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Gordonia Polyisoprenivorans Sp. Nov., a Rubber-Degrading Actinomycete Isolated from an Automobile Tyre

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Gordonia Polyisoprenivorans Sp. Nov., a Rubber-Degrading Actinomycete Isolated from an Automobile Tyre International Journal of Systematic Bacteriology (1 999): 49, 1785-1 791 Printed in Great Britain Gordonia polyisoprenivorans sp. nov., a rubber-degrading actinomycete isolated from an automobile tyre Alexandros Linos,’ Alexander Steinbuchel,’ Cathrin Sproer2 and Reiner M. Kroppenstedt’ Author for correspondence: Reiner M. Kroppenstedt. Tel: +49 531 2616 227. Fax: +49 531 2616 418. e-mail : [email protected] lnstitut fur Mikrobiologie, A rubber-degrading bacterium (strain KdZT)was isolated from fouling tyre Universitat Munster, water inside a deteriorated automobile tyre. The strain was aerobic, Gram- CorrensstraBe 3, 48149 Munster, Germany positive, produced elementary branching hyphae which fragmented into rodkoccus-li ke elements and showed chemotaxonomic markers which were 2 DSMZ - Deutsche Sammlung von consistent with the classification of Gordonia, i.e. meso-diaminopimelicacid, Mikroorganismen und N-glycolyl muramic acid, arabinose and galactose as diagnostic sugars, a fatty Zellkulturen, Mascheroder acid pattern composed of unbranched saturated and monounsaturated fatty Weg 1b, 381 24 B raunsc hweig, Germany acids with a considerable amount of tuberculostearic acid, and mycolic acids comprising 58-66 carbon atoms with two principal mycolic acids C,, and C,, counting for over 60%. Results of 16s rDNA analyses as well as chemotaxonomic results, led to the conclusion that Gordonia sp. strain KdZT (= DSM 443023 represents a new species within the genus Gordonia for which the name Gordonia polyisoprenivorans is proposed. Keywords: Gordonia polyisoprenivorans sp. nov., polyphasic taxonomy, rubber degradation INTRODUCTION and galactose in whole cell hydrolysates, cell wall type IV according to Lechevalier & Lechevalier (1970), Since the reclassification of Rhodococcus aichiensis and mostly N-glycolyl muramic acid in the peptidoglycan Nocardia amarae (Klatte et al., 1994b) in the genus wall (Uchida & Aida, 1977), menaquinones with a Gordonia, this taxon is now a well-defined genus partly saturated isoprenoid side chain (Kroppenstedt, among the so-called CMN group (Corynebacteriurn, 1982, 1985) and fatty acid patterns composed of Mycobacterium, Nocardia), i.e. bacteria which are able unbranched saturated and unsaturated fatty acids plus to synthesize mycolic acids (high-molecular-mass tuberculostearic acid (TBSA) (Kroppenstedt, 1985). alpha-branched 3-hydroxy fatty acids). The results of Although the overall chemical markers of members of 16s rDNA sequence analyses have shown that the the Corynebacterineae are roughly the same, the fine mycolic acid-producing bacteria, i.e. Corynebacterium, differences in the chemical markers can be used for Dietzia, Rhodococcus, Nocardia, Skermania, Coryn- genus and species differentiation (Klatte et al., 1994a, ebacterium, Tsukamurella and Mycobacterium, form 1994b, 1996). The best separation of Gordonia from one homogeneous cluster among actinomycetes. Based the other taxa is obtained using a combination of the on these results, the genera were combined into a new menaquinone type and the mycolic acid chain length. suborder of the order Actinomycetales, the Coryn- Gordonia synthesizes menaquinone MK-9(H2) and ebacterineae (Stackebrandt et al., 1997). Members of mycolic acids of a carbon atom chain length ranging this taxon have many other chemotaxonomic markers from C50 to c66 whereas Corynebacterium produces in common, meso-diaminopimelic acid and arabinose MK-8(H2),some MK-9(H2)and C22-C:36,Dietzia MK- 8(H2) and C34-C38, Rhodococcus MK-8(H2) and C34WC52, Nocardia MK-8(H4,w,,,,.) and C44-C60,Tsuk- Abbreviations: MlT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra- amurella MK-9 and C64-C68,and Mycobacterium MK- zolium bromide; TBSA, tuberculostearic acid. 9(H2) and C70-C90.The homogeneity of this taxon has The EMBL accession number for the 165 rDNA sequence of strain KdZT is also been proved by 16s rDNA sequence data (Rainey Y18310. et al., 1995). 