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1 Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, 53127 Bonn, Germany
2 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Institute of Marine Technology, Nan-Tzu, Kaohsiung 811, Taiwan, ROC
3 Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, 53121 Bonn, Germany
4 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Inhoffenstraße 7b, D-38124 Braunschweig, Germany
5 College of Agriculture and Natural Resources, Department of Soil and Environmental Sciences, National Chung Hsing University, Taichung 402, Taiwan, ROC
Correspondence
A. F. Yassin
yassin{at}mibi03.meb.uni-bonn.de
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for gliding organisms with high G+C contents (62–70.1 mol%) and with colonies that were very mucoid and cream, pink or yellow-brown in colour. Members of this genus are strongly proteolytic and characteristically lyse a variety of micro-organisms such as Gram-positive bacteria (including actinomycetes), fungi and green algae, as well as nematodes. Recently, 16S rRNA gene sequence analysis has revealed that members of the genus Lysobacter form a distinct phylogenetic line within the Gammaproteobacteria, being related to the genera Xanthomonas, Pseudoxanthomonas, Stenotrophomonas, Thermomonas and Xylella (Bae et al., 2005). In addition, analysis of the quinone system and the cellular fatty acids revealed that Lysobacter species contain ubiquinone Q-8 as the major respiratory quinone and have fatty acid profiles with a predominance of iso-branched fatty acids. At the time of writing, the genus Lysobacter encompasses eight species with validly published names: Lysobacter antibioticus, Lysobacter brunescens, Lysobacter concretionis, Lysobacter daejeonensis, Lysobacter enzymogenes, Lysobacter gummosus, Lysobacter koreensis and Lysobacter yangpyeongensis. In this paper we describe a bacterial strain that was isolated from a municipal solid waste dumping site at Udupi, India. On the basis of phylogenetic and phenotypic data, it is proposed that this strain (designated IMMIB APB-9T) be classified as a novel species of the genus Lysobacter.
Strain IMMIB APB-9T was isolated on nutrient agar from a soil sample taken from a municipal landfill site in Udupi, Karnataka State, India. The isolate was subsequently cultivated on brain-heart infusion (BHI) agar (no. 237100; Becton Dickinson) and tryptone soya agar (CM 131; Oxoid) to determine its morphological characteristics. Pigment production was determined by growing the strains at 37 °C for 7 days, with observations being made at 24 h intervals. The physiological properties of strain IMMIB APB-9T and of the type strains of recognized Lysobacter species were determined using tests to assess the hydrolysis of complex substrates, as described previously (Gordon, 1966, 1967; Gordon & Mihm, 1957). Tween 80 hydrolysis was performed as described by Riegel et al. (1994). Fermentation tests were performed using the API Coryne, API 20 Strep and API 20E systems (bioMérieux). Assimilation tests were performed using the API 20NE system (bioMérieux). Enzyme reactions and acid production from carbohydrates were read after 3 and 7 days incubation at 37 °C. Salt tolerance was determined by cultivating the organisms in tryptone soya broth supplemented with NaCl at final concentrations in the range 0.0–12.0 %.
Irrespective of previous chemotaxonomic analyses of Lysobacter species, all strains were re-examined to guarantee identical conditions. Chemotaxonomic characteristics of strain IMMIB APB-9T, as well as those for the type strains of recognized Lysobacter species, were determined by cultivating the organisms at 37 °C in shake flasks containing BHI broth for 1 week. After being checked for purity at maximum growth, the organisms were killed with formaldehyde (1 %, v/v), harvested by centrifugation, washed with distilled water and then freeze-dried. Lipids were extracted using acid methanolysis as described by Minnikin et al. (1980) and by saponification as described in the MIDI manual. Fatty acid methyl esters obtained after acid methanolysis were purified as described by Yassin (1988). After transesterification, extracts were purified by TLC (article no. 1.05554; Merck) using light petroleum benzene (boiling point, 60–80 °C)/acetone (95 : 5, v/v) as the solvent. The TLC plates were then sprayed with 2',7'-dichlorofluorescein and air-dried. Thereafter, the fatty acids were detected under UV light (366 nm) as bright yellow bands on a fluorescein background: the non-hydroxylated fatty acids migrated at an RF value of approximately 0.78 and the hydroxylated ones migrated at an RF value of approximately 0.13. The fatty acid bands were scraped off, extracted from the silica gel with 5 ml chloroform/methanol (2 : 1, v/v) by vortexing and then centrifuged at 3000 r.p.m. for 5 min. The dye was eliminated by washing the extract with 8 ml 0.5 M ammonium hydroxide solution. The green aqueous layer was discarded by aspiration using a Pasteur pipette and the extract was then washed with water to remove the rest of the dye. The water layer was aspirated using a Pasteur pipette and discarded. Finally, the extract was passed through a column (a cotton-plugged Pasteur pipette) filled to a height of approximately 3 cm with water-free sodium sulfate and then washed with diethyl ether. The eluant was collected in a fresh tube and then evaporated to dryness under a stream of nitrogen gas. The dry extract was dissolved in 50–100 µl methanol and 1–4 µl was used for GC-MS. The fatty acids were separated, identified and quantified by GC-MS using Shimadzu apparatus (QP2010) equipped with a 30.0 m capillary column (HP-1 Fa; Agilent) coated with a thin film of dimethylpolysiloxane as the stationary phase. The respiratory quinones were extracted and purified according to Collins et al. (1977). Mass spectral analyses of the quinones were recorded in positive-ion mode on a Q-TOF 2 mass spectrometer (Micromass) equipped with a nanospray source, as described by Yassin & Hupfer (2006). For the compounds under study, the major ions observed with electrospray were protonated pseudo-molecular ions, [M+Na]+. The identity of the ubiquinone was verified by observing the diagnostic ion at m/z 197, which represents the benzylium ion.
