Pseudonocardia Asaccharolytica Sp. Nov. and Pseudonocardia Sulfidoxydans Sp

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 the GC-MS transfer line temperature was 280 "C. of 653 polymorphic sites, were calculated using the programs Sulfate formation was detected photometrically after pre- NJFIND and NJBOOT. Final phylogenetic analysis was per- cipitation with BaC1, and acid production was measured formed with the closest phylogenetic neighbours using using a pH electrode. After several subcultivations with reference 16s rDNA and 16s rRNA sequences of the repeated sulfate production in the cultures, the subcultures following organisms (accession numbers are given in par- were transferred to enrichment medium agar slopes. Samples entheses) : Pseudonocardia saturnae DSM 43 195T(X78956) ; (0-1 ml) were taken from the subcultures and streaked on the Pseudonocardia thermophila DSM 43832T (X53 195); P. agar slopes. Slopes were made in the same tubes used for hydrocarbonoxydans DSM 4328 lT(X76955) ;P. autotrophica enrichment cultures. Methyl sulfides were added as pure DSM 535T(X54288); P. compacta DSM 43592T;Pseudono- liquid. Evaporation of the volatile substance resulted in a cardia petroleophila DSM 43 193T(X55608) ; Amycolatopsis methyl sulfide equilibrium between the gas and solid phases. fastidiosa DSM 43855T (X53200); Amycolatopsis meth-

~ ~~ 442 International Journal of Systematic Bacteriology 48 Two new Pseudonocardia spp.

anolica DSM 4409fjT (X54274) ; Amycolatopsis orientalis Morphology subsp. orientalis DSM 40040T (X76958) ; Amycolatopsis mediterranei ATCC 13695T (X6957) ; Amycolatopsis azurae All seven isolated strains showed a yellow substrate DSM 43854* (X53 199); and Saccharothrix australiensis mycelium, whereas the reference strains P. halophobica DSM 43800T (X53 193). Phylogenetic analyses were carried DSM 43089T and P. autotrophica DSM 535T showed out using the algorithm of De Soete (6). brown substrate mycelium and the mycelium of the DNA isolation and hybridization. DNA was isolated by strains P. compacta DSM 43592T and P. hydro- chromatography on hydroxyapatite by the procedure of carbonoxydans DSM 4328 1 was yellowish brown. All Cashion et al. (4). DNA-DNA hybridization was carried seven isolates and the four reference strains showed out according to De Ley et al. (5) with modifications as fragmentation of the substrate mycelium into rod and described by HUB et al. (12) and Escara et al. (8) using a coccoid elements, swollen hyphal segments, zigzag Gilford System 2600 spectrophotometer equipped with a mycelial growth, and longitudinal and transverse septa Gilford 2527-R thermoprogrammer and plotter. Renatur- in the mycelium. All isolates and reference strains ation rates were computed by the program TRANSFER.BAS showed white aerial mycelium. With the exception of (1 3). P. halophobica DSM 43089T, all isolates and reference Accession numbers. The 16s rRNA sequences are available strains had spore chains with more than ten spores. from EMBL under accession numbers YO8535 (P.alni DSM The thickness of the mycelium of the isolates was 44104T), YO8534 (P. halophobica DSM 43089T), YO8536 0.5-1.0 pm and hyphal segments were sometimes (Pseudonocardia asaccharolytica DSM 44247T) and YO8537 swollen up to 2 pm for strain 580 (= DSM 44247T) (Pseudonocardia suljidoxydans DSM 44248T). and up to 5 pm for strain 592 (= DSM 4424ST) (Fig. 1). The spore surface of isolate 580 was smooth (Fig. RESULTS 2). Isolation of methyl sulfide degrading strains Chemotaxonomic properties The enrichment procedure used led to the isolation of seven bacterial strains from the filter material samples. The composition of sugars and the diamino acid Four strains (576, 577, 578 and 580) were isolated pattern showed that all isolated strains had a type IV using DMDS as sole source of energy and sulfur and cell wall (meso-diaminopimelic acid, arabinose and were named group I. Three strains (59 1, 592 and 593) galactose). No mycolic acids were present. The pep- were isolated using DMS and were named group 11. tidoglycan was of the acetyl type. The principle

...... Fig. 1. Scanning electron micrograph of a 14-day-old culture of strain 592 (= DSM 442483 grown on TSB agar. Bar, 5 prn.

