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1 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, IFZ–Heinrich-Buff-Ring 26–32, D-35392 Giessen, Germany
2 Department of Genetics, Microbiology and Toxicology, S-106 91 Stockholm University, Stockholm, Sweden
3 CCUG, Culture Collection University of Göteborg, Göteborg, Sweden
4 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, Wien, Austria
Correspondence
Peter Kämpfer
peter.kaempfer{at}agrar.uni-giessen.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CCUG 49520T is AY837748.
A two-dimensional thin-layer chromatogram of the polar lipids of strain CCUG 49520T is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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). Subcultivation was performed on nutrient agar (Oxoid) at 37 °C for 24 h for up to 7 days and strain CCUG 49520T was recovered. On this agar, CCUG 49520T was able to grow at 10–37 °C, but not at 4 or 45 °C. Growth was slow in general. The organism was able to grow on nutrient agar, tryptone soy agar and R2A agar, and also weakly on MacConkey agar (all from Oxoid). Tests for growth were performed in Luria–Bertani broth at 10–50 °C (5 °C intervals) with shaking at 160 r.p.m. Growth occurred at 15–45 °C with an optimum at 30 °C. The generation time at 30 °C was 100 min. Gram-staining was performed as described by Gerhardt et al. (1994). Preparation of bacterial DNA for G+C content estimations was performed using the blood and cell culture DNA midi kit from Qiagen according to the manufacturer's instructions. G+C content was determined by HPLC (Svoboda & Harms-Ringdahl, 2002). Cell morphology was observed under a Zeiss light microscope at x1000, using cells that had been grown for 3 days at 37 °C on nutrient agar; results are given under the species description below.
The 16S rRNA gene was analysed as described previously (Kämpfer et al., 2003). Analysis of the sequence data was performed by using the software package MEGA version 2.1 (Kumar et al., 2001), after multiple alignments of the data using CLUSTAL X (Thompson et al., 1997). A distance matrix method (distance options according to the Kimura two-parameter model), including clustering by neighbour-joining (Fig. 1a), and a discrete character-based maximum-parsimony method (Fig. 1b) were used. In each case, bootstrap values were calculated based on 1000 replications. The 16S rRNA gene sequence of strain CCUG 49520T was a continuous stretch of 1498 bp. Sequence similarity calculations indicated that strain CCUG 49520T showed the greatest degree of similarity to Serratia rubidaea DSM 4480T (GenBank accession no. AJ233436; 92·1 %). Lower sequence similarities (<92 %) were found with all other species of the family Enterobacteriaceae.
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; Altenburger et al., 1996), polar lipids (Tindall, 1990), fatty acids (Kämpfer & Kroppenstedt, 1996) and polyamines (Busse & Auling, 1988; Busse et al., 1997). The fatty acid profile of strain CCUG 49520T (given in the species description) was similar to those of other species of the Enterobacteriaceae.
The detection of a polar lipid profile with the major compounds phosphatidylethanolamine and phosphatidylglycerol (see Supplementary Fig. S1 available in IJSEM Online) is in good agreement with characteristics of representatives of the Enterobacteriaceae and Vibrionaceae (for review see Wilkinson, 1988). However, species of the Enterobacteriaceae were reported also to contain significant amounts of diphosphatidylglycerol, while this lipid was shown to be present only in trace amounts in species of the Vibrionaceae. No diphosphatidylglycerol was detected in polar lipid extracts of strain CCUG 49520T, in contrast to results for Serratia (Wilkinson, 1988). The polar lipid profile of strain CCUG 49520T is more similar to those of members of the Vibrionaceae.