01073 0 1999 IUMS 1785 A. Linos and others Originally, the Gordonia species, i.e. Gordonia (Oxoid)] at 28 "C. For biochemical tests, strain Kd2T was aichiensis, Gordonia bronchialis and Gordonia sputi, harvested from GYM agar after 3 d. Morphological studies have been regarded primarily as opportunistic and colour examination were carried out after 1, 3 and 7 d. pathogens because they have been found in sputum For analysis of the fatty acid and mycolic acid composition, strain Kd2T was grown on TSB agar [3 YO(w/v) Trypticase of humans suffering from pulmonary lesions soy broth (BBL), 1.5 % (w/v) Bacto Agar (Difco)] for 4 d at (Tsukamura, 197 1, 1978, 1982). Later, gordoniae were 28 "C. For cell wall and quinone analysis, cells were grown in frequently isolated from the environment : Gordonia Trypticase soy broth for 4 d at 28 "C on a rotary shaker, rubropertincta (Heferan, 1904) and Gordonia terrae harvested by centrifugation and washed twice with distilled from soil (Tsukamura, 197 1) ; Gordonia rhizosphera water. from mangrove rhizosphere (Takeuchi & Hatano, 1998); Gordonia amarae and Gordonia sp. from foams Macroscopic and microscopic studies. Macroscopic and and in scumming activated sludge of sewage plants microscopic studies and the staining procedures followed the instructions of Lefford (1980). (Lechevalier & Lechevalier, 1974 ; Lemmer & Kroppenstedt, 1984; Sodell & Seviour, 1995); and Biochemical characterization. Carbon source ,utilization and Gordonia hydrophobica and Gordonia hirsuta from the quantitative enzyme tests were performed in standard packing material (tree bark compost) of biofilters used microtitration plates (F-form ; Greiner) as described by for the treatment of malodorous animal rendering Kampfer et at. (1990) with the following modifications: emission (Bendinger et al., 1995; Klatte et al., 1996). carbon source utilization test medium Yeast-Nitrogen-Base The isolation of several rubber-degrading gordoniae (DIFCO), yeast extract (0.02 g l-'), K,HPO, (1.74 g l-'), KH,PO, (0.36 g 1-l); enzyme test medium, 0.05 M Tris/HCl strains from tyre water inside a deteriorated auto- buffer, 0.05 % (w/v) Casamino acids (DIFCO), 0.05 % (wlv) mobile tyre and from soil of a rubber tree plantation yeast extract (DIFCO). The panels were inoculated with in Vietnam was previously reported (Linos & standardized bacterial suspension of 0.5-1.0 McFarlane Steinbuchel, 1996, 1998). The aim of this study was to units in 0.9% (w/v) NaCl, and then incubated for 24 h at clarify the taxonomic position of one of these strains, 28 "C. After addition of 50 pl filter-sterilized indicator Gordonia sp. strain Kd2T (= DSM 44310T). solution [4 mM MTT (3-[4,5-dimethylthiazol-2-y1]-2,5- diphenyltetrazolium bromide)] and phenazine methosulfate Like the other taxa of the suborder Corynebacterineae, (Sigma) to the auxanographic test and control wells, Gordonia plays an important role in bioremediation incubation continued for 24 h in the dark. The formation of and biodegradation of pollutants in the environment. the deep-blue coloured formazane was taken as an indication Some of the isolates have been used for the decon- for carbon source utilization (Kirchhof et al., 1992). Reading tamination of polluted soils; others are used for of the auxanographic test results was carried out photo- different biotechnological processes in industry (Bell et metrically by a Multiscan MCC340 MKII photometer (Flow al., 1998). Before these organisms can be grown on a Laboratories). Metabolism of a carbon source was con- greater scale, a risk assessment for these strains has to sidered positive if A,,, of the sample was greater than 0.129 be applied. A reliable taxonomy for this taxon is (control). The results of the enzyme tests were evaluated therefore essential to avoid any risk for humans and visually. the environment. Chemotaxonomic studies Analysis of cell wall amino acids and sugars. The amino acid and METHODS sugar analysis of whole-cell hydrolysates followed the described procedures of Stanek & Roberts (1974). Bacterial strain and culture conditions. During a screening programme for rubber-degrading micro-organisms, strain Determination of the acyl type of the peptidoglycan. The murein Kd2' was isolated from fouling tyre water inside a acyl type was determined by a modification of the colori- deteriorated automobile tyre on a farmer's field in Munster, metric method of Uchida & Aida (1977). In contrast to Germany as described previously (Linos & Steinbuchel, the original procedure, our whole-cell hydrolysate was 1998). The isolate was able to solubilize and mineralize neutralized by passing it through an ion-exchange column natural rubber substrates but also grow on synthetic cis- 1,4- (Analytichem Bond Elut SCX; Varian). polyisoprene after removal of antioxidants (added during manufacture against ageing) by organic solvent extraction. Extraction and analysis of isoprenoid quinones and polar lipids. It grew adhesively on a layer of natural latex concentrate Isoprenoid quinones were extracted and purified using the that was spread on mineral salt medium agar plate as a thin small-scale integrated procedure of Minnikin ( 1984). Dried layer, but not when latex was dispersed into the agar at a preparations were dissolved in 200 pl2-propanol and 1-10 p1 concentration of 0.02 Yo (w/v). Cells occurred suspended in amounts were separated by HPLC without further purifi- the medium when they were cultivated with squalene (trans- cation. The menaquinones were separated by HPLC on hexamer of isoprene), L-rhamnose or Standard I nutrient Lichrosorb RP-18 at 40 "C using acetonitrile/2-propanol
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