Tsukamurella spumae sp. nov., A Novel Actinomycete Associated with Foaming in Activated Sludge Plants

System. Appl. Microbiol. 26, 367–375 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam

Tsukamurella spumae sp. nov., A Novel Actinomycete Associated with Foaming in Activated Sludge Plants Sun-Woo Nam1, Jongsik Chun2, Seungbum Kim1, Wonyong Kim1,3, Jolanta Zakrzewska-Czerwinska4, and Michael Goodfellow1 1

School of Biology, King George VIth Building, University of Newcastle, Newcastle upon Tyne, United Kingdom School of Biological Sciences, Seoul National University, Kwanak-gu, Seoul, Republic of Korea 3 Department of Microbiology, Chung-Ang University College of Medicine, Dongjak-ku, Seoul, Republic of Korea 4 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland 2

Received: April 11, 2003

Summary A polyphasic taxonomic study was undertaken to establish the taxonomic position of six representative strains isolated from activated sewage sludge foam. The organisms were found to have chemical and morphological properties consistent with their assignment to the genus Tsukamurella. DNA:DNA relatedness studies showed that five out of the six isolates formed a distinct genomic species, the remaining strain was most closely associated with this taxon. The five isolates had a unique phenotypic profile that served to distinguish them from representatives of the validly described species of Tsukamurella. The combination of the genotypic and phenotypic data indicated that the five strains should be classified as a new species in the genus Tsukamurella. The name proposed for this taxon is Tsukamurella spumae, the type strain is N1171T (= DSM 44113T = NCIMB 13947T). It was also shown that some of the reference strains were misclassified as Tsukamurella paurometabola. Key words: Tsukamurella spumae sp. nov. – polyphasic taxonomy – mycolic acids – 16S rDNA

Introduction The most commonly used method for the treatment of wastewater is the activated sludge process [25, 41]. The formation of scum or foam on the surface of aeration basins and secondary clarifiers in activated sludge sewage treatment plants is often, though not exclusively associated with filamentous mycolic acid-containing actinomycetes. Such foams cause several operational problems in activated sludge plants, notably the reduction of effluent quality, and may represent a public health hazard because of the potential spread of pathogens through the generation of aerosols. Foaming problems are common, have been reported in many countries, and are difficult to control. Engineering solutions to the problem require an understanding of the taxonomic diversity, functional roles and numbers of the causal organisms [8, 18, 46]. The application of polyphasic taxonomy has led to marked changes in the classification and identification of mycolic acid-containing bacteria, including those associated with foams [17, 18, 19, 40, 46]. These actino-

mycetes have many features in common, form a distinct phylogenetic line in the 16S rDNA tree, and are classified in the genera Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocardia, Rhodococcus, Skermania, Tsukamurella and Williamsia [20]. Recent selective isolation and characterisation studies have revealed the presence of several of these taxa in foams, including Gordonia [8, 10, 20], Mycobacterium [44], Rhodococcus [45], Skermania [2, 8, 43] and Tsukamurella [19, 40]. However, the emphasis on conspicuous filamentous morphologies means that unicellular and pleomorphic forms, including mycolic acid-containing strains, are often overlooked. The primary aim of the present investigation was to resolve the taxonomic position of representative strains that had been isolated from activated sewage sludge foam and provisionally assigned to the genus Tsukamurella as “Tsukamurella spumae” [19]. To this end, six representative isolates were compared with marker strains of validly described Tsukamurella species using a polyphasic taxonomic approach. 0723-2020/03/26/03-367 $ 15.00/0

