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1 Kuwait University, Department of Biological Sciences, Microbiology Division, P.O. Box 5969, 13060 Safat, Kuwait
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, 38124 Braunschweig, Germany
Correspondence
Reiner M. Kroppenstedt
kdt{at}dsmz.de
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). Martin & So (1969) isolated a strain of myxobacteria that could use autoclaved feathers and wool as sole C and N sources, but it was unable to attack untreated material. Among the actinomycetes, members of the genera Cellulomonas (Goodfellow, 1971), Streptomyces (Jensen, 1930) and Pilimelia (Kane, 1966) show keratinolytic activities. Species of the genus Pilimelia are well known for their ability to degrade keratin, as they are frequently isolated from soil using the hair bait technique. Species of this genus are easily differentiated from all other actinomycetes by their typical sporangia, which release zoospores when the mycelium is flooded with water (Vobis et al., 1986). Until now, there have been no reports concerning keratin-degrading Amycolatopsis spp. The aim of this study was to clarify the taxonomic position of a novel keratin-degrading Amycolatopsis isolate, strain D2T, by morphological, physiological, chemotaxonomical and molecular biological methods.
Strain D2T was isolated from marsh soil in Kuwait that had been baited with sterilized and defatted wool; the strain was deposited with the DSMZ as Amycolatopsis keratiniphila DSM 44409T. Determinations of morphological traits, colour of the aerial and substrate mycelia, and of soluble pigments were performed as described previously (Shirling & Gottlieb, 1966). Biochemical tests were done as described by Lechevalier et al. (1986). Strain D2T showed the typical macroscopic and microscopic appearance of a member of the genus Amycolatopsis (Henssen et al., 1987; Kothe et al., 1989; Embley, 1992; Holt et al., 1994), having dirty-white aerial mycelium which became light-grey in ageing cultures grown on GYM medium (4 g glucose l-1, 4 g yeast extract l-1, 10 g malt extract l-1). No specific pigments were produced by the strain. In older cultures, aerial hyphae disintegrated into spore-like structures.
Cell material used for chemotaxonomic analyses was obtained in trypticase soy broth (BBL) after 4 days incubation of strain D2T at 28 °C on a rotary shaker. The cell material was harvested by centrifugation and washed twice with distilled water. Analyses of amino acids and sugars were carried out using the methods of Staneck & Roberts (1974). Menaquinones and polar lipids were extracted following the procedure of Minnikin et al. (1984). Polar lipids and menaquinones were analysed by TLC (Minnikin et al., 1977) and HPLC (Kroppenstedt, 1982, 1985), respectively. Analyses of fatty acids were performed following the methods of Miller (1982) and Sasser (1990). The presence of mycolic acids was checked by the method of Minnikin et al. (1975). Whole-cell hydrolysates of strain D2T contained meso-diaminopimelic acid as the diamino acid of the peptidoglycan and galactose and arabinose as the major cell-wall sugars. Menaquinone with a tetrahydrogenated isoprenoid chain of nine isoprene units [MK-9(H4)] was found as the dominant isoprenoid quinone. The polar lipid pattern was composed of phosphatidylinositol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine and diphosphatidylglycerol. This pattern matched quite well with the patterns for other Amycolatopsis spp. reported by Lechevalier et al. (1986). The fatty acid pattern was composed mainly of iso-/anteiso-branched fatty acids. Of diagnostic value is the combination of 10-methyl-branched iso-branched fatty acids together with 2-hydroxy-branched fatty acids (fatty acid pattern 3f sensu Kroppenstedt & Evtushenko, 2002), i.e. iso-C14 : 0 (13·9 %), iso-C15 : 0 (8·0 %), iso-C16 : 0 (33·5 %), iso-C17 : 0 (0·8 %), anteiso-C15 : 0 (1·9 %), anteiso-C17 : 0 (1·3 %), 10-methyl-iso-C17 : 0 (1·0 %), 2-hydroxy-iso-C16 : 0 (7·4 %), 2-hydroxy-anteiso-C15 : 0 (0·9 %), C15 : 0 (7·1 %), C16 : 0 (2·0 %), C16 : 1 (4·9 %), C17 : 0 (7·6 %) and C17 : 1 (9·9 %). Mycolic acids were not detected. All chemotaxonomic properties of strain D2T were consistent with its classification in the genus Amycolatopsis (Lechevalier et al., 1986; Henssen et al., 1987; Yassin et al., 1993).
