International Journal of Systematic and Evolutionary Microbiology (2000), 50, 35–41 Printed in Great Britain

Description of 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: j49 711 6855489. Fax: j49 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 Control, strains into the genus Sphingomonas. Both strains are representatives of one Swiss Federal Institute of species according to the level of DNA relatedness (707%). 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 (l DSM 6383T) and 4 Institut fu$ r Angewandte N,N (l DSM 8566). Mikrobiologie, Justus- Liebig-Universita$ t Giessen, Senckenbergstr. 3, 35390 Giessen, Germany Keywords: Sphingomonas xenophaga sp. nov., , 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 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- GjC content. Isolation of genomic DNA and spectro- monas et al photometric determination of the GjC 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 (1n3–4n0 µmin N,N belong to a new species of the genus Sphingo- length and 0n7–1n2 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 70n7%. 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 40n0%. 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 GjC content of DNA analysed by using a Hewlett Packard model HP6890A gas The GjC 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 62n1p0n2 and described previously (Ka$ mpfer & Kroppenstedt, 1996). 63n3p0n3% 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 0n5 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 (1000i) using cells grown for 3 d were observed in respect to hydrolysis of p- at 30 mC on Nutrient Agar. nitrophenyl-β--xylopyranoside, assimilation of -

36 International Journal of Systematic and Evolutionary Microbiology 50 Sphingomonas xenophaga sp. nov.

Table 1. Physiological characteristics of different Sphingomonas species which are characterized by spermidine as the predominant compound in the polyamine pattern ...... Test results were read after 72 h incubation at 30 mC. The following strains were investigated: S. macrogoltabidus IFO 15033T; S. terrae IFO 15098T; S. chlorophenolica ATCC 33790T; S. yanoikuyae DSM 7462T; S. rosa IFO 15208T; S. capsulata IFO 12533T; S. stygia SMCC B0712T; S. subterranea SMCC B0478T; S. aromaticivorans SMCC F199T; S. herbicidovorans DSM 11019T; S. subarctica KF1 HAMBI 2110T (SMCC Subsurface Microbial Culture Collection, Florida State University, FL, USA; IFO Institute of Fermentation, Osaka, Japan). Data for S. macrogoltabidus, S. terrae, S. chlorophenolica, S. yanoikuyae, S. rosa, and S. capsulata were taken from Ka$ mpfer et al. (1997). Only those substrates which gave a uniform reaction for strains BN6T and N,N are reported. All strains hydrolysed -alanine-p-nitroanilide but none of the strains grew with adonitol, i-inositol, -mannitol, -sorbitol, putrescine, trans-aconitate, 4-aminobutyrate, itaconate, mesaconate, oxoglutarate, β-alanine, -ornithine, -serine, 3-hydroxybenzoate or phenylacetate. Abbreviations: xenop, xenophaga; macro, macrogoltabidus; chloro, chlorophenolica; yanoi, yanoikuyae; capsu, capsulata; subter, subterranea; aroma, aromaticivorans; herbic, herbicidovorans; subarc, subarctica; pNP, p-nitrophenyl; pNA, p-nitroanilide. Symbols: j, Positive; k, negative; (j) weakly positive. Data are in accordance with those published by: a, Yabuuchi et al. (1990); b, Takeuchi et al. (1993); c, Takeuchi et al. (1994); d, Balkwill et al. (1997); e; Nohynek et al. (1996).

