International Journal of Systematic Bacteriology (1998), 48, 441-449 Printed in Great Britain Pseudonocardia asaccharolytica sp. nov. and Pseudonocardia sulfidoxydans sp. nov., two new dimethyl disulf ide-degrading actinomycetes and emended description of the genus Pseudonocardia Katrin Reichert,’ Andre Lipski,’ Silke Pradella,2 Erko Stackebrandt2 and Karlheinz Altendorf’ Author for correspondence: Andre Lipski. Fax: +49 541 969 2870. e-mail : Lipski@sfbbiol .biologie.uni-osnabrueck.de 1 Abteilung Mikrobiologie, Seven bacterial strains capable of oxidizing methyl sulfides were isolated from Universitat Osnabruck, experimental biofilters filled with tree-bark compost. The isolates could be Fachbereich BiologieKhemie, D-49069 divided into two groups according to their method of methyl sulfide Osnabruck, Germany degradation. Four isolates could use only dimethyl disulfide as the sole source * Deutsche Sammlung von of energy and three strains were able to use dimethyl sulfide and dimethyl Mikroorganismen und disulfide. Oxidation of the methyl sulfides by both groups led to the Zellkulturen GmbH, stoichiometric formation of sulfate. Chemotaxonomic, morphological, Mascheroder Weg 1b, D-38124 Braunschweig, physiological and phylogenetic properties identified all isolates as members of Germany the genus Pseudonocardia. The absence of phosphatidylcholine from the polar lipid pattern, as well as results of 16s rDNA analyses, led to the proposal of two new species, Pseudonocadia asaccharolytica sp. nov. and Pseudonocardia sulfidoxydans sp. nov. The type strains are P. asaccharolytica DSM 44247Tand P. sulfidoxydans DSM 44248T.With respect to the characteristic polar lipid pattern and the ability to oxidize sulfides, an emended description of the genus Pseudonocardia is proposed. Keywords : Pseudonocardia asaccharolytica sp. nov., Pseudonocardia sulJidoxydans sp. nov., methyl sulfide-degrading actinomycetes, biofilters INTRODUCTION enrichment, isolation and investigation of degrading microorganisms is a prerequisite for an understanding Volatile organic sulfur compounds such as dimethyl of the degradation mechanism and provides pure disulfide (DMDS), dimethyl sulfide (DMS) and cultures for inoculation approaches. Previous efforts methanethiol are widely distributed in various mi- for enrichment of methyl sulfide degraders led to the crobial environments. They are produced by microbial isolation of strains of the genera Thiobacillus (15,29), degradation of sulfur-containing amino acids or by Comamonas (36) and Hyphomicrobium (26). Up to thermal decomposition. Because methyl sulfides have now, all known methyl sulfide-degrading bacteria are a low odour threshold, the occurrence of these sub- members of the class Proteobacteria. The approach stances in industrial emissions causes serious odour used in this study led to the isolation of methyl sulfide- problems for these industries. To remove odorous oxidizing actinomycetes with morphological and compounds, biological deodorization processes like chemotaxonomic properties of the family Pseudo- biofiltration have been developed and are well es- nocardiaceae. This family was defined on the basis of tablished. Removal of methyl sulfides by this technique 16s rRNA sequence analyses for organisms with type is possible, but the rates are sometimes low (34). The IV cell walls (meso-diaminopimelic acid and sugar pattern A with arabinose and galactose as diagnostic Abbreviations: DMDS, dimethyl disulfide; DMS, dimethyl sulfide; FAMEs, sugars) without mycolic acids (7). It encompasses fatty acid methyl esters. the genera Actinopolyspora, Amycolatopsis, Kib- The EMBL accession numbers for the sequences reported in this paper are delosporangium, Pseudonocardia, Saccharomonospora YO8534-YO8537. and Saccharopolyspora (35). The names Amycolata 00666 0 1998 IUMS 441 K. Reichert and others and Pseudoamycolata (1) were shown to be junior The slopes were incubated at 25 "C until growth could be syn-onyms of the name Pseudonocardia based on seen visually. Colonies were subcultivated on agar slopes. 