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Methods The GenBank accession numbers for the 16S rDNA sequences of Tsukamurella spumae strain N1173, Tsukamurella sp. N1176 and Tsukamurella tyrosinosolvens DSM 44234T are AY 238512, AY 238513 and AY 238514. Strains and cultivation conditions The source and histories of the representative “Tsukamurella spumae” strains are shown in Table 1 together with those of the marker Tsukamurella strains. The organisms were maintained as glycerol suspensions (20%, v/v) at –20 °C and as glucose yeast extract slopes (GYEA; [22]) at room temperature. Biomass for the chemotaxonomic and molecular systematic studies was prepared in shake flasks of modified Sauton’s broth [35] and glucose yeast extract broth [22], respectively, as described by Goodfellow et al. [19]. Chemotaxonomy Strains N1171 to N1176 were examined for key chemical properties, using appropriate control organisms. Standard procedures were used for the extraction and analysis of the isomeric form of diaminopimelic acid (A2pm: 47), whole-organism sugars [38] and muramic acid residues [51]. Menaquinones were extracted from freeze-dried biomass (50 mg) using the small-scale procedure of Minnikin et al. [34]; the purified menaquinones were separated by HPLC, as described by Chun and Goodfellow [5]. Fatty acid methyl esters (FAMES) from all of the strains were prepared by acid methanolysis after Minnikin et al. [33], and analysed using a Shimadzu Mini-3 chromatograph fitted with a SPB-1 fused silica capillary column (Supelco Ltd.); the column was programmed to operate from 150 to 250 °C with increases of 4 °C per minute, using nitrogen as the carrier gas. Individual peaks were identified by comparing with a standard FAMES mixture (Supelco Ltd.) and fatty acid methyl esters extracted from Nocardia asteroides ATCC 19247T. Mycolic acid methyl esters (MAMES) were pyrolysed at 310 °C and the resultant products separated and detected using a Hewlitt Packard GC-MS (model 5890) equipped with a Hewlitt Packard capillary column (HP-5MS cross-linked 5% Ph Me silicone) programmed from 150 to 250 °C with increases

of 5 °C per minute, using helium as the carrier gas. The composition of the purified MAMES from strain N1171 were determined using electron-impact mass spectrometry according to Collins et al. [6]. 16S rDNA sequencing and phylogenetic analysis Extraction of chromosomal DNA, PCR amplification and the isolation, cloning and sequencing of the amplified DNA from strains N1173, N1176 and T. tyrosinosolvens N1274T were carried out as described by Chun and Goodfellow [5]. The resultant 16S rDNA sequences were aligned manually with corresponding sequences of Tsukamurella strains and representatives of the mycolic acid containing genera retrieved from the EMBL, GenBank and RDP databases using the PHYDIT program [4]. Evolutionary trees were inferred using the leastsquares [15], maximum-likelihood [12] and neighbour-joining [39] algorithms from the PHYLIP suite of programs [14]. Evolutionary distance matrices were generated for the neighbour-joining method, as described by Jukes and Cantor [26]. The resultant unrooted tree topology was evaluated in a bootstrap analysis [13] of the neighbour-joining method based on 1000 resamplings using the SEQBOOT and CONSENSE programs from the PHYLIP software [14]. Ribotyping Ribotyping was carried out using SalI digests of genomic DNA according to Kim et al. [28]. A digoxigenen labelled DNA fragment containing the 16S, 23S and 5S genes of the rRNA operon of Streptomyces lividans TK21 [57] was used to visualise the ribosome patterns. DNA base composition Chromosomal DNA was extracted from wet biomass of T. paurometabola N663 and strain N1171 after Mordarski et al. [36]. The thermal denaturation procedure of Marmur and Doty [31] was used to determine the guanine (G) plus cystosine (C) content of the DNA preparations, using the following equation: mol. % G+C = (Tm – 53.9) 2.44, where Tm is the melting temperature. Each preparation was examined either three or four times in 0.1 × SSC (1 × SSC is 0.15M NaCl plus 0.015M – trisodium citrate, pH 7.0), using DNA taken from Micrococcus luteus ATCC 4698 (72 mol% GC) as standard.

Table 1. Test strains. N1238T

Tsukamurella inchonensis, A.F.Yassin, Institute of Medical Microbiology and Immunology, University of Bonn, Bonn, Germany, IMMIB D.771; blood cultures of a patient who had ingested hydrochloric acid.