Genomic DNA extraction, PCR-mediated amplification of the 16S rDNA and purification of PCR products were carried out as described previously (Rainey et al., 1996). The AE2 editor (Maidak et al., 1999) was used to align the almost-complete 16S rDNA sequence of strain D2T (1439 nt) against the 16S rDNA gene sequences of representatives of the main actinobacterial lineages and then against members of the genus Amycolatopsis. Phylogenetic analyses (De Soete, 1983; Felsenstein, 1993) followed described methods. The almost-complete 16S rDNA sequence of strain D2T, consisting of 1439 nt, was compared to 16S rDNA sequences of members of the class Actinobacteria. With intrageneric relationships ranging between 99·4 and 94·3 % sequence similarity, strain D2T was most closely related to members of the genus Amycolatopsis, especially to Amycolatopsis japonica (99·4 % sequence similarity). A phylogenetic dendrogram (Fig. 1) was constructed from a distance matrix using the treeing algorithm of De Soete (1983). Distance-matrix and maximum-likelihood analyses gave consistent results, in that strain D2T clustered with the type strains of A. japonica, Amycolatopsis azurea and Amycolatopsis orientalis (Fig. 1).
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) and could grow in the presence of 5 % NaCl. The strain grew at 10 °C but not at 45 °C; the optimal growth temperature was between 25 and 28 °C. In addition, strain D2T could grow on feathers as sole C and N sources, and tolerated 25 % NaCl for growth. Based on these properties, strain D2T could be distinguished from its closest phylogenetic relatives (Table 1).
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. DNA–DNA hybridization was carried out as described by De Ley et al. (1970), with the modifications described by Huss et al. (1983) and Escara & Hutton (1980), using a Gilford System model 2600 spectrometer equipped with a Gilford model 2527-R thermoprogrammer and plotter. Renaturation rates were computed with the TRANSFER.BAS program (Jahnke, 1992). The DNA–DNA relatedness of strain D2T to A. japonica was 59·5 %, indicating that they were distinct species based on the criteria set forth by Wayne et al. (1987) for strains of the same species. Based on phenotypic and genotypic data, we conclude that strain D2T merits separate species status within the genus Amycolatopsis. We, therefore, propose the name Amycolatopsis keratiniphila for strain D2T, the sole strain of this species.
Description of Amycolatopsis keratiniphila sp. nov.
Amycolatopsis keratiniphila (ke.rat.i.ni'phi.la. N.L. n. keratinum keratin; Gr. adj. philos loving; N.L. adj. keratiniphila keratin-loving, referring to the ability of the species to degrade keratin).
Aerobic, Gram-positive, non-motile actinomycete, which forms light-grey aerial mycelium. No specific endo- or exopigments are produced. In ageing cultures, aerial and substrate mycelia disintegrate into spore-like structures. Optimal growth is obtained on GYM medium (4 g glucose l-1, 4 g yeast extract l-1, 10 g malt extract l-1) at 28 °C. Whole-cell hydrolysates contain meso-diaminopimelic acid, arabinose and galactose. The predominant menaquinone is MK-9(H4). The polar lipids are phosphatidylinositol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine and diphosphatidylglycerol. Major fatty acids are iso-C16 : 0 (33·5 %), iso-C14 : 0 (13·9 %), C17 : 1 (9·9 %), iso-C15 : 0 (8·0 %), C17 : 0 (7·6 %), 2-hydroxy-iso-C16 : 0 (7·4 %) and C15 : 0 (7·1 %); minor fatty acids are C16 : 1 (4·9 %), C16 : 0 (2·0 %), anteiso-C15 : 0 (1·9 %), anteiso-C17 : 0 (1·3 %), 10-methyl-iso-C17 : 0 (1·0 %), 2-hydroxy-anteiso-C15 : 0 (0·9 %) and iso-C17 : 0 (0·8 %). Mycolic acids are not synthesized. Other characteristics of the species can be found in Table 1
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The type strain of Amycolatopsis keratiniphila is D2T (=DSM 44409T=NRRL B24117T). Isolated from agricultural soil in Kuwait using animal wool as bait.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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