Test Sphingomonas

xenop macro terrae chloro yanoi rosa capsu stygia subter aroma herbic subarc

Hydrolysis of: Aesculin jjb kkja (j) kjd jd jd jje pNP-β--Galactopyranoside (j) jkk(j)a jc ja k (j) jkk pNP-β--Glucuronide kkkkjjjkjjkk pNP-α--Glucopyranoside jkjkja jja jjjjj pNP-β--Glucopyranoside jjkkja jja jjjjj bis(pNP) Phosphate jjjjjjj(j)(j) jkj pNP-Phenylphosphonate jjjjjjjkjjkj pNP-Phosphorylcholine jjjkjjjkk(j) kk 2-Deoxythymidine-5h-pNP-phosphate jjjjjjjjjjkj -Glutamate-γ-3-carboxy-pNA jjjjjkjkjjjj -Proline-pNA kjjjkkkkkjkk Assimilation of: N-Acetyl--glucosamine kkkkja (j)c ja kkjkj -Arabinose jkkkja jc (j)a kd jd kd kje -Fructose kkb kb kkjc kkkkkke -Galactose kkb kkja kjkkkkje -Glucose jjkjjc jc jkjjjj -Mannose kkb kb k (k)a jc (j)a kjjkk -Maltose jk(k) kja jc ja kjjkje α--Melibiose kkkkjc jc kkjkkk -Rhamnose kkkkjc jc jkjjjje -Ribose kkb kb jka kka kkkkk Sucrose jkkkjc jc jkjjkk Maltitol kkkkjkkkkkkk Acetate (j) jkjjc kkkkjjje Azelate kjkkkkkkkjkk Citrate kkkkjc kc kc kkkkke Fumarate jjb jjjc jjkkkjj Glutarate kkkkkkkkkjkk -3-Hydroxybutyrate jjjjjkkkjjjje -Lactate kkkkjc kc kkkkkje Pyruvate kkkjjkkkjjjj Suberate kjkjkjkk(j) jkk -Aspartate kjkkjkjkkjjje -Histidine kjkkjjjkkkkj -Leucine kjkjjkjkkjkje -Phenylalanine kjjkjkkkkkkk -Tryptophan kjkkkkkkkkkk

International Journal of Systematic and Evolutionary Microbiology 50 37 A. Stolz and others

Table 2. Major fatty acid composition of different Sphingomonas species showing spermidine as the major compound in the polyamine pattern ...... The following strains were investigated: S. xenophaga BN6T (DSM 6383T) and N,N (DSM 8566); S. macrogoltabidus IFO 15033T; S. terrae IFO 15098T; S. chlorophenolica ATCC 33790T; S. yanoikuyae DSM 7462T; S. rosa IFO 15208T; S. capsulata IFO 12533T; S. stygia SMCC B0712T; S. subterranea SMCC B0478T; S. aromaticivorans SMCC F199T; S. herbicidovorans DSM 11019T; S. subarctica KF1 HAMBI 2110T. Data for S. macrogoltabidus, S. terrae, S. chlorophenolica, S. yanoikuyae, S. rosa and S. capsulata are taken from Ka$ mpfer et al. (1997). Abbreviations are defined in the legend to Table 1.

Fatty acid* xenop macro terrae chloro yanoi rosa capsu stygia subter aroma herbic subarc

