International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1831–1837 Printed in Great Britain

Xenophilus azovorans gen. nov., sp. nov., a soil NOTE bacterium that is able to degrade azo dyes of the Orange II type

1 Institut fu$ r Mikrobiologie, Silke Blu$ mel,1 Hans-Ju$ rgen Busse,2,3 Andreas Stolz1 and Peter Ka$ mpfer4 Universita$ t Stuttgart, D-70569 Stuttgart, Germany Author for correspondence: Peter Ka$ mpfer. Tel: j49 641 99 37352. Fax: j49 641 99 37359. 2 Institut fu$ r Bakteriologie, e-mail: peter.kaempfer!agrar.uni-giessen.de Mykologie und Hygiene, Veterina$ rmedizinische Universia$ t, A-1210 Vienna, The of strain KF46FT, which was isolated previously after an aerobic Austria enrichment with the azo compound 1-(4’-carboxyphenylazo)-2-naphthol as the 3 Institut fu$ r Mikrobiologie sole source of energy and carbon, was investigated by a polyphasic approach. und Genetik, Universita$ t The organism contained a quinone system with ubiquinone Q-8 and 2- Wien, A-1030 Vienna, Austria hydroxyputrescine and putrescine as the major polyamines, suggesting that strain KF46FT belonged to the β-subclass of the . The polar 4 Institut fu$ r Angewandte Mikrobiologie, Justus- profile consisted mainly of and minor amounts of Liebig-Universita$ t Giessen, phosphatidylglycerol and diphosphatidylglycerol. Sequencing of the 16S rRNA D-35392 Giessen, Germany gene supported its placement in the family , but the sequence similarities to the most closely related species of the genera Hydrogenophaga, , and were only in the range 950to961%. Different methods for the construction of phylogenetic trees showed the separate position of strain KF46FT ‘between’ the genera Hydrogenophaga, Variovorax, Comamonas and Xylophilus. Analysis of the fatty acids revealed an unusual profile, with the presence of 8:0 3-OH, 10:0 3-OH, 16:1 2-OH, 16:0 2-OH and 18:1 2-OH in addition to 17:0 cyclo, which is unique among the previously described genera of the family Comamonadaceae. Thus, a new taxon is proposed for strain KF46FT, with the name azovorans gen. nov., sp. nov.

Keywords: gen. nov., sp. nov., β-Proteobacteria, polyphasic taxonomy

with simple carboxylated azo compounds has been studied in some detail. Overney (1979) isolated a Synthetic azo compounds are used in large quantities ‘Flavobacterium’ that was able to grow aerobically as dyes for textiles, paper, leather, cosmetics, food and with the simple model compound 4,4h-dicarboxyazo- various other materials. These xenobiotic compounds . Later, it was shown that a mixed bacterial are generally not degraded in conventional aerobic culture that degraded 4,4h-dicarboxyazobenzene could wastewater-treatment systems and therefore are of be adapted to the degradation of more complex azo significant concern as pollutants of the environment compounds such as 1-(4h-carboxyphenylazo)-4-naph- (Clarke & Anliker, 1980; Pagga & Brown, 1986). Over thol (‘carboxy-Orange I’) or 1-(4h-carboxyphenylazo)- the last 10 years, some reports have been published 2-naphthol (‘carboxy-Orange II’). From these ad- that demonstrated the ability of certain to aptation processes in continuous cultures, strain decolorize azo dyes under aerobic conditions (e.g. ‘’ K22 was obtained after cultivation Banat et al., 1996; Blu$ mel et al., 1998; Coughlin et al., with carboxy-Orange I and strain KF46FT from an 1999; Dykes et al., 1994; Heiss et al., 1992; Jiang & enrichment with carboxy-Orange II (Kulla, 1981; Bishop, 1994; Ogawa et al., 1986; Shaul et al., 1991). Kulla et al., 1983, 1984; Zimmermann et al., 1982, The adaptation of environmental bacteria to grow 1984). The aerobic azoreductases from both strains ...... were purified, characterized and shown to differ The EMBL accession number for the 16S rRNA gene sequence of strain significantly in their structure and substrate specificity KF46FT is AF285414. (Zimmermann et al., 1982, 1984).