JC7T

Tsukamurella paurometabola, DSM 20162T (Corynebacterium paurometabolum). Isolated from the mycetomes and ovaries of the bed bug (Cimex lectularus).

M333

T. paurometabola, R.E. Gordon, Institute of Microbiology, Rutgers University, New Brunswick, USA, IMRU 1238; G. Altmann, 4479; human eye

M337

T. paurometabola, R.E. Gordon, IMRU 1505; C. Mc Durmont, R161

N663

T. paurometabola, NCTC 10741 (Rhodococcus aurantiacus); M. Tsukamura, 3462; A. Kruse; sputum T

N1240

T. pulmonis, A.F. Yassin, IMMIB D-1321; sputum from a patient with pulmonary tuberculosis.

N1171-N1176

“Tsukamurella spumae”, J. Chun, Department of Microbiology, University of Newcastle, Newcastle upon Tyne, UK; activated sludge foam, Stoke Bardolph Water Reclamation Works, near Nottingham, UK

N1275 T

Tsukamurella strandjordae, Dr. L.C. Carlson, Department of Laboratory Medicine, University of Washington, Seattle, USA; blood from a five year old girl with acute mycelogenous leukaemia

N1274T

Tsukamurella tyrosinosolvens, A.F. Yassin, IMMIB D-1397T; from a blood culture of a patient with a cardiac pacemaker implant.

Tsukamurella spumae sp. nov., A Novel Actinomycete Associated with Foaming in Activated Sludge Plants DNA:DNA hybridization studies The fluorometric microplate method [11], as modified by Goris et al. [23], was used. Photobiotin-labelled DNA from T. paurometabola JC7T, T. paurometabola N663 and T. spumae N1173 was individually hybridized with single-stranded unlabelled DNA, non-covalently bound to microtitre wells. The hybridization experiments were carried out under highly stringent conditions in 50% formamide at an optimal temperature (43 °C). Fluorescent intensities were measured using a Fluroskan CF fluorimeter (Thermo Lab Systems Inc., Beverley, MA, USA) at a wavelength of 360 mm for excitation and 450 mm for emission. Hybridizations were performed in triplicate and mean percentage DNA relatedness values determined. DNA-DNA relatedness studies were also carried out using the nitrocellulose filter method [28]. Phenotypic tests All of the strains were examined for a range of phenotypic tests (Table 4) known to be of value in the classification of tsukamurellae [4]. Unless otherwise stated, the tests were carried out at 30 °C and read weekly for up to 21 days. Most of them were performed using a multipoint inoculator (Denley Instruments Ltd., UK); the exceptions were the cultural and morphological tests which were determined on GYEA plates [22] after incubation for 5 days at 30 °C. After incubation, colonies were examined both with the naked eye and microscopically (×400 magnification). Smears from GYEA plates were prepared, Gram-stained using Hucker’s modification [42] and examined for cellular morphology. Smears were also stained using a modification of the Ziehl-Neelson method [21] and the degree of acid alcohol fast-

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ness noted. The degradation and tolerance tests were carried out using GYEA as the basal medium. The degradation of hypoxanthine (0.4%, w/v), tyrosine (0.5%, w/v), uric acid (0.5%), xanthine (0.4%, w/v) and xylan (0.%, v/v) were performed using standard procedures [16, 24], as were aesculin [53] and urea [37] hydrolysis. The ability of the organisms to grow on a range of sole carbon compounds was examined using the basal medium of Boiron et al. [3].

Results and Discussion The chemotaxonomic and morphological data obtained from the examination of the six strains confirmed and extended those from a pilot study [19]. The isolates are aerobic, Gram-positive, partially acid-alcohol fast, non-motile and nonsporeforming, form straight to slightly curved rods on GYEA, contain meso-A2pm, arabinose and galactose in whole-organism hydrolysates (wall chemotype IV sensu 30), N-glycolyl residues in the glycan moiety of the cell walls, major proportions of straight chain monounsatured and tuberculostearic acids (Table 2; fatty acid type 1b sensu 29), predominant amounts of fully unsaturated menaquinones with nine isoprene units, and highly unsaturated long chain mycolic acids with 68 to 76 carbon atoms. All of these properties are consistent with the classification of the isolates in the genus Tsukamurella [7, 27, 54, 55, 56].

