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1 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, Veterinärplatz 1, A-1210 Vienna, Austria
2 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany
Correspondence
Hans-Jürgen Busse
hans-juergen.busse{at}vu-wien.ac.at
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Sphingomonas abaci C42T and Sphingomonas panni C52T are AJ575817 and AJ575818, respectively.
The fatty acid content, polar lipid profiles, protein patterns and genomic fingerprints of strains C42T and C52T are available as supplementary data in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). From protein-similarity groups consisting of at least three strains, a representative was characterized in more detail. On the basis of a preliminary classification, selected representatives of these protein-similarity groups were identified as members of the yeast genus Rhodotorula, the bacterial genera Pseudomonas (Hauser et al., 2004), Acinetobacter, Staphylococcus, Bacillus and Sphingomonas, and coliforms. Here we report on the taxonomic characterization of the orange-pigmented strain C42T and the yellow-pigmented strain C52T preliminarily identified as members of the genus Sphingomonas. Strain C42T was isolated from a treatment table after inoculation on PYE agar (l–1: 3·0 g peptone from casein, 3·0 g yeast extract, 15·0 g agar; pH 7·2). Strain C52T was isolated from a wipe in the treatment room after cultivation on PYE agar. Single colonies were visible after cultivation at 28 °C for 48 h.
The 16S rRNA gene was amplified and analysed as described previously (Zlamala et al., 2002). The 16S rRNA gene sequences of strains C42T and C52T were continuous stretches of 1407 and 1408 nt, respectively, showing 94·7 % similarity to each other. Sequence comparisons (ungapped) performed using FASTA3 (Pearson & Lipman, 1988) indicated that the closest relatives of the orange-pigmented strain C42T are Sphingomonas aquatilis KCTC 2881T and Sphingomonas melonis DSM 14444T (both showing 97·7 % sequence similarity). Only moderate sequence similarities were found with respect to the orange-pigmented species (Busse et al., 2003) Sphingomonas faeni MA-olkiT (96·2 %), Sphingomonas aurantiaca MA101bT (95·8 %) and Sphingomonas aerolata NW12T (95·7 %) and to selected other species of the genus Sphingomonas (96·7–95·3 %). Strain C52T shared the greatest levels of sequence similarity with Sphingomonas koreensis KCTC 2882T (97·2 %), S. aquatilis KCTC 2881T (97·1 %) and S. melonis DSM 14444T (97·0 % sequence similarity). Sequence similarities with other species of the genus were below 96·8 %. Phylogenetic analyses were performed using the PHYLIP package (Felsenstein, 1995). In all of the phylogenetic trees, strain C42T grouped with S. aquatilis KCTC 2881T and S. melonis DSM 14444T, but the level of bootstrap support varied greatly according to the algorithm applied (maximum likelihood and maximum parsimony, <25 %; neighbour-joining, 72 %). Strain C52T was always placed next to S. koreensis KCTC 2882T with high levels of bootstrap support (Fig. 1), confirming the results from sequence comparisons.
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). Temperature tolerance was examined on PYE agar, while NaCl tolerance was tested on PYE agar supplemented with different concentrations of NaCl. Growth on TSA, R2A and MacConkey agar was examined as well. Growth under microaerobic conditions (gas mixture 95 % N2, 5 % CO2, 1·5–2 % remaining O2) was examined on PYE agar. Obvious differences could be observed among the physiological properties of C42T, C52T and their close relatives, S. aquatilis KCTC 2881T, S. koreensis KCTC 2882T and S. melonis DSM 14444T, which were analysed in parallel. The reactivity of our isolates was much lower than the reactivity of the reference strains. A high degree of similarity in terms of physiological characteristics (88 %) was found for S. aquatilis KCTC 2881T and S. melonis DSM 14444T. Detailed results are listed in Table 1 and in the species descriptions below.
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and Altenburger et al. (1996), respectively, polyamines were analysed as described by Busse & Auling (1988) and Busse et al. (1997) and fatty acids were determined as described by Kämpfer et al. (1997). The results are given in the species descriptions and in Supplementary Table S1 (available in IJSEM Online).
The detection of quinone system Q-10 in C42T and C52T corresponded with species of the genus Sphingomonas sensu stricto and the family Sphingomonadaceae (Busse et al., 1999; Kosako et al., 2000). The polyamine patterns of the two strains showed the predominance of the compound sym-homospermidine, the key characteristic of Sphingomonas sensu stricto (Busse et al., 1999; Takeuchi et al., 2001), and minor amounts of spermidine. The polar lipid profiles of both strain C42T and strain C52T (see Supplementary Fig. S1, available in IJSEM Online) contained phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, sphingoglycolipid and phosphatidyldimethylethanolamine, which is in excellent agreement with the characteristics in the profiles of other species of the genus Sphingomonas sensu stricto (Busse et al., 1999). However, their unique polar lipid profiles distinguished the two strains from each other and from other species of the genus Sphingomonas sensu stricto (Busse et al., 1999).