01811 # 2001 IUMS 1831 S. Blu$ mel and others

In order to clarify the taxonomic position of bacterial were also detected. A similar profile has been strains with the ability to degrade azo compounds described for the genera belonging to the family aerobically, we characterized strain KF46FT from the Comamonadaceae sensu Willems et al. (1991b). original enrichments performed by Kulla (1981) and Willems et al. (1989) reported fatty acid profiles for the Kulla et al. (1984). genera Hydrogenophaga, Acidovorax, Variovorax and Comamonas. For these genera, the major fatty acids Strain KF46 was isolated from a soil inoculum after a were 16:1ω7c,18:1ω7c,18:1ω9t and\or 18:1ω12t, prolonged enrichment with carboxy-Orange II as the and 16:0. The presence of 8:0 3-OH and 10:0 3-OH sole source of carbon and energy (Kulla et al., 1984). was detected in almost all species of these genera, but For the present study, strain KF46FT was used, which 16:0 2-OH was detected only in some species of is a non-mucoid variant of strain KF46, a strain that Comamonas and Hydrogenophaga (Willems et al., has been preserved freeze-dried for the last 25 years 1989). The fatty acid 16:1 2-OH, which was found to (T. Leisinger, personal communication). The strain constitute 4 4% of the total fatty acids of strain was kindly provided by T. Leisinger (ETH Zu$ rich, n KF46FT, has been found only in representatives of the Switzerland). genus Comamonas (Stead, 1992) and 18:1 2-OH, which The Gram reaction was tested as described by also constitutes 4% in KF46FT, has not previously Gerhardt et al. (1994). Cell morphology was observed been detected in representatives of the family Coma- under a light microscope (1000i) with cells grown for monadaceae. 3 d at 30 C on . On nutrient agar, strain m Analysis of the respiratory quinone of KF46FT by KF46FT formed visible colonies (diameter of about HPLC (Tindall, 1990) gave only one characteristic 1 mm) within 3 d at 30 C. No growth was observed m peak, which corresponded to ubiquinone Q-8. Analysis within 14 d at 4, 37 or 42 C. The colonies were m of the polyamines (Busse & Auling, 1988; Busse et al., yellowish and opaque. Often, colonies were not de- 1997) revealed a pattern for strain KF46FT that is tectable as single entities because of the production of characteristic for taxa of the β-Proteobacteria (Busse & extracellular slimy substances. The strain was oxidase- Auling, 1988). Strain KF46FT displayed the major and -positive. Cells were motile, non-- " compounds 2-hydroxyputrescine (13 54 µmol g− dry forming rods (about 2 µm in length) and stained Gram- n " weight) and putrescine (11 71 µmol g− dry weight) negative. The strain was able to grow on various n " and moderate amounts of spermidine (3 24 µmol g− nutrient-rich media, like Luria–Bertani or nutrient n dry weight), whereas 1,3-diaminopropane, cadaverine, broth, at 30 C, but lost the ability to degrade Orange m homospermidine and spermine were detected only II rapidly under these conditions. −" in minor amounts (0n04, 0n03, 0n03 and 0n30 µmol g Physiological characterization was done as described dry weight, respectively). previously (Ka$ mpfer et al., 1991). Strain KF46FT Polar were determined by two-dimensional TLC, showed no (or very weak) production of acid from as described previously (Ventosa et al., 1993). The various and related compounds, but -, polar lipid profile of strain KF46FT consisted of four - and -maltitol were utilized as sole sources compounds. Phosphatidylethanolamine was detected of carbon. In addition, several organic acids, including as the major lipid and phosphatidylglycerol and amino acids, and some aromatic compounds were diphosphatidylglycerol were detected as minor spots utilized (for details, see species description below). after two-dimensional TLC. In addition, an unknown Most of the p-nitrophenyl derivatives tested were not polar lipid (ninhydrin-negative, molybdenum blue- hydrolysed, but hydrolysis of -alanine p-nitroanilide positive and alpha-naphthol-negative staining) was and 2-deoxythymidine 2 -p-nitrophenylphosphate h detected. were positive. Isolation of genomic DNA was performed as described Fatty acid methyl esters were extracted and prepared by Ausubel et al. (1996) and the determination of the by the standard protocol of the Microbial Identifi- G C content was carried out by HPLC according to cation System (MIDI; Microbial ID Inc.). Extracts j Mesbah & Whitman (1989). Genomic DNA of Hydro- were analysed by using a Hewlett Packard model genophaga palleronii DSM 63T (G C content 67 5 HP6890A gas chromatograph equipped with a flame- j n mol%; Aragno & Schlegel, 1992) and subtilis ionization detector, an automatic sampler, an inte- DSM 618 (G C content 43 mol%; Slepecky & grator and a computer, as described previously j Hemphill, 1992) were used as standards. The G C (Ka$ mpfer & Kroppenstedt, 1996). Strain KF46FT j content of the genomic DNA of strain KF46FT was contained the fatty acids 16:0 (35 1%), summed n determined to be 70 4 0 3 mol% (mean of four feature 4 (16:1ω7c and iso 15:0 2-OH, 11 6%), n p n n measurements). summed feature 7 (18:1ω7c,18:1ω9t and\or 18:1ω12t,20n5%) and 17:0 cyclo (14n2%) in major The gene for the 16S rRNA was amplified by PCR amounts. Furthermore, it was interesting that several using different universal primers (Gerhardt et al., 1994) hydroxylated fatty acids were produced (8:0 3-OH, and sequenced. Phylogenetic analysis was performed 1n2%; 10:0 3-OH, 3n7%; 16:1 2-OH, 4n4%; 16:0 2- using the  software package (Ludwig & Strunk, OH, 1n5%; 18:1 2-OH, 4n0%), some in significant 1997). Distance-matrix, maximum-parsimony and amounts. The fatty acids 10:0 (2n8%) and 17:0 (1n2%) maximum-likelihood methods were applied for tree