International Journal of Systematic Bacteriology 48 443 K. Reichert and others

...... Figrn2. Scanning electron micrograph of a 14-d-old culture of strain 580 (= DSM 44247') grown on TSB agar. Bar, 5 pm,

Table 1. Cellular fatty acid composition (%) of the new strains and Pseudonocardia reference strains

Fatty acid abbreviation examples: 10: 0, decanoate; 12 :0 iso, iso-branched dodecanoate; 15: 0 anteiso, anteiso-branched pentadecanoate ; 15 : 1, pentadecenoate ; 16 : 1 cis9, cis-9-hexadecenoate; 16 :0 10-methyl, 10-methyl hexadecanoate ; 16 :0 is0 2-OH, iso-branched, 2-hydroxyhexadecanoate.

~ Fatty acid P. asaccharolytica P. su&doxydans P. halophobica P. autotrophica P. compacta P. hydrocarbonoxydans (group I, n = 4) (group 11, n = 3) DSM 43089' DSM 539 DSM 43592T DSM 4328IT

8:O 06-1.0 0.3 lo:o 0.243 12:O is0 03 12:o 0.143 0.3 13:O is0 0.1-0.3 02 13.0 0+0.2 14:O is0 I &2.0 1'1-3.4 08 0.3 5.7 3.6 14:O 1'7-3'0 06-3.0 0.3 1.1 3.3 15:O is0 8.2-1 1.4 11.2-164 2% 3.8 2.6 10.9 15 :0 anteiso 0.8- 1'3 0.5-1.7 0.9 0.4 1.6 15: 1 0.6 0.4 0.2 15:O 1.2-26 1.3-2.9 2.0 0.8 1.9 2.2 16: 1 is0 cis9 05-1.6 064.3 4.7 6.0 5.5 0.7 16:O is0 213-28.1 24.8-32'3 52.1 52.1 51.0 300 16: 1 cis9 24-3.3 1.3-6.0 7.5 2.4 5.1 5.2 16:O 193-23.3 15.0-303 6.5 4.4 11.8 2.54 15 :0 10,14-dimethyl 0.1-6.9 0.7 0.6 08 0.5 17: 1 is0 cis9 OM3 09-1.0 0.8 3.4 0.4 1.1 16:O 10-methyl 08-1'4 1.44.1 4.6 2.5 2.9 2.5 17:O is0 11.0-12.7 1'9-9.3 2.4 6.9 1.3 2.7 17 :0 anteiso 4+6.1 3.64.7 6.1 2.1 1.2 2.3 17: 1 cis9 1.7-2.3 0.64.5 2.4 52 2.4 1.4 16:O is0 2-OH 0.M1 0.0-2.8 0.6 17:O 3.3-63 2.5-6.4 1.9 143 3.5 2.7 16:O 10,15-dimethyl 04-0.8 18: 1 is0 0&0.2 0.2 17:O 10-methyl 0.1-06 0.0-2.5 0.9 5.5 0.7 0.4 18:0 is0 06-1.0 0.0-0.3 0.6 04 0.3 0.2 18: 1 cis9 1.0-1-5 0.1 1.2 18:O 1.7-3'3 0.0-1.1 0.3 0.5 0.8 18:O 10-methyl 02

444 International Journal of Systematic Bacteriology 48 Two new Pseudonocardia spp.

Table 2. Polar lipid pattern of the isolates and reference strains of the genus Pseudonocardia ...... DPG, diphosphatidylglycerol ; PME, phosphatidylmethylethanolamine; PE, phosphatidylethanolamine ; PG, phosphatidylglycerol ; PI, phosphatidylinositol ; PIM, phosphatidylinositol mannoside ; PL, phospholipid ; OH-PE, hydroxyphosphatidylethanolamine;PC, phosphatidylcholine.