The fatty acid profiles of strains C42T and C52T (Supplementary Table S1) showed the predominance of C18 : 1
7c and the relatively high levels of C16 : 0 that are shared with the majority of members of the ‘Alphaproteobacteria’. The presence of 2-hydroxy myristic acid (2-OH C14 : 0) and the lack of 3-hydroxy fatty acids are important characteristics of members of the family Sphingomonadaceae (Busse et al., 1999; Takeuchi & Hiraishi, 2001; Takeuchi et al., 2001). However, our isolates differed from the reference strains both qualitatively and quantitatively with regard to certain acids.
Comparison of protein patterns after SDS-PAGE was performed with C42T, C52T, S. aquatilis KCTC 2881T, S. koreensis KCTC 2882T and S. melonis DSM 14444T as described previously (Altenburger et al., 1996). The protein patterns clearly distinguished both isolates from their closest relatives, S. aquatilis KCTC 2881T, S. koreensis KCTC 2882T and S. melonis DSM 14444T. In contrast, almost identical protein patterns (Supplementary Fig. S2) were detected for S. aquatilis KCTC 2881T (Lee et al., 2001) and S. melonis DSM 14444T (Buonauria et al., 2002).
Genomic fingerprints (Supplementary Fig. S3) of C42T, C52T, S. aquatilis KCTC 2881T, S. koreensis KCTC 2882T and S. melonis DSM 14444T obtained after ERIC-PCR (Wieser & Busse, 2000) clearly differentiated the five strains, but obvious similarities between S. melonis DSM 14444T and S. aquatilis KCTC 2881T, as might have been expected from their similar protein patterns, were not observed. Analysis of DNA relatedness (Ziemke et al., 1998; Kämpfer et al., 2003) between the pairs C42T/S. melonis DSM 14444T (19 %, reciprocal 12 %), C42T/S. aquatilis KCTC 2881T (17 %, reciprocal 17 %), S. aquatilis KCTC 2881T/S. melonis DSM 14444T (56 %, reciprocal 53 %), C52T/S. melonis DSM 14444T (23 %, reciprocal 17 %), C52T/S. aquatilis KCTC 2881T (18 %, reciprocal 15 %) and C52T/S. koreensis KCTC 2882T (23 %, reciprocal 24 %) did not reveal any relatedness at the species level.
The results of the analysis of 16S rRNA gene sequences, polyamine patterns, quinone system, fatty acids and polar lipids prove that strain C42T and strain C52T are both members of the genus Sphingomonas sensu stricto. The two isolates can be distinguished from each other on the basis of their 16S rRNA gene sequences and phenotypic traits (chemotaxonomically and physiologically) and from their close relatives by their genomic fingerprints, protein patterns, fatty acid composition and numerous physiological characteristics. On the basis of these results, we conclude that strains C42T and C52T represent two novel species of the genus Sphingomonas sensu stricto, for which we propose the names Sphingomonas abaci sp. nov. and Sphingomonas panni sp. nov., respectively.
Although S. aquatilis KCTC 2881T and S. melonis DSM 14444T show almost undistinguishable protein patterns and 16S rRNA gene sequences as well as relatively high degrees of similarity with respect to their fatty acid profiles and physiological traits, their DNA relatedness (
55 % similarity) does not suggest that they should be placed in a single species. However, this observation demonstrates that comparison of protein patterns is not suitable for the identification of strains as members of S. melonis or S. aquatilis.
Description of Sphingomonas abaci sp. nov.
Sphingomonas abaci (a'ba.ci. L. gen. n. abaci of/from a table, referring to the fact that the type strain was isolated from a treatment table).