BN6T N,N

14:0 kkkkk0n51n5 kk0n90n5 kk 12:0 2-OH kkkkkkkk0n6 k 1n0 kk 13:0 2-OH kkkkkkkk0n6 k 2n6 kk 14:0 2-OH 6n710n13n00n89n47n112n814n714n913n016n85n57n6 Summed feature 4 23n927n431n88n09n616n322n86n723n120n120n013n412n6 16:0 8n09n312n84n59n511n210n98n12n54n72n47n410n2 Summed feature 7 55n247n241n213n460n156n052n064n849n348n237n768n361n1 17:1ω6c 2n53n04n548n16n43n2 kk4n16n410n21n73n3 18:1ω5c 1n61n50n90n62n52n1 kk0n8 kk2n0 k 17:0 kkk3n8 kkkkkkkkk 16:1ω5c 2n02n02n92n12n42n7 k 1n70n6 k 0n71n62n9 16:0 2-OH k 1n02n9 kk0n9 kkkkkk2n1 15:0 kkk5n9 kkkkkk0n9 kk 17:1ω8c kkk7n9 kkkk0n81n02n6 kk 15:0 2-OH kkk4n9 kkkk2n53n34n5 kk 18:0 kkkkkkkkk3n2 kkk * The position of the double bond in unsaturated fatty acids can be located by counting from the methyl (ω) end of the carbon chain. Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system. Summed feature 4 contained one or more of the following fatty acids: 16:1ω7c and 15:0 iso 2-OH. Summed feature 7 contained one or more of the following isomers: 18:1ω7c,18:1ω9t and\or 18:1ω12t (cis and trans isomers are indicated by the suffixes c and t, respectively). cellobiose, -mannose, 4-aminobutyrate, -alanine, - Quinones and polyamines aspartate, -leucine and 4-hydroxybenzoate. The extracted isoprenoid quinones from both strains Where possible, a detailed comparison was made with gave one characteristic spot by HPLC analysis which all previously published data (Takeuchi et al., 1993, corresponded to ubiquinone Q-10 upon comparison 1994; Yabuuchi et al., 1990; Balkwill et al., with authentic standards. In both strains spermidine −" 1997; Nohynek et al., 1996). Table 1 shows that the was the major polyamine [31n7–38n3 µmol (g dry wt) ]. majority of test results which could be compared In strain BN6T small amounts of spermine [3 5 µmol (g " n directly with data from previously published papers dry wt)− ] were detected. The combination of these two were confirmed. Table 1 shows also that the new features is in accordance with the characteristics of species proposed in this paper, Sphingomonas other species of the genus Sphingomonas such as xenophaga sp. nov., can be differentiated from all other Sphingomonas capsulata and S. yanoikuyae (Segers et Sphingomonas species on the basis of several tests. al., 1994). Additional physiological and biochemical charac- S xenophaga teristics of . are given below. Whole-cell fatty acid composition and polar lipid pattern from strains BN6T and N,N Characterization of the yellow pigment Strains BN6T and N,N contained the fatty acids 16:0, Strains BN6T and N,N formed bright yellow colonies summed feature 7 (18:1ω7c, 18:1ω9t and\or on agar plates. The yellow pigment of ‘Pseudomonas 18:1ω12t), 14:0 2-OH and summed feature 4 (16:1ω7c paucimobilis’ has been identified as the carotenoid and\or 15:0 iso 2-OH) (Table 2), as found in other nostoxanthin [(2R, 3R, 2hR, 3hR)-β,β-carotene- Sphingomonas species (Takeuchi et al., 1993, 2,3,2h,3h-tetrol] (Jenkins et al., 1979). The extracted 1994; Yabuuchi et al., 1990; Ka$ mpfer et al., 1997). 3- pigments of strains BN6T and N,N showed the same Hydroxy acids were not detected. Table 2 shows that spectral characteristics in acetone (λmax l 453 and the type strains of Sphingomonas species containing 480 nm) as nostoxanthin. spermidine as the major polyamine exhibit hetero-

38 International Journal of Systematic and Evolutionary Microbiology 50 Sphingomonas xenophaga sp. nov.

and N,N are closely related to each other and should be considered as different strains of the same species. P Nevertheless, strain BN6T is not able to grow with N,N-DMA and strain N,N could not oxidize naphthalenesulfonates. It was recently shown that the L ability to degrade naphthalenesulfonates is a plasmid- encoded trait in strain BN6T (J. Klein, personal PL3 communication). Therefore, these degradative abilities presumably do not have importance for the taxonomic PL4 DPG classification of these strains. PME The 16S rDNA gene sequence, the GjC content of the DNA, the detection of ubiquinone Q-10, 2- PE PG hydroxymyristic acid in the fatty acid profile and the sphingoglycolipid in the polar lipid pattern clearly PDE T SGL place strains BN6 and N,N into the genus Sphingo- monas as it is currently defined (Yabuuchi et al., 1990). PC The 16S rDNA gene sequence placed strain BN6T in the neighbourhood of S. yanoikuyae, Sphingomonas chlorophenolica and ‘S. paucimobilis EPA505’. Ac- cording to the 16S rRNA gene sequence comparison, a

