Description of Sphingomonas Xenophaga Sp. Nov. for Strains BN6T and N,N Which Degrade Xenobiotic Aromatic Compounds
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International Journal of Systematic and Evolutionary Microbiology (2000), 50, 35–41 Printed in Great Britain Description of Sphingomonas xenophaga sp. nov. for strains BN6T and N,N which degrade xenobiotic aromatic compounds Andreas Stolz,1 Christian Schmidt-Maag,2† Ewald B. M. Denner,3 Hans-Ju$ rgen Busse,3,5 Thomas Egli2 and Peter Ka$ mpfer4 Author for correspondence: Andreas Stolz. Tel: 49 711 6855489. Fax: 49 711 6855725. e-mail: andreas.stolz!po.uni-stuttgart.de 1 Institut fu$ r Mikrobiologie, The taxonomic position of two bacterial strains, BN6T and N,N, with the ability Universita$ t Stuttgart, to degrade xenobiotic aromatic compounds (naphthalenesulfonates or N,N- Allmandring 31, 70569 Stuttgart, Germany dimethylaniline) was investigated. The 16S rRNA gene sequence, the GMC content of the DNA (62–63 mol%) and the detection of ubiquinone Q-10, 2- 2 Swiss Federal Institute for Water Resources and hydroxymyristic acid and the sphingoglycolipid present clearly placed the two Water Pollution Control, strains into the genus Sphingomonas. Both strains are representatives of one Swiss Federal Institute of species according to the level of DNA relatedness (70<7%). The strains could be Technology, 8600 Du$ bendorf, Switzerland separated from all validly described taxa of the genus Sphingomonas, according to the 16S rRNA gene sequence (the highest sequence similarity 3 Institut fu$ r Mikrobiologie und Genetik, Universita$ t observed was 96% to Sphingomonas yanoikuyae), the pattern of the polar Wien, Dr Bohr-Gasse 9, lipids and physiological characteristics. Therefore, the new species Sphingomonas A-1030 Vienna, Austria xenophaga is proposed to accommodate strains BN6T (¯ DSM 6383T) and 4 Institut fu$ r Angewandte N,N (¯ DSM 8566). Mikrobiologie, Justus- Liebig-Universita$ t Giessen, Senckenbergstr. 3, 35390 Giessen, Germany Keywords: Sphingomonas xenophaga sp. nov., taxonomy, degradation, naphthalenesulfonate, dimethylaniline 5 Institut fu$ r Bakteriologie, Mykologie und Hygiene, Veterina$ rmedizinische Universita$ t Wien, Veterina$ rplatz 1, A-1210 Vienna, Austria INTRODUCTION can be expected. Therefore, a reliable taxonomic description of known Sphingomonas strains is urgently The genus Sphingomonas is becoming increasingly needed. interesting because it contains various xenobiotic- T degrading bacterial isolates. Members of this genus are The bacterial isolate ‘Pseudomonas’ sp. BN6 has been able to degrade compounds such as biphenyl, naph- studied intensively because of its ability to degrade thalene and pyrene (Balkwill et al., 1997; Khan et al., naphthalenesulfonates. The strain was isolated from 1996; Ye et al., 1996), diphenylether and dibenzo-p- the River Elbe as a member of a bacterial consortium which completely degraded 6-aminonaphthalene-2- dioxin (Schmidt et al., 1992; Moore et al., 1993), T herbicides and pesticides (Feng et al., 1997; Nagata et sulfonate. It was found that strain BN6 oxidized a al., 1997; Nickel et al., 1997), polyethylene glycols wide range of (substituted) naphthalenesulfonates (Takeuchi et al., 1993) and chlorinated phenols to the corresponding (substituted) salicylates (Karlson et al., 1995; Nohynek et al., 1995, 1996). (No$ rtemann et al., 1986). Different enzymes of the Because of the high physiological versatility within degradative pathway for naphthalenesulfonates have members of the genus Sphingomonas, the isolation of been purified and characterized (No$ rtemann et al., new Sphingomonas strains with interesting properties 1994; Kuhm et al., 1991, 1993a, b). Different extradiol dioxygenases from this strain have also been studied ................................................................................................................................................. (Heiss et al., 1995, 1997; Riegert et al., 1998). † Present address: AB Biotechnologie, TU Hamburg-Harburg, 21071 T Hamburg, Germany. Furthermore, strain BN6 reduced the azo bond of Abbreviation: DMA, dimethylaniline. sulfonated azo dyes under anaerobic conditions and 01075 # 2000 IUMS 35 A. Stolz and others has been used in an anaerobic}aerobic process for the Physiological and biochemical characterization. The two mineralization of sulfonated azo compounds (Haug strains were characterized on the basis of 66 biochemical and et al., 1991; Keck et al., 1997; Kudlich et al., 1996, physiological characteristics as described previously 1997). (Ka$ mpfer et al., 1997). During a comparative study with different Sphingo- GC content. Isolation of genomic DNA and spectro- monas et al photometric determination of the GC content were carried strains, Nohynek . (1996) demonstrated out according to Auling et al. (1986). that strain BN6T