Cells are Gram-negative (by staining and in the KOH test), non-spore-forming rods with rounded poles and are 0·4–0·5x1·5–3·0 µm in size. Cells occur singly or in pairs (rarely). Oxidase-negative and catalase-positive; growth occurs under aerobic and microaerobic conditions but not under anaerobic conditions. No motility is observed under the light microscope. Good growth occurs on PYE agar, TSA, 1 % (w/v) NaCl and at temperatures between 15 and 28 °C on PYE agar. No growth occurs on MacConkey agar or at 4, 37 and 42 °C on PYE agar. On PYE agar, orange, shiny, circular, leathery, dry colonies approximately 0·5 mm in diameter form within 48 h. After 5 days incubation, colonies on PYE agar reach diameters of approximately 2 mm. The quinone system of the type strain consists of Q-10 (86 %), Q-9 (9 %) and Q-8 (5 %). The polyamine pattern primarily consists of sym-homospermidine [48·1 µmol (g dry weight)–1] with moderate amounts of spermidine [12·0 µmol (g dry weight)–1]. The predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and sphingoglycolipid. Additionally, moderate amounts of phosphatidylcholine, three unidentified glycolipids and an unknown polar lipid, small amounts of phosphatidyldimethylethanolamine, phosphatidylmonomethylethanolamine and three unknown phospholipids are present. The fatty acids comprise C18 : 17c (64·8 %), 2-OH C14 : 0 (12·9 %), C16 : 0 (12·4 %), summed feature 3 (C16 : 17c and/or 2-OH C15 : 0 iso) (8·3 %) and C17 : 16c (1·6 %). Acid production from sugars, carbon-source utilization and hydrolysis of chromogenic substrates are listed in Table 1
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The type strain, C42T (=LMG 21978T=DSM 15867T), was isolated from the treatment table in the Medical Clinic for Small Animals, University for Veterinary Medicine, Vienna, Austria.
Description of Sphingomonas panni sp. nov.
Sphingomonas panni (pan'ni. L. gen. n. panni of/from a piece of cloth, or by extension a wipe, referring to the fact that the type strain was isolated from a wipe).
Cells are Gram-negative (by staining and in the KOH test), non-spore-forming rods with rounded poles and are approximately 0·5–0·7x1·2–2·0 µm in size. Cells occur singly or in pairs (rarely). Oxidase-negative and catalase-positive; growth occurs under aerobic and microaerobic conditions but not under anaerobic conditions. No motility is observed under the light microscope. Good growth occurs on TSA, R2A, at 1 % (w/v) NaCl and at temperatures between 15 and 37 °C on PYE agar. No growth occurs on MacConkey agar or at 4 and 42 °C on PYE agar. Yellow, shiny, circular colonies approximately 0·3 mm in diameter form within 48 h on PYE agar. After 5 days incubation, colonies reach diameters of about 1·5 mm. The quinone system of the type strain consists of Q-10 (95 %), Q-9 (3 %) and Q-8 (1 %). The polyamine pattern primarily consists of sym-homospermidine [36·0 µmol (g dry weight) –1] with minor amounts of spermidine [1·3 µmol (g dry weight)–1]. The predominant polar lipids are phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and sphingoglycolipid. Additionally, small amounts of phosphatidyldimethylethanolamine, diphosphatidylglycerol and an unidentified phospholipid are present. The fatty acids comprise C18 : 17c (51·3 %), C16 : 0 (17·8 %), summed feature 3 (C16 : 17c and/or 2-OH C15 : 0 iso) (17·1 %), 2-OH C14 : 0 (5·3 %), C17 : 16c (3·6 %), C16 : 15c (2·1 %), C15 : 0 (1·1 %), C14 : 0 (1·1 %) and C18 : 15c (0·7 %). Acid production from sugars, carbon-source utilization and hydrolysis of chromogenic substrates are listed in Table 1.
The type strain, C52T (=LMG 21979T=DSM 15761T), was isolated from a wipe in the Medical Clinic for Small Animals, University of Veterinary Medicine, Vienna, Austria.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Buonauria, R., Stravato, V., Kosako, Y., Fujiwara, N., Naka, T., Kobayashi, K., Cappelli, C. & Yabuuchi, E. (2002). Sphingomonas melonis sp. nov., a novel pathogen that causes brown spots on yellow Spanish melon fruits. Int J Syst Evol Microbiol 52, 2081–2087.[Abstract]
Busse, H.-J. & Auling, G. (1988). Polyamine pattern as a chemotaxonomic marker within the Proteobacteria. Syst Appl Microbiol 11, 1–8.
Busse, H.-J., Bunka, S., Hensel, A. & Lubitz, W. (1997). Discrimination of members of the family Pasteurellaceae based on polyamine patterns. Int J Syst Bacteriol 47, 698–708.[CrossRef]
Busse, H.-J., Denner, E. & Kämpfer, P. (1999). Chemotaxonomic characterisation of Sphingomonas. J Ind Microbiol Biotechnol 23, 242–251.[CrossRef][Medline]
Busse, H.-J., Denner, E., Buczolits, S., Salkinoja-Salonen, M., Bennasar, A. & Kämpfer, P. (2003). Sphingomonas aurantiaca sp. nov., Sphingomonas aerolata sp. nov. and Sphingomonas faeni sp. nov., air- and dustborne and Antarctic, orange-pigmented, psychrotolerant bacteria, and emended description of the genus Sphingomonas. Int J Syst Evol Microbiol 53, 1253–1260.