1832 International Journal of Systematic and Evolutionary Microbiology 51 Xenophilus azovorans gen. nov., sp. nov.

...... Fig. 1. showing the relationship between Xenophilus azovorans strain KF46FT (l NCIMB 13707T) and selected strains from other bacterial taxa. The tree was constructed using the maximum-parsimony method. GenBank accession numbers are given in parentheses. construction as implemented in the  software KF46FT harbours two large , of about 100 package. and 350 kb. The 16S rRNA sequence of strain KF46FT, containing The detection of ubiquinone Q-8 as the single quinone, a continuous stretch of 1484 bp (approximately the presence of hydroxyputrescine and putrescine, the positions 28–1521 according to the Escherichia coli large amounts of 16:0 and 17:0 cyclo fatty acids and numbering), was used to search the GenBank and the absence of 16:0 3-OH, as well as the GjC content Ribosomal Database Project libraries. Sequence of the DNA and the sequence of the 16S rRNA, clearly searches showed that strain KF46FT was phylo- placed strain KF46FT in the family Comamonadaceae. genetically most closely related to the β-subclass of the This family consisted originally of the genera Coma- Proteobacteria. The results of sequence similarity monas, Acidovorax, Variovorax, Hydrogenophaga and calculations indicated that the nearest relatives of Xylophilus and some misclassified Aquaspirillum T strain KF46F are Xylophilus ampelinus (96n1% species (Willems et al., 1991b). In later studies, further sequence similarity), Aquaspirillum metamorphum species belonging to the genus Acidovorax were (96n0%) and Hydrogenophaga palleronii (95n4%). Only detected (Willems et al., 1992; Schulze et al., 1999) and slightly lower sequence similarities (92–95% sequence also further genera were described (Hiraishi, 1994; identity) were found to different species from the Hiraishi et al., 1995; Irgens et al., 1996; Wen et al., genera Variovorax, Comamonas, Brachymonas, Delftia 1999). The phylogeny of the family was investigated in and Acidovorax. From these results, it was evident more detail by Wen et al. (1999) and, on the basis of that strain KF46FT belonged to the family Coma- this study, the family comprises the 10 genera included monadaceae. Dendrograms were generated by using in Table 1. Strain KF46FT is clearly different from all the maximum-parsimony, maximum-likelihood and other previously described genera belonging to the neighbour-joining methods from the  program family Comamonadaceae. package. The results of all these trees (Fig. 1 shows the The main reason for creating a new genus for strain maximum-parsimony analysis) showed a separate and KF46FT was the rather large degrees of sequence almost equidistant position of strain KF46FT between divergence of the 16S rRNA gene from the corre- the genera mentioned above. sponding sequences of different genera of the family Extrachromosomal DNA was detected by a modi- Comamonadaceae. Furthermore, strain KF46FT can fication of the method of Barton et al. (1995). Pulsed- be clearly separated from the most closely related field gel electrophoresis demonstrated that strain genera, Hydrogenophaga, Comamonas and Xylophilus,