Table 2. Percentage fatty acid composition of the Tsukamurella strains.

4.4 1.0 0.8 32.8 – – 1.4 – 33.2 1.1 9.2 1.4 14.2 – –

– 25.4 – – 1.3 – 23.5 2.4 17.9 6.0 16.7 – 1.1 –

7.2 1.3 1.1 32.9 – – 1.7 – 27.9 1.3 8.2 1.5 17.1 – 1.1

– – 2.2 ± 0.1 1.1 ± 0.1 1.8 ± 0.3 29.7 ± 0.4 0.8 ± 0.0 – 2.1 ± 0.1 – 33.5 ± 0.4 2.0 ± 0.2 9.3 ± 0.8 3.3 ± 0.9 10.7 ± 1.2 – 0.7 ± 0.1

0.3 – 3.8 1.9 0.7 34.2 1.2 – 2.1 – 34.4 0.8 8.1 1.1 10.6 0.4 –

– – 4.9 1.0 – 40.3 – – 1.7 – 23.7 2.1 16.5 1.6 7.8 – –

A, C16:1w7 or C15–iso–20H; B, C18:1w7/C18:1w9t or C18:1w12t; C, C19:1w11 or unknown C18.756; – not present. Abbreviations for fatty acid methyl esters are exemplified by the following examples: C16:0, straight chain hexadecanoic acid; C18:1w9, oleic acid; and C18:0 10 methyl, 10-methyl-octadecanoic acid.

– 0.4 ± 0.1 10.3 ± 3.7 0.9 ± 0.1 1.8 ± 0.1 26.5 ± 4.3 – – 0.7 ± 0.1 – 26.9 ± 0.6 0.6 ± 0.1 17.5 ± 0.3 – 15.7 ± 0.5 – –

Tsukamurella. sp. N1176

3.8 1.5 0.9 31.4 1.1 – 2.0 – 34.9 1.2 8.5 1.3 13.5 – –

– – 3.6 1.1 0.8 31.3 1.2 – 2.0 – 27.4 1.8 14.6 1.0 13.9 – 0.83

“T. spumae” (5 strains)

– –

T. strandjordae N1275 T

–

T. tyrosinosolvens N1274T

– –

0.6 3.2 0.6

T. pulmonis (3 strains)

T. paurometabola N663

–

T. inchonensis N1238T

T. paurometabola M337

C10:0 C12:0 C14:0 C15:0 C16:1w9 C16:0 C17:1w8 C17:1w5 C17:0 C10:0 10 methyl C18:1w9 C18:0 C18:0 10 methyl C20:1w9 A B C

T. paurometabola M333

Mean ± SD fatty acid content (%) –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– T. paurometabola JC7T

Fatty acid

– – 4.4 1.1 0.9 34.1 – – 0.7 – 33.9 5.7 – – 19.5 –

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Comparison of the almost complete 16S rDNA nucleotide (nt) sequences of “T. spumae” strains N1171, N1173 and N1176 with corresponding sequences of representatives of the suborder Corynebacterineae provided further evidence that these isolates are correctly assigned to the genus Tsukamurella. It is evident from Fig. 1 that the three isolates are more closely related to one another than to the representatives of recognised species of Tsukamurella, an observation that is supported by the products of all three treeing algorithms and a high bootstrap value in the neighbour-joining analysis. The two

most closely related “T. spumae” isolates, strains N1171 and N1173, have identical 16S rDNA sequences at the 1474 compared sites. In contrast “T. spumae” strains N1171 and N1176 share a 16S rDNA nt similarity of 99.7%, a value which corresponds to 5 nt differences at 1474 sites. The high mean 16S rDNA similarities found between isolates N1171, N1173 and N1176 and the reference Tsukamurella strains, namely 99.35%, is not unexpected as it is known that the 16S rDNA molecules of the type strains of Tsukamurella species are highly conserved [27, 54, 55, 56]. The taxonomic integrity of the

