed described methods.
Cells of strain 17A3T were Gram-positive and acid-fast. The scotochromogenic, rough colonies were yellow, which intensified after exposure to light. In liquid media, the cells clumped together or showed biofilm formation on glass or PAH crystal surfaces. This is probably due to their hydrophobic cell-wall surface. On TSB (trypticase soy broth) agar plates, growth appeared within 7 days at 35 °C. In the semi-quantitative catalase test, the foam height was >45 mm. The nitrate reduction test showed a weak positive reaction. Strain 17A3T was not able to hydrolyse Tween 80 within 10 days. Mannitol could be used for growth. Xylose, trehalose and sorbitol could not be assimilated. The results of physiological tests are listed in Table 1.
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Separation of whole-organism acid methanolysates by TLC developed in two dimensions produced a multispot pattern composed of alpha-, epoxy and omega-dicarboxy mycolates plus a spot of alcohols (wax-ester mycolates). This pattern was shared by M. aurum, M. austroafricanum, M. vaccae and a few other mycobacteria (Vincent Lévy-Frébault & Portaels, 1992; Häggblom et al., 1994; Hinrikson & Pfyffer, 1994) (data not shown).
Analyses of mycolic acids by HPLC revealed a characteristic UV-HPLC chromatogram of Mycobacterium species that have separated, triple-peak clusters with prominent peaks in the early cluster that emerge prior to 5·0 min. Strain 17A3T shares this pattern with its relatives M. aurum, M. austroafricanum, M. vaccae (Butler & Guthertz, 2001) and M. vanbaalenii (Khan et al., 2002). Strain 17A3T can easily be separated from these species by quantitative differences (Table 2).
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). Taking the qualitative and quantitative composition of the fatty acids into consideration, this pattern resembles those of M. aurum/M. austroafricanum. The fatty acid pattern of strain 17A3T was identified by the Microbial Identification System (MIDI Inc.) as M. aurum but with only a low correlation.
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; Garcia-Martinez et al., 1999). These spacers can be easily amplified by means of universal PCR primers with similarity to the conserved flanking sequences. RISA can be used for both molecular characterization of a bacterial species and ecological studies of the diversity of prokaryotic communities (e.g. Ranjard et al., 2000; Sigler et al., 2002). Length polymorphisms occur to a minor degree within a species. The diversity of spacer length is greater if different species or genera are compared, although distantly related species may produce the same RISA pattern (Sonderkamp et al., 2001). Fig. 1 shows the patterns of the intergenic rRNA gene spacer amplificates from strain 17A3T, M. vanbaalenii DSM 7251T, M. aurum DSM 43999T and M. austroafricanum DSM 44191T.
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; distance-matrix tree). The presence of clear differences in genomic (RISA, 16S rRNA gene sequence), chemotaxonomic (mycolic acids, fatty acids) and physiological properties in the five members of the M. aurum sublineage of the genus Mycobacterium and the finding that 16S rRNA gene similarity values determined for these five type strains are similar or even lower than those found for several other pairs of type strains of Mycobacterium species (e.g. M. aurum DSM 43999T and M. vanbaalenii DSM 7251T, 98·7 %; M. vaccae DSM 43292T and M. vanbaalenii DSM 7251T, 99·3 %; or Mycobacterium fortuitum DSM 46621T and Mycobacterium senegalense DSM 43656T, 99·4 %) justifies the description of a novel species for strain 17A3T, for which the name Mycobacterium pyrenivorans sp. nov. is proposed.
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Cells are strictly aerobic, Gram-positive, acid-fast rods. The rough colonies show a yellow colour, which intensifies after exposure to light. In liquid media, the cells clump together or show biofilm formation on glass. On TSA, growth appears within 7 days at 35 °C. Cells grow well between 24 and 37 °C but not at 42 °C. Classified as a scotochromogenic, rapidly growing mycobacterium. Catalase-positive. Nitrate reduction test shows a weak reaction. Does not hydrolyse Tween 80 within 10 days. Mineralizes phenanthrene, fluoranthene and pyrene but not anthracene or benzo[a]pyrene. Can use mannitol as a sole source of carbon but can not grow on xylose, trehalose or sorbitol. The fatty acid pattern from whole-cell methanolysates is composed of tetradecanoic acid (3 %), cis-7-hexadecenoic acid (3 %), palmitic acid (25 %), oleic acid (31 %), stearic acid (3 %), tuberculostearic acid (8 %) and eicosanoic acid (1 %). Significant amounts of alcohols 2-octadecanol (13 %) and eicosanol (6 %) are also present. TLC of mycolic acid methanolysates reveals alpha-mycolates, epoxy-mycolates and omega-carboxymycolates plus 2-eicosanol (wax-ester mycolates). The mycolic acid HPLC elution profile is unique and can be used for differentiation from the closely related species M. aurum, M. austroafricanum, M. vaccae and M. vanbaalenii and all other mycobacteria.
The type strain, 17A3T (=DSM 44605T=NRRL B-24244T), was isolated from soil of a former coking plant at Übach-Palenberg, Germany.
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ACKNOWLEDGEMENTS |
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