belongs to the genus Sphingomonas, according to its 16S rRNA gene sequence. They DNA reassociation experiments. Genomic DNA was isolated suggested that strain BN6T represents a yet un- by chromatography on hydroxyapatite by the procedure of described species within the genus Sphingomonas.A Cashion et al. (1977). DNA–DNA hybridization studies T were carried out by using the thermal renaturation method taxonomic description of strain BN6 was hampered (Yassin et al., 1993). by the fact that only this single isolate of the presumed new species was available. Schmidt (1994) isolated another Sphingomonas strain (designated N,N), after RESULTS enrichment with N,N-dimethylaniline (N,N-DMA) as Morphological and cultural characteristics sole source of carbon and energy, which resembled T strain BN6T in many aspects. Therefore, we compared Cells of strains BN6 and N,N were aerobic Gram- these strains and suggest that strain BN6T and strain negative, non-spore-forming rods (1±3–4±0 µmin N,N belong to a new species of the genus Sphingo- length and 0±7–1±2 µm in diameter). Both strains monas for which we propose the name Sphingomonas formed intense yellow colonies on solid medium. No xenophaga sp. nov. growth was observed at 4 or 42 mC on agar plates. After reaching the stationary growth phase in liquid METHODS cultures, both strains rapidly lost the ability to form colonies on solid medium. Bacterial strains. Strain BN6T was isolated from the River Elbe as a member of a 6-aminonaphthalene-2-sulfonate- degrading mixed bacterial culture (No$ rtemann et al., 1986). DNA reassociation It has been deposited at the Deutsche Sammlung von According to the 16S rDNA gene sequence, strain Mikroorganismen und Zellkulturen (DSMZ), Braun- T schweig, Germany, as DSM 6383T. Strain N,N was BN6 showed the highest degree of similarity to obtained from a mixed sample from wastewater treatment Sphingomonas yanoikuyae (96% similarity) (Nohynek et al., 1996). Therefore, DNA reassociation studies plants, contaminated soil, river sediments and rotten wood T after a continuous enrichment with a simultaneous supply of were performed with strains BN6 , N,N and S. " T 2,4-, 2,5- and 2,6-DMA and N,N-DMA (50 mg l− each). yanoikuyae DSM 7462 . The level of DNA relatedness T The strain was isolated from the enrichment culture after between strains BN6 and N,N was 70±7%. In con- plating on agar plates with N,N-DMA as sole source of trast, the level of DNA relatedness between strain T T carbon, nitrogen and energy. Repeated transfers to liquid BN6 and S. yanoikuyae DSM 7462 was only 40±0%. cultures with N,N-DMA and solid complex media resulted These results suggested that strains BN6T and N,N are in a pure culture of strain N,N (Schmidt, 1994). It has been indeed members of the same species but do not belong deposited as strain DSM 8566. to the species S. yanoikuyae. Cellular fatty acids. Fatty acid methyl esters were extracted and prepared by the standard protocol of the Microbial Identification System (MIDI; Microbial ID). Extracts were GC content of DNA analysed by using a Hewlett Packard model HP6890A gas The GC contents of the genomic DNA from strains chromatograph equipped with a flame-ionization detector, T an automatic sampler, an integrator and a computer, as BN6 and N,N were determined to be 62±1³0±2 and described previously (Ka$ mpfer & Kroppenstedt, 1996). 63±3³0±3% mol%, respectively. These values fall within the range observed for members of the genus Polyamines. Polyamines were extracted and analysed ac- cording to Busse & Auling (1988). Sphingomonas (Yabuuchi et al., 1990). Polar lipids and quinones. The quinone system and polar lipids were determined by TLC as described previously Physiological and biochemical characteristics (Tindall, 1990; Auling et al., 1993). The results obtained from further tests demonstrated Extraction and analysis of the pigment. Lyophilized cells that strains BN6T and N,N reacted very similarly to (about 0±5 g) were stirred for 3 h at 4 mC in acetone (5 ml). each other but showed characteristics different from S. Cells were removed by centrifugation and the dissolved T pigment in the supernatant was analysed by UV}visible yanoikuyae. Strains BN6 and N,N did not produce spectroscopy (IMA Gilford Analysentechnik). Spectra were acids from various sugars and sugar alcohols tested recorded between 350 and 520 nm against acetone as under aerobic conditions. Only a limited number of reference. carbohydrates as well as simple organic acids were utilized as sources of carbon and energy (Table 1). Morphology. Gram reaction was tested as described by T Gerhardt et al. (1994). Cell morphologies were observed Distinguishing results between strains BN6 and N,N under a light microscope (1000¬) using cells grown for 3 d were observed in respect to hydrolysis of p- at 30 mC on Nutrient Agar. nitrophenyl-β--xylopyranoside,