...... previously described strain (C7) with the ability to Fig. 1. Polar lipids of Sphingomonas sp. BN6T after separation decolorize sulfonated azo compounds under aerobic conditions shared the highest sequence similarity by two-dimensional TLC. PE, phosphatidylethanolamine; T PME, phosphatidylmonomethylethanolamine; PG, phosphati- (99%) with strain BN6 (Govindaswami et al., 1993). dylglycerol; DPG, diphosphatidylglycerol; PDE, phosphatidyl- Unfortunately, we could not obtain strain C7 and dimethylethanolamine; PC, phosphatidylcholine; SGL, sphingo- therefore it was not included in this study. The glycolipid; PL3 and PL4, unidentified phospholipids; L, T unidentified polar lipid; P, pigment. allocation of strain BN6 in close proximity to S. yanoikuyae was also indicated by the presence of spermidine as the major polyamine. The members of the genus Sphingomonas can be divided into two major geneity in their fatty acid profiles, which correlates groups on the basis of their polyamine pattern: with the heterogeneity found in the polar lipid profiles. spermidine is the main compound in Sphingomonas However, the presence of 2-hydroxymyristic acid (14:0 aromaticivorans, S. capsulata, S. chlorophenolica, 2-OH) as the dominant hydroxylated acid and the Sphingomonas herbicidovorans, Sphingomonas macro- absence of 3-hydroxylated fatty acids are consistent goltabidus, Sphingomonas rosa, Sphingomonas stygia, with the description of the genus Sphingomonas and Sphingomonas subterranea, Sphingomonas terrae, S. confirm the results of other studies (Takeuchi et al., yanoikuyae,‘S. paucimobilis EPA505’ and three so- 1993, 1994; Yabuuchi et al., 1990; Ka$ mpfer et al., called Sphingomonas–Rhizomonas-like strains. Other 1997). member species which can be considered as represen- The examination of total polar lipids revealed the tative of the genus, Sphingomonas adhaesiva, Sphingo- presence of identical patterns with eight compounds in monas asaccharolytica, Sphingomonas mali, Sphingo- strains BN6T and N,N (Fig. 1). Using authentic monas parapaucimobilis, S. paucimobilis, Sphingo- standards or in comparison with a lipid extract from monas pruni, Sphingomonas sanguinis and Sphingo- Sphingomonas paucimobilis, phosphatidylethanol- monas trueperi, are characterized by the presence of amine (PE), phosphatidyldimethylethanolamine sym-homospermidine as the major compound in their (PDE), phosphatidylglycerol (PG), sphingoglycolipid polyamine pattern (Busse & Auling, 1988; Hamana & (SGL), diphosphatidylglycerol (DPG) and an un- Matsuzaki, 1992; Segers et al., 1994; Ka$ mpfer et al., identified phospholipid (PL4) were identified as the 1997; Takeuchi et al., 1994; H. J. Busse, unpublished major lipids. Phosphatidylmonomethylethanolamine data). (PME), phosphatidylcholine (PC), an unidentified Various traits demonstrated that strains BN6T and phospholipid (PL3) and an unidentified polar lipid (L) N,N are different from all other previously described were detected in minor amounts. This polar lipid Sphingomonas species. This was already evident from pattern clearly distinguishes strains BN6T and N,N T the dendrograms obtained by Nohynek et al. (1996) from S. yanoikuyae DSM 7462 (Ka$ mpfer et al., for the comparison of 16S rRNA gene sequences and 1997). the whole-cell fatty acid composition of different Sphingomonas strains, including BN6T. The 16S rRNA T DISCUSSION gene of strain BN6 showed only 96% similarity to the most related validly described species (S. yanoikuyae), The results from DNA–DNA hybridization and all which suggested that this strain is a representative other experiments demonstrated that strains BN6T of a new Sphingomonas species. A comparison of the

International Journal of Systematic and Evolutionary Microbiology 50 39 A. Stolz and others phenotypical characteristics obtained in the present ethanolamine, phosphatidylglycerol, phosphatidyl- study for strains BN6T and N,N with previously choline and an unknown phospholipid are detected published data for other Sphingomonas species in the polar lipid pattern. Type strain is BN6T T T (Ka$ mpfer et al., 1997) also demonstrated significant (l DSM 6383 ). Strain BN6 was isolated from differences. Also, the pattern in the polar lipids was river water (River Elbe, Germany). The 16S rRNA different from other sphingomonads. While strains gene sequence is deposited at the EMBL database BN6T and N,N displayed identical polar lipid patterns, under the accession number X94098 (Nohynek et al., they were clearly distinguishable from their nearest 1996). phylogenetic neighbour, S. yanoikuyae, because of the lack of an unknown glycolipid (GL1) described for S. yanoikuyae (Ka$ mpfer et al., 1997). Taking these results ACKNOWLEDGEMENTS T into consideration, we propose strains BN6 and N,N P.K. is indebted to the German Ministry of Education and as representatives of a new species of the genus Research (BMBF) for research and personal grants Sphingomonas, Sphingomonas xenophaga sp. nov. (BEO31\0311768).

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