DNA was isolated using an Ultra Clean microbial DNA isolation kit (MO BIO Laboratories) by using the method described in the manufacturer's protocol. The DNA G+C contents were determined by HPLC (Mesbah et al., 1989) using 8 phage as the reference. Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene, and purification of the PCR products were carried out using procedures described previously (Rainey et al., 1996). The purified PCR products were sequenced using a Taq DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems) as described in the manufacturer's protocol. An Applied Biosystems 310 DNA Genetic Analyzer was used for the electrophoresis of the sequence reaction products. The 16S rRNA gene sequences of recognized Lysobacter species (retrieved from GenBank) were added to the ARB database (Ludwig et al., 2004) and aligned using the respective tool of the ARB package. The resulting alignment was corrected manually and then evolutionary trees were inferred using maximum-parsimony (Fitch, 1971), neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981) methods. An evolutionary distance matrix was calculated using the correction of Jukes & Cantor (1969). The topologies of the resulting tree were evaluated in bootstrap analyses (Felsenstein, 1985) of the neighbour-joining method, based on 1000 resamplings.
Strain IMMIB APB-9T consisted of Gram-negative, non-spore-forming, rod-shaped cells. On BHI agar, nutrient agar and tryptone soya agar, colonies were mucoid and yellow in colour. The micro-organism produced a brown pigment that became more marked in older cultures, and is able to swarm and spread, by gliding, on agar plates incubated for 1 week. The micro-organism grew aerobically and was found to be catalase- and oxidase-positive. It hydrolysed casein, elastin, gelatin and hippurate, but not adenine, chitin, aesculin, guanine, hypoxanthine, keratin, starch, testosterone, tyrosine, Tween 80 or xanthine. The physiological properties of strain IMMIB APB-9T are given in the species description below. Biochemical characteristics (determined in this study) that can be used to distinguish strain IMMIB APB-9T from recognized Lysobacter species are given in Table 1.
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. The novel isolate forms a distinct subline within the genus Lysobacter, branching together with the type strains of L. concretionis and L. daejeonensis, an association underpinned by all of the tree-making algorithms and by a bootstrap percentage of 99 % in the neighbour-joining analysis. Strain IMMIB APB-9T displayed the highest sequence similarities with respect to L. concretionis (96.0 %) and L. daejeonensis (96.1 %). However, sequence divergence values of >3 % with respect to recognized lysobacterial species show unequivocally that isolate IMMIB APB-9T represents a hitherto unknown species (Stackebrandt & Goebel, 1994).
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5c (0.14 %), iso-C15 : 0 (40.87 %), n-C15 : 0 (0.83 %), iso-C16 : 0 (19.25 %), n-C16 : 17c (0.83 %), n-C16 : 0 (2.92 %), iso-C17 : 19c (5.84 %), iso-C17 : 0 (11.14 %), n-C17 : 0 (0.76 %) and n-C18 : 17c (0.56 %) as the major non-hydroxylated fatty acid methyl esters. In addition, analysis of the hydroxylated fatty acid fraction revealed the presence of iso-C11 : 0 3-OH (7.21 %), characterized through the fragment at m/z 103. These fatty acids, with some minor differences, were also detected in recognized members of the genus Lysobacter (Table 2a and b). Mass spectral analysis of the main isoprenoid components isolated from strain IMMIB APB-9T showed a strong peak at m/z 749.45 attributable to [M+Na]+ in the high-mass region. This corresponds to an ubiquinone with eight isoprene units (Q-8).