Polar lipid P. asaccharolytica P. sullfdoxydans P. halophobica P. autotrophica P. compacta P. hydrocarbonoxydans (group I, n = 4) (group II, n = 3) DSM 43089T DSM 535' DSM 43592' DSM 43281'

DPG + + + + + PME + + + + + PE + + - + - PG + + + + + PI + + + + + PIM + + + + + Ninhydrin-positive PL + + - - - OH-PE + + - - - PC - - + + + Phospholipid type* I1 I1 111 I11 I11 * According to Lechevalier et al. (19). t Two PIMs present.

Table 3. Physiological properties of the isolates and Pseudonocardia reference strains

Characteristic P. asaccharolytica P. sullfdoxydans P. halophobica P. autotrophica P. compacta P. hydrocarbonoxy&s (group I, n = 4) (group 11, n = 3) DSM 43089T DSM 535' DSM 43592' DSM 43281'

Urease production - - - + - - Acid production from: D( + )Glucose - + + + + + L( -)Rhamnose - - + - - + - D( + )xylOSe - + + + + D( -)FWCtOX - + + + + - D( + )Trehalose - + - + - + Cellobiose - - - + + + D( - )Mannitol - - + + + + D( - )Sorbit01 - - + + - - iso-Erythritol - - - + - - Maltose - - - + - - D( +)Lactose - - - - - + L( + )Arabinose - - - + - - Adonitol - - - + - - Salicin - - - - - + Decomposition of: Adenine - - - + - - L-Tyrosine - +* - - - + Xanthine - + + - - - Hypoxanthne - + + - - - Dimethyl sulfide + + - - - - Dimethyl disulfide - + - - - - Growth on: 3% NaCl - + + + - - 4% NaCl - - + + - - 5% NaCl - - weak + - - * Production of a brown pigment observed. menaquinone was tetrahydrogenated with eight iso- present in group I isolates. 16: 0 10,15-Dimethyl was prene units [MK-8(H4)].The fatty acid pattern was of not detected in any of the reference strains, whereas the iso/anteiso-branched type in which the iso- 15 :0 10,14-dimethyl was present in all reference branched and straight-chain hexadecanoate pre- strains. Small amounts of a 2-hydroxy fatty acid (16 :0 dominated. Saturated, unsaturated and 1O-methyl- is0 2-OH) were also detected. The phospholipid branched fatty acids were also present (Table 1). Small composition is shown in Table 2. The phospholipid amounts of tuberculostearic acid (18 :0 10-methyl) pattern of the isolated strains was type I1 according to were present only in group I. Two dimethyl fatty acids Lechevalier et al. (19) showing phosphatidylethanol- were found in strains of group 11, which could be amine and/or hy droxy pho spha tid y lethanolamine. identified by their mass spectra as 15 :0 10,14-dimethyl Phosphatidylcholine was not detected in the isolates or and 16:O 10,15-dimethyl. These fatty acids were not P. halophobica DSM 43089T, but was present in all

International Journal of Systematic Bacteriology 48 445 K. Reichert and others

Physiological properties Physiological properties are shown in Table 3. Twenty- - four properties have been studied. The isolates of eudonocardia petroleophila DSM 431 93T group I did not produce acid from any of the

- Pseudonocardia hydrocarbonoxydans DSM 43281 carbohydrates. The isolates of group I1 produced acid on D( +)glucose, D( +)xylose, D( -)fructose and udonocardia sulfidoxydans DSM 44248T D( + )trehalose. L-Tyrosine, xanthine and hypoxan- Pseudonocardia halophobica DSM 43089l thine were decomposed. The isolates of group I Pseudonocardia auto trophica DSM 535T degraded only DMS, whereas the isolates of group I1 -100 degraded both DMS and DMDS. None of the ref- Pseudonocardia compacta DSM 43592l -93 erence strains degraded any methyl sulfide studied. Pseudonocardia alni DSM 441 04T