Fig. 1. Neighbour-joining tree [39) based on nearly complete 16S rDNA sequences of strains N1173 and N1176 and corresponding sequences of representatives of the genus Tsukamurella and other genera classified in the suborder Corynebacterineae. Asterisks indicate branches of the tree that were also found using the least-squares [15] and maximum-likelihood [14] treeing algorithms; the symbols F and L indicate branches recovered using the leastsquares and maximum-likelihood methods, respectively. The numbers at the nodes indicate the level of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets; only values above 50% are given. The scale bar indicates 0.1 substitutions per nucleotide position. T, type strain.

Fig. 2. Ribotyping patterns generated from Sal I genomic DNA digests hybridized with the digoxigenin-labelled rDNA probe. Lanes: 1) T. spumae N1171T; 2) T. paurometabola M334; 3) T. paurometabola N663, and 4) T. paurometabola JC7T.

Tsukamurella spumae sp. nov., A Novel Actinomycete Associated with Foaming in Activated Sludge Plants

species proposed by these investigators was based on DNA:DNA relatedness and phenotypic data. The minimum level of DNA:DNA relatedness between strains required to delineate a genomic species was recommended as 70% by Wayne et al. [52]. It is, therefore, clear that five out of the six “T. spumae” isolates form a well defined genomic species based on the DNA:DNA relatedness studies carried out using the fluorometric microplate method (Table 3). The remaining strain, isolate N1176, shared a relatively high DNA:DNA relatedness value, namely 46% with the reference “T. spumae” strain. It is evident from Table 4 that the five highly related T. spumae isolates have many phenotypic properties in common; the latter serve to distinguish them from all of the other tested strains, notably from isolate N1176 and the type strains of the recognised species of Tsukamurella. The DNA:DNA similarity between selected strains was also studied using the nitrocellulose filter method. The hybridization values obtained with this method are lower than those found using the fluorometric microplate method (Table 3) but even so the correlation found between the corresponding data sets is good, indicating that the microplate method can be used as a reliable and rapid taxonomic tool. It is clear from the genotypic and phenotypic data that strains N1171 to N1175 form a new species within the genus Tsukamurella. It is, therefore, proposed that these organisms be assigned to the genus Tsukamurella as Tsukamurella spumae. Further comparative taxonomic work is needed to determine the taxonomic status of isolate N1176 which was originally designated “T. spumae” [19]. The present data also help to clarify the taxonomy of T. paurometabola, a species proposed by Collins et al.

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[7] to accommodate the type strains of Corynebacterium paurometabolum Steinhaus [48] and Rhodococcus aurantiacus [49] Tsukamura and Yano [50]. Subsequent studies based on a range of criteria indicated that these strains belonged to different species [1, 32]. The original type strain of R. aurantiacus was eventually shown to be a typical strain of T. inchonensis based on DNA:DNA relatedness and phenotypic data; corresponding data from the present study underpin this classification (see Tables 3 and 4). It is also clear that T. paurometabola strains M333 and M337 are misclassified as these organisms show relatively low DNA similarities with reference DNA from T. paurometabola JC7T (see Table 3). The present DNA relatedness data also suggest that the assignment of strain M337 to the same genomic species as strain N333 [17] is more apparent than real. The phenotypic data (Table 4) suggest that strains M333 and M337 may belong to the same species though further comparative studies are needed to resolve the taxonomic position of these strains within the genus Tsukamurella. The ribotyping analysis (Fig. 2) shows that “T. spumae” N1171T has a distinct ribotype pattern, a result in good agreement with the DNA:DNA relatedness data. The remaining analysed strains, T. paurometabola M334, T. paurometabola N663 and T. paurometabola JC7T also gave distinct DNA profiles and had little DNA:DNA relatedness in common (less than 20% [nitrocellulose method]; data not shown). It can also be concluded from the ribotyping analysis that the “T. spumae” N1171T genome contains four ribosomal operons. The copy number of rRNA operons amongst actinomycete genomes varies from one (eg. Mycobacterium tuberculosis) to seven (Streptomyces venezuelae) [http://rrndb.cme.msu.edu/rrndb/servlet/controller].