International Journal of Systematic and Evolutionary Microbiology 51 1833 S. Blu$ mel and others ] d d  k k   10:0 ...... Aquaspirillum metamorphum [ flagella at only one pole , variable . (1996) . (1985), ); Palleroni  et al et al k k kk         Q-8 Rods Spirilla or curved rods One polar Bipolar tufts or 1–2 Acidovorax ); Irgens ); Bradbury (1984) and ). ) k k kk k k kk k j     ( or short rods Variovorax . (1990, 1992) ( Brachymonas metamorphum et al were taken from: De Vos RQ-8 ] kk k k k        8:0 10:0 j . (1991b) ( 16:0, 16:1 16:0, 16:1 16:1, 16:0, 18:1 16:0, 16:1 . (1995) ( et al et al Xenophilus k kj k k kj k k k k                  Aquaspirillum -Glutamate Biotin, thiamin ......  ), weak reaction; d, 11–89% of strains positive; ); Hiraishi j Comamonadaceae d d d d d kkkk k              . (1995) and Willems Rhodoferax ); Palleroni (1984) and Willems et al dd d k k jjjk jjjjkj kk k j Comamonas , absent from all species; ( and k ddd d dd d dd d d d d dd kk jjk k jjjjkkj kkk One polar One polar Peritrichous One polar One polar Delftia and other genera in the family . (1992a, b) and Sakane & Yokota (1994) ([ , no data available. Data for genera other than  et al d kk kkk k k k k k k . (1991a) ( Polar or bipolar tufts . (1991) and Busse & Auling (1988) ( ...... Xenophilus et al et al ); Kersters & De Ley (1984), Urakami , Present in all species; j d dd d d dddd d d d d kk kk Polar or bipolar tufts ); Hiraishi . (1999). )d )d d et al . (1994), Krieg (1984), Pot Hydrogenophaga j k jjk jjkj kkk j kkkkjk kkkkkkkkkj kk k jkkkjjjk kkkjkk j kkkkkkkjkk jjjjjjkjjj           . (1987) and Willems 4 (HPLC) 67–69 63–66 67–70 65–69 66–68 68–69 59–61 (HPLC) 63–65 (HPLC) 52–57 56–62 n Xenophilus Delftia Comamonas Acidovorax Hydrogenophaga Variovorax Xylophilus Rhodoferax Brachymonas Polaromonas et al Xylophilus et al # . (1989) ( et al ); and Hamana . (1987, 1991b) ( Differential characteristics of the genus C content (mol%) 70 et al j )-Tartrate j -Tryptophan -Mannitol -Fructose -Glucose -( -Tryptophan ( -Alanine    Phenylacetate  Maleate 3-Aminobutyrate   Glycerol ( Malonate 2-Aminobutyrate Acetamide β Major quinone systemMajor 3-OH acids2-OH acids detectedDNA G Q-8 16:0, 16:1, 10:0, 18:1 8:0 Q-8 – 10:0, 8:0 Q-8 16:0, 10:0 16:1 10:0, Q-8 – 8:0 8:0, (10:0) Traces of 16:0 Q-8 10:0 14:0 Q-8 Q-8 Q-8 Major cellular fatty acid(s) 16:0, 16:1, 18:1 16:0, 16:1, 18:1 16:0, 16:1, 18:1 16:0, 16:1, 18:1 16:0, 16:1 16:1, 16:0, 18:1 Flagella Character Cell morphology Rods Rods Rods or spirilla Rods Rods Rods Rods Curved rods Coccobacilli Psychrophilic growth Growth factors Denitrification Carbon source used for growth: Pigments Phototrophy Oxidase Chemolithotrophic growth with H Polaromonas Willems reaction depending on the method used; RQ, rhodoquinone; Palleroni (1984), Tamaoka (1984) and Willems ( Table 1...... The table was adapted from Wen