Detection of the quinone system Q-8 is in agreement with the phylogenetic affiliation of CCUG 49520T because Q-8 has been reported to be present in all representatives of the Enterobacteriaceae and Vibrionaceae (Yokota et al., 1992). The detection of a polyamine pattern with the single major compound 1,3-diaminopropane clearly distinguishes strain CCUG 49520T from species of the Vibrionaceae, which were reported characteristically to contain sym-norspermidine (Yamamoto et al., 1983, 1991; Hamana, 1997). Among more than 70 analysed species of Enterobacteriaceae, a similar polyamine pattern has only been reported for five strains of Erwinia amylovora (Zherebilo et al., 2001) and Serratia marcescens IAM 12142T [only when grown on a highly specified cell culture medium (Hamana, 1996)]. However, the polyamine pattern of this latter strain also contained moderate amounts of putrescine and cadaverine (>10 % of the total polyamine content). On a more common medium (nutrient broth), the same Serratia strain showed a polyamine pattern with the predominant compound cadaverine and moderate to major amounts of 1,3-diaminopropane and putrescine. All Serratia species examined by Hamana (1996) exhibited as a common feature in the polyamine pattern the presence of moderate to major amounts of cadaverine (>10 % of the total polyamine content) and most of them also contain at least moderate amounts of putrescine. In the polyamine pattern of CCUG 49520T, by contrast, cadaverine was completely lacking and putrescine could only be detected in trace amounts. Hence, the polyamine pattern alone allows differentiation of strain CCUG 49520T from the indicated relatives of the family Enterobacteriaceae, including the genus Serratia.
Results of the physiological characterization of strain CCUG 49520T are given in the species description, determined using the methods of Kämpfer (1990) and Kämpfer et al. (1991). Strain CCUG 49520T was able to produce acid from various carbohydrates, but carbon substrate utilization tests showed weak results, even after incubation for several days. On the basis of the results presented we propose that strain CCUG 49520T constitutes a member of a new genus and species, for which the name Thorsellia anophelis gen. nov., sp. nov. is proposed.
Description of Thorsellia gen. nov.
Thorsellia (Thor.sel'li.a. N.L. fem. n. Thorsellia named in honour of Walborg Thorsell, a pioneer on mosquito repellent research in Sweden).
Cells are Gram-negative, facultatively anaerobic, motile and rod-shaped. Growth on all tested media is slow. Growth occurs after 48 h incubation on tryptone soy agar and nutrient agar at 37 °C; good growth also occurs at 30 °C. Growth occurs in Luria–Bertani broth at 15–45 °C with an optimum at 30 °C, at which the generation time is 100 min. 1,3-Diaminopropane is the characteristic compound in the polyamine pattern, ubiquinone Q-8 is the single quinone and in the polar lipid profile phosphatidylethanolamine and phosphatidylglycerol are predominant. The major fatty acids are C16 : 0, C18 : 1
7c and C14 : 0. The DNA G+C content is 46 mol%. The type species is Thorsellia anophelis.
Description of Thorsellia anophelis sp. nov.
Thorsellia anophelis (a.no'phe.lis. N.L. gen. n. anophelis from a mosquito of the genus Anopheles).