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). On the basis of both phenotypic and phylogenetic evidence, the isolate represents a novel species within the genus Lysobacter, for which the name Lysobacter defluvii sp. nov. is proposed.
Description of Lysobacter defluvii sp. nov.
Lysobacter defluvii [de.flu'vi.i. L. gen. neut. n. defluvii from outflow (sewage)].
Cells are non-motile, non-spore-forming rods (approximately 1–2 µm in length). Gram-negative and oxidase- and catalase-positive. Colonies are mucoid and yellow in colour. Produces a brown pigment that becomes more marked in older cultures. Growth occurs at temperatures in the range 22–37 °C. Salt concentrations in the range 0.0–6.0 % (w/v) NaCl are tolerated. Growth occurs on tryptone soya agar and nutrient agar but the micro-organism grows preferentially on BHI agar. Has the ability to swarm and spread on the surface of agar plates by means of gliding. Shows the salient chemotaxonomic characteristics of members of the genus Lysobacter. Fatty acids mainly comprise saturated and unsaturated fatty acids with straight and branched chains of the iso/anteiso type. The main hydroxylated fatty acid is iso-C11 : 0 3-OH. Hydrolyses casein, elastin, gelatin and hippurate, but not adenine, chitin, aesculin, guanine, hypoxanthine, keratin, starch, testosterone, tyrosine, Tween 80 or xanthine. Does not produce acid from L-arabinose, starch, amygdalin, D-glucose, glycogen, inositol, inulin, D-lactose, D-maltose, D-mannitol, D-melibiose, D-raffinose, D-rhamnose, D-ribose, D-sorbitol, D-sucrose, D-trehalose or xylose. Shows alkaline phosphatase activity, but is negative for arginine dihydrolase, 
-glucosidase, 
-glucosidase, -galactosidase, -galactosidase, -glucuronidase, N-acetyl--glucosaminidase, leucine aminopeptidase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, pyrazinamidase, pyrrolidonyl arylamidase, nitrate reductase and urease activities. Negative for acetoin, indole and H2S production. Assimilates trisodium citrate but not D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid or phenylacetic acid. The DNA G+C content of the type strain is 67.1 mol%.
The type strain, IMMIB APB-9T (=CCUG 53152T=DSM 18482T), was isolated from a soil sample taken from a municipal landfill site in Udupi, Karnataka State, India.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Christensen, P. & Cook, F. D. (1978). Lysobacter, a new genus of nonfruiting, gliding bacteria with a high base ratio. Int J Syst Bacteriol 28, 367–393.
Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef][Medline]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.[Abstract]
Gordon, R. E. (1966). Some criteria for the recognition of Nocardia madurae (Vincent) Blanchard. J Gen Microbiol 45, 355–364.
Gordon, R. E. (1967). The taxonomy of soil bacteria. In The Ecology of Soil Bacteria, pp. 293–321. Edited by T. R. G. Gray & B. Parkinson. Liverpool: Liverpool University Press.
Gordon, R. E. & Mihm, J. M. (1957). A comparative study of some strains received as nocardiae. J Bacteriol 73, 15–27.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Minnikin, D. E., Hutchinson, I. G., Caldicott, A. B. & Goodfellow, M. (1980). Thin-layer chromatography of methanolysates of mycolic acid-containing bacteria. J Chromatogr 188, 221–223.[CrossRef]
Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsiaceae fam. nov. Int J Syst Bacteriol 46, 1088–1092.
Riegel, P., de Briel, D., Prévost, G., Jehl, F. & Monteil, H. (1994). Genomic diversity among Corynebacterium jeikeium strains and comparison with biochemical characteristics. J Clin Microbiol 32, 1860–1865.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA–DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Yassin, A. F. (1988). Chemotaxonomische Untersuchungen zur vereinfachten Differenzierung und Identifizierung von aeroben Aktinomyzeten und Mykobakterien. Inaugural-Dissertation zur erlangung des Doktorgrades der Mathematische-Naturwissenschaftlichen, Fakultät der Rheinischen, Friedrich-Wilhelms-Universität, Bonn (in German).
Yassin, A. F. & Hupfer, H. (2006). Williamsia deligens sp. nov., isolated from human blood. Int J Syst Evol Microbiol 56, 193–197.
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