Amycolatopsis fastidiosa DSM 438SST 93 Phylogenetic analyses Amycolatopsis methanolica DSM 44096l - -Amycolatopsis orientalis DSM 40040T The almost complete 16s rDNA sequences of P. - halophobica DSM 43089T, P. alni DSM 44104T, and those of isolate 580 (= DSM 44247T) and isolate 592 (= DSM 44248T),consisting of 1455 and 15 15 nucleotides, respectively, were compared to the sequences of the members of the genera Pseudonocardia and Fig. 3. Phylogenetic dendrogram obtained by distance matrix Amycolatopsis (Fig. 3). These organisms were deter- analysis showing the position of Pseudonocardia mined to be the closest phylogenetic neighbours. The asa ccha rolytica DS M 44247T and Pseudonoca rdia sulfidoxydans DSM 44248T among members of the genus Pseudonocardia and 16s rDNA sequences of P. alni DSM 44104T and P. the phylogenetically neighbouring genus Amycolatopsis. halophobica DSM 43089T were determined to provide Bootstrap values above 85% (expressed as percentages of 300 the database with more complete data than that replications) are shown at branch points. Bar, 0.02 estimated available for these strains. Sequence analyses were nucleotide changes per position. restricted to a stretch of about 1150 nucleotides [position 200-1 355, Escherichia coli nomenclature (3)] because several reference sequences lacked inform- other reference strains. An additional characteristic ation at the 5’ and 3’ termini. The phylogenetic position feature of the isolates and P. halophobica DSM 43089T of isolates 580 and 592 indicate that they are members was the presence of an unknown ninhydrin-positive of the genus Pseudonocardia. Binary similarity values phospholipid. Moreover, all strains contained diphos- of the 16s rDNA between the isolates and other p ha tid ylgl ycerol, pho spha tid ylme t hy le t hanolamine, members of the genus Pseudonocardia are 95-97 %, phosphatidylglycerol, phosphatidylinositol and phos- whereas those found between the two isolates and phatidylinositol mannoside. No glucosamine-contain- members of the neighbouring genus Amycolatopsis ing phospholipids were detected. were 052.8% lower. The binary value for the two

Table 4. Chemotaxonomic characteristics of the family Pseudonocardiaceae ...... No genus has mycolic acids; all genera have whole-sugar pattern type A (galactose and arabinose present), meso-diaminopimelic acid as the cell-wall diamino acid and the acetyl type of peptidoglycan. Data are from Lechevalier et al. (20), Akimov et al. (1) and this study.

Group or genus Major menaquinone(s) Fatty acids* Phospholipid type?

Groups I and I1 MK-8(H4) s, A, 1, M I1 Amycolatopsis MK-9(H,, H4) s, A, I, M I1 Actinopolyspora MK-9(H4) s, A, I, M I11 Kibdelosporangium ND A, 1 I1 Pseudonocardia MK-8(H4) s, A, I, M III/II Saccharomonospora MK-9(H4) s, A, 1 I1 Saccharopolyspora MK-9(H4) s, A, I, M I11 * S, Saturated and unsaturated fatty acids; A, anteiso-branched fatty acids; I, iso-branched fatty acids; M, methyl-branched fatty acids. t Type I1 phospholipid pattern, phosphatidylethanolamine or derivatives present ; type I11 phospholipid pattern, phosphatidylcholine present. ND, Not determined.

~ ~~ ~ 446 International Journal of Systematic Bacteriology 48 Two new Pseudonocardia spp.