Exhibits the following properties in addition to those given in the genus description. Slow growth occurs after 24 h incubation on tryptone soy agar and nutrient agar at 37 °C. The fatty acid profile comprises C16 : 0 (32·9 %), C18 : 17c (35·9 %), C14 : 0 (12·2 %), C18 : 0 (1·0 %), C12 : 0 3-OH (0·8 %), summed feature 2 (C14 : 0 3-OH/iso I C16 : 1, 5·4 %) and summed feature 3 (C16 : 17c/iso-C15 : 0 2-OH, 8·0 %). The polar lipid profile contains moderate to minor amounts of two unknown aminophospholipids, an unknown phospholipid and two unknown polar lipids; when the organism is grown under strictly aerobic conditions, the quinone system consists exclusively of ubiquinone Q-8; the polyamine pattern consists of the predominant compound 1,3-diaminopropane [94·1 µmol (g dry weight)–1], spermidine [1·5 µmol (g dry weight)–1] and traces [<1·0 µmol (g dry weight)–1] of putrescine and sym-homospermidine. Aesculin is hydrolysed. Positive for acid production from glucose (O/F positive after 48 h incubation), lactose, sucrose, D-mannitol, dulcitol, salicin, adonitol, inositol, rhamnose, maltose, trehalose, cellobiose, D-arabitol and D-mannose. No acid is produced from sorbitol, L-arabinose, raffinose, D-xylose, methyl D-glucoside, erythritol or melibiose. The following carbon sources are utilized (only after prolonged incubation, 7 days): p-arbutin, D-gluconate, D-glucose, sucrose, D-trehalose, D-xylose, adonitol, myo-inositol, maltitol, D-mannitol, acetate, propionate, cis-aconitate, trans-aconitate, adipate, fumarate, DL-3-hydroxybutyrate, DL-lactate, L-malate, mesaconate, pyruvate, L-alanine and L-leucine. The following substrates are not utilized as carbon sources: N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, salicin, D-sorbitol, putrescine, 4-aminobutyrate, azelate, citrate, glutarate, itaconate, oxoglutarate, suberate, 
-alanine, L-aspartate, L-histidine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzoate and phenylacetate. Negative for hydrolysis of p-nitrophenyl (pNP) -D-glucopyranoside, o-nitrophenyl (oNP) -D-galactopyranoside, pNP -D-glucuronide, pNP 
-D-glucopyranoside, pNP -D-xylopyranoside, bis-pNP phosphate, pNP phenylphosphonate, pNP phosphorylcholine, 2-deoxythymidine-5'-pNP phosphate, L-alanine p-nitroanilide (pNA), L-glutamate-
-3-carboxy pna and L-proline pNA.
The type strain is CCUG 49520T (=CIP 108754T), isolated from the midgut contents of the mosquito Anopheles arabiensis.
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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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.
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
Hamana, K. (1996). Distribution of diaminopropane and acetylspermidine in Enterobacteriaceae. Can J Microbiol 42, 107–114.[Medline]
Hamana, K. (1997). Polyamine distribution patterns within the families Aeromonadaceae, Vibrionaceae, Pasteurellaceae, and Halomonadaceae, and related genera of the gamma subclass of the Proteobacteria. J Gen Appl Microbiol 43, 49–59.
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Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.
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Kämpfer, P., Dreyer, U., Neef, A., Dott, W. & Busse, H.-J. (2003). Chryseobacterium defluvii sp. nov., isolated from wastewater. Int J Syst Evol Microbiol 53, 93–97.
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Svoboda, P. & Harms-Ringdahl, M. (2002). Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2'-deoxyguanosine in solutions of free 2'-deoxyguanosine and calf thymus DNA. Biochim Biophys Acta 1571, 45–54.[Medline]
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Tindall, B. J. (1990). Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66, 199–202.
Wilkinson, S. G. (1988). Gram-negative bacteria. In Microbial Lipids, vol. 1, pp. 299–488. Edited by C. Ratledge & S. G. Wilkinson. New York: Academic Press.
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Yamamoto, S., Chowdhury, M. A. R., Kuroda, M., Nakano, T., Koumoto, Y. & Shinoda, S. (1991). Further study on polyamine compositions in Vibrionaceae. Can J Microbiol 37, 148–153.[Medline]
Yokota, A., Akagawa-Matsushita, M., Hiraishi, A., Katayama, Y., Urakami, T. & Yamasato, K. (1992). Distribution of quinone systems in microorganisms: Gram-negative eubacteria. Bull JFCC 8, 136–171.
Zherebilo, O., Kucheryava, N., Gvozdyak, R. I., Ziegler, D., Scheibner, M. & Auling, G. (2001). Diversity of polyamine patterns in soft rot pathogens and other plant-associated members of the Enterobacteriaceae. Syst Appl Microbiol 24, 54–62.[CrossRef][Medline]
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