1834 International Journal of Systematic and Evolutionary Microbiology 51 Xenophilus azovorans gen. nov., sp. nov. by its unique fatty acid pattern and several physio- -proline, -tryptophan, 3-hydroxybenzoate and 4- logical features. On the basis of these observations, we hydroxybenzoate. The following compounds are not propose strain KF46FT as representative of a new utilized as sole sources of carbon: N-acetylgalactos- species of a new genus, Xenophilus azovorans gen. nov., amine, N-acetylglucosamine, -arabinose, -arbutin, sp. nov. -cellobiose, -galactose, gluconate, -glucose, - maltose, -mannose, α--melibiose, -rhamnose, - Description of Xenophilus gen. nov. ribose, -sucrose, salicin, -trehalose, -xylose, adon- itol, i-inositol, -sorbitol, putrescine, cis-aconitate, Xenophilus (xe.nohphi.lus. Gr. adj. xenos foreign; Gr. trans-aconitate, 4-aminobutyrate, citrate, mesaconate, masc. n. philos friend; N.L. masc. n. Xenophilus friend -alanine, β-alanine, -ornithine, -phenylalanine, - of foreign compounds, referring to the isolation of the serine and phenylacetate. The major non-polar fatty type species by enrichment on azo dyes). acids are 16:0, summed feature 4 (16:1ω7c and iso 15:0 2-OH) and summed feature 7 (18:1ω7c,18:1ω9t Cells are straight to slightly curved rods, 0 5–1 µmby n and\or 18:1ω12t). In addition, 8:0 3-OH, 10:0 3-OH, 1–3 µm. Cells occur singly or in pairs and are motile. 16:0 2-OH, 16:1 2-OH, 18:1 2-OH and 17:0 cyclo are Gram-negative, oxidase-positive and possesses an oxi- detected in significant amounts. dative metabolism. The GjC content of DNA is T T 70n4 mol% (HPLC method). Xenophilus belongs to The type strain is strain KF46F (l DSM 13620 l the family Comamonadaceae and, on the basis of 16S NCIMB 13707T). Strain KF46FT was isolated from rRNA sequences, is equidistantly related to represen- soil after continuous enrichment with 1-(4h-carboxy- tatives of the genera Xylophilus and Hydrogenophaga. phenylazo)-2-naphthol. The 16S rRNA gene sequence Ubiquinone Q-8 is the only quinone type; 2-hydroxy- has been deposited in the EMBL database under the putrescine and putrescine are the major polyamines. accession number AF285414. The polar lipid profile is characterized by the pres- ence of phosphatidylethanolamine (major lipid) and References phosphatidylglycerol and diphosphatidylglycerol in addition to an unknown polar lipid (minor lipids). The Aragno, M. & Schlegel, H. G. (1992). The mesophilic hydrogen- type species is Xenophilus azovorans. oxidizing (Knallgas) bacteria. In The Prokaryotes, 2nd edn, vol. 1, pp. 344–384. Edited by A. Balows, H. G. Tru$ per, M. Dworkin, W. Harder & K.-H. Schleifer. Berlin: Springer. Description of Xenophilus azovorans sp. nov. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidmam, Xenophilus azovorans (a.zo.vohrans. N.L. neut. azo J. G., Smith, J. A. & Struhl, K. (editors) (1996). Current Protocols from azo compounds; L. v. vorare to devour; N.L. in Molecular Biology. New York: Wiley. part. adj. azovorans azo-devouring). Banat, I. M., Nigam, P., Singh, D. & Marchant, R. (1996). Microbial decolorization of textile-dye-containing effluents: a review. The description is the same as that given above for the Bioresour Technol 58, 217–227. genus. On nutrient agar, colonies are circular, opaque, Barton, B. M., Harding, G. P. & Zuccarelli, A. J. (1995). A general slightly raised and pale-yellowish with entire margins. method for detecting and sizing large plasmids. Anal Biochem Colonies are often not detectable as single entities 226, 235–240. $ because of the production of extracellular slimy sub- Blumel, S., Contzen, M., Lutz, M., Stolz, A. & Knackmuss, H.-J. stances. 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