16s rRNA data (23, 35). Recently, the family Isolation of single strains was performed and strain purity Pseudonocardiaceae was extended by the genera was checked on TSB agar. Actinosynnema, Kutzneria, Lentzea, Saccharothrix, Reference strains. Strains Pseudonocardia autotrophica DSM Streptoalloteichus and Thermocrispum based on char- 535T, Pseudonocardia compacta DSM 43592T, Pseudono- acteristic 16s rDNA signature nucleotides (30). The cardia halophobica DSM 43089Tand Pseudonocardia hydro- genera of this family vary in morphology and several carbonoxydans DSM 4328 lT were used for comparing chemotaxonomic markers. Based on these properties chemotaxonomic, morphological and physiological proper- sequences, the ties. Reference strains for phylogenetic analyses are listed and analysis of the 16s rDNA isolates below. were identified as new species of the genus Pseudo- nocardia. Morphology and physiology. Morphological characteristics of the isolates and reference strains were observed by phase- contrast microscopy of mycelia grown for 14 d on TSB agar. METHODS A Cambridge Stereoscan model 250 microscope was used for scanning electron microscopy. Physiological charac- Media. The basal medium that was used for enrichment, teristics were studied as described by Gordon et al. (10) isolation and methyl sulfide degradation experiments con- except for tolerance of NaCl which was determined on tained : NH,Cl, 1.0 g 1-l; MgC1,. 6H,O, 0.5 g 1-l; KH,PO, .- peptone/yeast extract agar (9) after incubation for 21 d. 3H,O, 0.4 g 1-1 ; K,HPO, .7H,O, 0.6 g 1-1 ; CaCl, .2H,O, 0.2 g 1-l; FeC1,. 6H,O, 20 mg 1-l; Na,-EDTA, 50 mg 1-l; Chemotaxonomic analyses. For the analysis of whole-cell pH 6.6. Yeast extract (0.01 YO)was added as an additional fatty acids, cells were grown for 14 days at 30 "C in TSB and substrate for enrichment cultures. For isolation, the medium harvested by filtration. Wet cell material (100 mg) was was solidified with 15-0g 1-1 agar. For degradation experi- saponified, methylated and the fatty acid methyl esters ments, yeast extract was replaced by 1 ml 1-1 trace element (FAMEs) were extracted according to Sasser (28). Identi- solution (Na,-EDTA, 3.0 g 1-1 ; MnC1,. 2H,O, 50 mg 1-1 ; fication of the FAMEs was performed as described pre- CoC1,. 6H,O, 190 mg 1-1 ;ZnCl,, 4 1 mg 1-1 ;H,BO,, 6 mg 1-1 ; viously (21). Extraction of polar lipids and isoprenoid NiCl, .6H,O, 24 mg l-l, CuCl, .2H,O, 2 mg 1-l; Na,MoO, . - quinones and analysis of the polar lipids by TLC were 2H,O, 18 mg 1-l; pH 6.0) and 5 ml 1-1 vitamin solution performed using the method of Minnikin et al. (25). (thiamin, 10 mg 1-l; nicotinic acid, 20 mg 1-l; pyri- Phosphatidylcholine was identified using Dragendorff s re- doxine. HCl, 20 mg 1-1 ; p-aminobenzoic acid, 10 mg 1-1 ; agent (31). The menaquinone extract (40 p1) was analysed riboflavin, 20 mg 1-l; pantothenic acid, 20 mg 1-l; biotin, using a Hewlett-Packard 1050 series model HPLC as 1 mg 1-l; cyanocobalamin, 1 mg 1-l; pH 7-0). DMDS described by Kroppenstedt (16). Mycolic acids were (1.0 mM) or DMS (03 mM) were also added. The isolated extracted and analysed by the method of Minnikin et al. strains were maintained on Trypticase soy agar (TSB agar ; (24). Whole-cell hydrolysates were analysed for diamino- Becton Dickinson). pimelic acid isomers as described by Becker et al. (2) and for sugars using the method of Lechevalier (18) modified by Enrichment and isolation procedure. For the enrichment of Staneck & Roberts (32). The acyl type of the peptidoglycan methyl sulfide-degrading bacteria, tree-bark compost from was analysed by the method of Uchida & Aida (33). biofilters was used as an inoculum. These biofilters were supplied with methyl sulfide containing off-gas from an 165 rDNA sequencing. Extraction of genomic DNA from animal-rendering plant. Tubes (50 ml) sealed with Teflon- Pseudonocardia alni DSM 44104T, P. halophobica DSM lined screw caps were filled with 15 ml mineral salts medium 43089Tand the isolates 580 (deposited as DSM 44247T)and as described above. The tubes were incubated at 25 "C for 592 (deposited as DSM 44248T)and amplification of the 16s several weeks and cultures were then transferred to fresh rDNA were carried out as described previously (27). PCR media. Aliquots of the media were used for subsequent products were sequenced directly using the Taq DyeDeoxy cultivation and tested for growth and sulfate formation. The Terminator Cycle Sequencing kit (Applied Biosystems), sulfate-producing cultures were subcultivated and tested for according to the manufacturer's protocol. The sequence methyl sulfide degradation and sulfate and acid production. reactions were electrophoresed using the Applied Biosystems Degradation of methyl sulfides was tested by headspace GC- 373A DNA Sequencer. Sequences were manually aligned MS with a Hewlett-Packard model 5890 series I1 gas with published sequences from representatives of the main chromatograph equipped with a 5 YOphenyl methyl silicone actinomycete sublines of descent included in the Ribosomal capillary column (0.25 mm x 30 m) and a model 5972 mass Database Project (22) and from entries in the DSMZ selective detector. Helium was used as carrier gas and the database of 16s rDNA sequences. Pairwise evolutionary injection volume was 100 pl headspace gas. The injector distances were computed using the correction of Jukes & temperature was 120 "C, the column temperature was 50 "C Cantor (14). Bootstrap values, based on analysis of 300 trees and