Table 3. DNA-DNA hybridization of Tsukamurella strains. Values for DNA:DNA hybridizations are means with standard deviations given in brackets. Strain

DNA:DNA hybridization with DNA from ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– T. paurometabola T. paurometabola Strain Strain JC7T N663 N1173 N1171T

T. inchonensis N1238T T. paurometabola JC7T T. paurometabola M333 T. paurometabola M337 T. paurometabola N663* T. pulmonis N1240T T. strandjordae N1275 T T. tyrosinosolvens N1274T Strain N1171 Strain N1172 Strain N1173 Strain N1174 Strain N1175 Strain N1176

33 100 18 18 25 28 56 28 18 19 20 17 18 15

(1.6) (2.4) (0.5) (3.1) (0.5) (6a) (1.9) (0.8) (1.2) (0.6) (2a) (0.7) (1.1) (1.1) (0.2) (0.8)

95 25 40 32 100 42 55 51 28 21 30 21 24 23

(0.3) (0.1) (11a) (0.2) (0.1) (0.3) (0.2) (1.4) (0.2) (0.1) (16a) (0.1) (0.1) (0.1) (0.1) (0.1)

32 20 34 37 25 27 17 30 72 80 100 86 83 46

DNA:DNA hybridization data obtained using the nitrocellulose filter method (35% formamide, 3 × SSC, 60 °C) * The mean DNA base composition of this strain was 71.7 mol % G+C.

a

(0.3) (0.2) (0.4) (2.0) (0.5) (1.5) (0.7) (0.2) (1.8) (0.6) (5.1) (1.9) (1.0) (0.7)

(14a) (6a) (7a) (8) (10a) (100a)

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T. inchonensis N1238T

JC7T

M333

M337

N663

T. pulmonis N1240T

N1171

N1172

N1173

N1174

N1175

N1176

T. standjordae N1275T

T. tyrosinosolvens N1274T

Table 4. Phenotypic properties of Tsukamurella strains†.

Biochemical tests: Aesculin hydrolysis Urea hydrolysis

+ +

+ +

+ +

+ +

+ +

+ +

– –

– +

– +

– +

– +

+ –

+ +

+ –

Colony properties: Orange/red colonies White/cream colonies Edge of colonies irregular Elevation of colonies irregular Colony size (< 5 mm)

– + + + +

– + –* –* –*

– + + + +

– + + + +

– + + + +

– + + + +

+ – + + +

+ – + + +

+ – + + +

+ – + + +

+ – + + +

+ – + + +

– + + + +

– + + + +

Degradation tests: Hypoxanthine Tyrosine Xanthine

+ – –

– – –

+ + –

+ + –

+ + –

+ – –

+ + –

+ + –

+ + –

+ + –

+ + –

+ + –

– – –

+ + –

Growth at: 10 °C

–

+

+

+

–

–

–

+

+

+

+

+

–

–

Growth on sole carbon sources (1%, w/v): D(–) arabinose L(+) arabinose D(+) arabitol D(+) cellobiose Dulcitol Meso-erythritol D(+) fucose Maltose D(–) mannitol D(+) melezitose Melibiose D(–) ribose Salicin D(–) sorbitol D(+) xylose