isolates was 96-2%, which falls into the range deter- or may not be present. Both types of mycelium exhibit mined for other Pseudonocardia species (95.0- cell division in different directions and, in some species, the hyphae are covered by an electron-dense outer 98-5% similarity). Isolate 580 is equally related to the layer. Spore sizes vary and spores are normally other species of the genus Pseudonocardia (94-9-96-2YO smooth; chains of spores are formed by acropetal similarity), while isolate 592 is closely related to budding or septation from the substrate or aerial Pseudonocardia halophobica DSM 43089T (98.6 % mycelium. Non-motile. Biochemically versatile. Some similarity). To determine whether these two strains are species are facultative autotrophs and some strains can members of the same species, the extent of DNA oxidize hydrocarbons. Some species can oxidize similarity was determined by DNA-DNA hybridi- methyl sulfides to sulfate. P. thermophila can degrade zation; the value of 39% indicated that both strains cellulose. The major menaquinone is tetrahydro- belonged to different genospecies. genated with eight isoprene units [MK-8(H4)]. Iso- branched fatty acids are predominant. The main DISCUSSION compound is iso-branched hexadecanoic acid. Straight-chain hexadecanoic acid and 1O-methyl hexa- Seven bacterial strains were isolated from biofilters decanoic acid are also present. Phosphatidylmethyl- used for waste gas treatment of animal-rendering plant ethanolamine is present, occurrence of phosphatidyl- emissions. Their morphological, phenotypic, chemo- ethanolamine and phosphatidylcholine is variable taxonomic and phylogenetic features were analysed between the species. Cell wall type IV (arabinose, and all the strains were shown to be members of the galactose and meso-diaminopimelic acid as diagnostic family Pseudonocardiaceae. The genera of this family constituents). Mycolates are absent. The G + C content vary in morphology, polar lipid and menaquinone of the DNA is 68-79 mol %. Members of this genus composition, and cellular fatty acid pattern. These form a coherent group on the basis of 16s rRNA variations allow identification of the isolated strains at sequence data. The genus contains the species P. alni, the genus level. Diagnostic differences are shown in P. asaccharolytica, P. autotrophica, Pseudonocardia Table 4. The isolates of both groups have branching azurea, P. compacta, P. halophobica, P. hydro- hyphal structures with septa. Some hyphal segments carbonoxydans, P. petroleophila, P. saturnae, Pseudo- show intercalar or apical swellings and therefore nocardia spinosa, P. sulfdoxydans and P. thermophila. resemble the genus Pseudonocardia in morphology. The type species is P. thermophila. Also, the major quinone is menaquinone type MK- 8(H,), which is characteristic of the genus Pseudo- nocardia. The genus Kibdelosporangium differs in mor- Description of Pseudonocardia asaccharolytica sp. phology [formation of sporangia (1 7)]. Chemo- nov. taxonomic data were confirmed by analysis of the 16s rDNA sequence which placed the isolates into the Pseudonocardia asaccharolyt ica (a. sac.cha. ro .I y’ti .ca. genus Pseudonocardia. Within the genus Pseudo- Gr. pref. a not; M.L. n. saccharum sugar; Gr. adj. nocardia, only P. halophobica shows identical chemo- lyticum dissolving; M.L. fem. adj. asaccharolytica taxonomic characteristics to the isolated strains. The referring to the failure to produce acid from carbo- absence of phosphatidylcholine differentiated the iso- hydrates). lates from all other Pseudonocardia species. However, Forms a yellow vegetative mycelium on TSB agar that the 16s rDNA data and DNA-DNA hybridization fragments easily into rod-shaped and coccoid elements. data clearly differentiated the isolates from P. halo- Aerial mycelium is white with long chains of oval phobica. The results indicate that isolates 580 and 592 spores. Spore surface is smooth. Formation of swollen belong to the genus Pseudonocardia but are different hyphal segments with up to 2 pm diameter. Does not from previously described species. Therefore, two new decompose adenine, L-tyrosine, xanthine and hy- species, Pseudonocardia asaccharolytica and Pseudo- poxanthine. No growth with 3% NaCl. No acid nocardia sulfdoxydans, are proposed. The description production from glucose, rhamnose, xylose, fructose, of two new species of this genus which lack phospha- trehalose, cellobiose, mannitol, sorbitol, erythritol, tidylcholine supports the suggestion of McVeigh et al. maltose, lactose, arabinose, adonitol and salicin. (23) to emend the description of the genus Pseudo- Urease-negative. Oxidation of DMDS. The main nocardiawithrespectto thephosphatidylcholine-lacking menaquinone is MK-8(H4). Phosphatidylmethyl- species. Based on the descriptions of Warwick et al. ethanolamine, hydroxyphosphatidylethanolamine, (35) and our own data, we therefore suggest an phosphatidylinositol, phosphatidylinositol manno- emended description of this genus. side, diphosphatidylglycerol and phosphatidylglycerol are present. Major fatty acids are iso-branched hexa- Emended description of the genus Pseudonocardia decanoate and hexadecanoate. Small amounts of 10- Henssen 1957 (11) methyl fatty acids (16 :0 10-methyl, 17 :0 1O-methyl and 18 :O 10-methyl) are present. Strains 576, 577, 578 The vegetative mycelium varies in thickness (0.4- and 580 were isolated from tree-bark compost bio- 2.0 pm) and in the degree of branching. Some strains filters from an animal-rendering plant. The type strain form swollen hyphal segments. Aerial mycelium may is isolate 580 (= DSM 44247T).