Felsenstein, J. (1995). PHYLIP (phylogeny inference package), version 3.57c. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Hall, T. A. (1999). BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
Hauser, E., Kämpfer, P. & Busse, H.-J. (2004). Pseudomonas psychrotolerans sp. nov. Int J Syst Evol Microbiol 54, 1633–1637.
Kämpfer, P., Steiof, M. & Dott, W. (1991). Microbiological characterisation of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria. Microb Ecol 21, 227–251.
Kämpfer, P., Denner, E. B. M., Meyer, S., Moore, E. R. B. & Busse, H.-J. (1997). Classification of "Pseudomonas azotocolligans" Anderson 1955, 132, in the genus Sphingomonas as Sphingomonas trueperi sp. nov. Int J Syst Bacteriol 47, 577–583.[CrossRef][Medline]
Kämpfer, P., Buczolits, S., Albrecht, A., Busse, H.-J. & Stackebrandt, E. (2003). Towards a standardized format for the description of a novel species (of an established genus): Ochrobactrum gallinifaecis sp. nov. Int J Syst Evol Microbiol 53, 893–896.
Kosako, Y., Yabuuchi, E., Naka, T., Fujiwara, N. & Kobayashi, K. (2000). Proposal of Sphingomonadaceae fam. nov., consisting of Sphingomonas Yabuuchi et al. 1990, Erythrobacter Shiba and Shimidu 1982, Erythromicrobium Yurkov et al. 1994, Porphyrobacter Fuerst et al. 1993, Zymomonas Kluyver and van Niel 1936, and Sandaracinobacter Yurkov et al. 1997, with the type genus Sphingomonas Yabuuchi et al. 1990. Microbiol Immunol 44, 563–575.[Medline]
Lee, J.-S., Shin, Y. K., Yoon, J.-H., Takeuchi, M., Pyun, Y.-R. & Park, Y.-H. (2001). Sphingomonas aquatilis sp. nov., Sphingomonas koreensis sp. nov. and Sphingomonas taejonensis sp. nov., yellow-pigmented bacteria isolated from natural mineral water. Int J Syst Evol Microbiol 51, 1491–1498.[Abstract]
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
Pearson, W. & Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 2444–2448.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Takeuchi, M. & Hiraishi, A. (2001). Taxonomic significance of 2-hydroxy fatty acid profiles of the species in the genus Sphingomonas and related taxa. IFO Res Commun 20, 72–82.
Takeuchi, M., Hamana, K. & Hiraishi, A. (2001). Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51, 1405–1417.[Abstract]
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Tindall, B. J. (1990). A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13, 128–130.
Wieser, M. & Busse, H.-J. (2000). Rapid identification of Staphylococcus epidermidis. Int J Syst Evol Microbiol 50, 1087–1093.[Abstract]
Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48, 179–186.[CrossRef][Medline]
Zlamala, C., Schumann, P., Kämpfer, P., Rosselló-Mora, R., Lubitz, W. & Busse, H. J. (2002). Agrococcus baldri sp. nov., isolated from the air in the ‘Virgilkapelle’ in Vienna. Int J Syst Evol Microbiol 52, 1211–1216.[Abstract]
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-D-glucopyranoside, pNP phenylphosphonate, 2-deoxythymidine-5'-pNP phosphate, L-alanine p-nitroanilide (pna) and assimilation of L-malate. None of the strains was able to grow at 4 or 42 °C, on PYE agar supplemented with 5, 7·5 or 10 % NaCl or on MacConkey agar. All strains were negative for acid production from glucose, lactose, sucrose, D-mannitol, dulcitol, salicin, adonitol, inositol, sorbitol, L-arabinose, raffinose, maltose, D-xylose, trehalose, cellobiose, methyl D-glucoside, erythritol, melibiose, D-arabitol, D-mannose, hydrolysis of L-proline pna or assimilation of n-acetyl-D-galactosamine, 
-D-melibiose, D-ribose, salicin, adonitol, i-inositol, maltitol, D-mannitol, D-sorbitol, putrescine, acetate, propionate, cis-aconitate, trans-aconitate, adipate, 4-aminobutyrate, azelate, itaconate,