International Journal of Systematic Bacteriology 48 447 K. Reichert and others

Description of Pseudonocardia sulfidoxydans sp. nov. mycetes and description of Pseudonocardiaceae fam. nov. Syst Appl Microbiol 11, 44-52. Pseudonocardia sulJidoxydans (sul.fid. ox’ y .dans. M .L. 8. Escara, J. F. & Hutton, 1. R. (1980). Thermal stability and fem. adj . sulJidoxydans oxidizing sulfides). renaturation of DNA in dimethylsulphoxide solutions : acceleration of renaturation rate. Biopolymers 19, 13 15- Forms a yellow vegetative mycelium and a white aerial 1327. mycelium with long chains of oval spores on TSB agar. 9. Evtushenko, L. I., Akimov, V. N., Dobritsa, 5. V. &Taptykova, Mycelium fragments into coccoid and rod-shaped 5. D. (1989). A new species of actinomycete, Amycolata alni. elements. Hyphal segments are sometimes swollen up Int J Syst Bacteriol39, 72-77. to 5 pm. Decomposes L-tyrosine, xanthine and hy- 10. Gordon, R. E., Barnett, D. A., Handerhan, 1. E. & Pang, poxanthine. No decomposition of adenine. Acid pro- C. H.-N. (1 974). Nocardia coeliaca, Nocardia autotrophica, duction from glucose, xylose, fructose and trehalose. and the nocardin strain. Int J Syst Bacteriol24, 54-63. No acid from rhamnose, cellobiose, mannitol, sorbitol, erythritol, maltose, lactose, arabinose, adonitol and 11. Henssen, A. (1957). Beitrage zur Morphologie und Systematik der thermophilen Actinomyceten. Arch Mikro- salicin. Growth on 3 % NaC1. Oxidation of DMS and biol26, 374414. DMDS. The main menaquinone is MK-8(H4). Phos- p ha tid ylme t h yle t hanolamine, p ho sphat id yle t hanol- 12. HUB, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization amine, hydroxyphosphatidylethanolamine, phos- from renaturation rates. Syst Appl Microbiol4, 184-192. phatidylinositol, phosphatidylinositol mannoside, diphosphatidylglycerol and phosphatidylglycerol are 13. Jahnke, K.-D. (1992). BASIC computer program for evalu- present. The major fatty acids are iso-branched hexa- ation of spectroscopic DNA renaturation data from Gilford System 2600 spectrophotometer on a PC/XT/AT type decanoate and hexadecanoate. Small amounts of personal computer. J Microbiol Methods 15, 6 1-73. 1O-methyl hexadecanoate and 10,14-dimethyl pentadecanoate are present. Strains 591, 592 and 593 were 14. Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein isolated from tree-bark compost biofilters from an molecules. In Mammalian Protein Metabolism, pp. 2 1-1 32. animal-rendering plant. The type strain is isolate 592 Edited by H. N. Munro. New York: Academic Press. (= DSM 44248T). 15. Kanagawa, T. & Kelly, D. P. (1986). Breakdown of dimethyl sulphide by mixed cultures and by Thiobacillus thioparus. FEMS Microbiol Lett 34, 13-19. 16. Kroppenstedt, R. M. (1985). Fatty acid and menaquinone ACKNOWLEDGEMENTS analysis of actinomycetes and related organisms. 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