+ – – – – – – + + + + + + + +

– – – – – – + – + – + + + – +

+ + + – + + + – + + + + + + +

+ + + – + + + – + + + + + + +

– – – – – – – + + + + + + + +

+ + + + + + + + – – + + + – +

– + + – + + + + + + + + – + +

+ + + – + + + + + + + + – + +

+ + + – + – + + + + + + – + +

+ + + – + – + + + + + + – + +

+ + + – + – + + + + + + – + +

+ + + – – – + + – + – + – – –

– – + – – – – – + – + – + + –

+ + + + + + + + + + + + + + +

Resistance to antibiotics (µg ml–1) Clindamycin [2] Cotrimoxazole [25] Erythromycin [5] Fusidic acid [10] Tetracycline hydrochloride [10]

+ + + + +

– + + – –

+ – – + +

+ + + + +

+ + + + +

+ + – + +

+ + – + –

+ + – – –

+ + – + –

+ + – + –

+ + – – –

+ + – + +

+ + + + +

+ + + + +

T. paurometabola

Isolates

* T. paurometabola JC7T produces small (>2 mm), convex colonies with entire edges. a All of the strains degraded Tween 80 and uric acid, grew on (D(+) galactose, β (–) gentiobiose, D (+) glucose, meso-inositol, α (–) lactose, α (–) lactose, D (+) mannose, α-L-rhamnose, D (+) sucrose, D (+) trehalose, D (+) turanose and D (–) xylitol (at 1%, w/v) as sole sources of carbon for energy and growth, grew at 18 °C, 25 °C and 37 °C, and were resistant to colistin (25 µg ml–1), naladixic and (5 µg ml–1), novobiocin (5 µg ml–1) and penicillin (1 international unit). None of the organisms degraded xylan, used D (+) rhamnose (1%, w/v) as a sole carbon source or were resistant to ciprofloxacin (5 µg ml–1).

Tsukamurella spumae sp. nov., A Novel Actinomycete Associated with Foaming in Activated Sludge Plants

Description of Tsukamurella spumae sp. nov. Tsukamurella spumae (spu’mae, L.gen.n. spumae of foam denoting the presence of the organism in the foam of activated sewage sludge plants). The description given below is based upon data from both the present and earlier studies [4, 19]. Aerobic, Gram positive, partially acid-alcohol fast, non-motile, non-sporeforming actinomycetes that form straight to slightly curved rods and a few long filaments, but which do not differentiate into substrate or aerial hyphae. Colonies on glucose yeast extract agar are large (<5 mm), orange to red with margins and elevation irregular. Grows at 25 °C and 37 °C, but not at 45 °C. Amyl alcohol, butane-2,3-diol, sodium citrate and sodium pyruvate are used as sole carbon sources but not adonitol, p-aminosalicylic acid, butane-1,4-diol, ethanolamine, D(–)galacturonic acid, D(+) glucosamine, D(–) glucuronic acid, methanol, DL-norleucine, resorcinol, sodium benzoate or sodium tartrate. L-asparagine, Lphenylalanine and L-serine are used as sole carbon and nitrogen sources but not L-histidine, L-lysine, succinamide or L-valine. Resistant to crystal violet (0.001%, w/v), 5-fluorouracil (20 µg ml–1), bekanamycin (16, 32 and 64 µg ml–1), erythromycin (2, 4 and 8 µg ml–1), gentamicin sulphate (16 and 32 (g ml–1), kanamycin sulphate (4, 8, 16 and 64 µg ml–1), neomycin sulphate (4, 8, 16 and 32 µg ml–1), novobiocin (16 µg ml–1), oleandomycin phosphate (16, 32 and 64 µg ml–1), rifampicin (0.5 and 2 µg ml–1) and vancomycin (1, 2 and 4 µg ml–1). Susceptible to chlortetracycline hydrochloride (2 and 8 µg ml–1), erythromycin (16 µg ml–1), novobiocin (64 µg ml–1), penicillin G (16, 32 and 64 µg ml–1) and rifamipicin (8 and 16 µg ml–1). Additional phenotypic properties are shown in Table 3. MK-9 is the major menaquinone, minor amounts of MK8 and MK-10 are also present. The organism contains predominant amounts of hexadecanoic and tuberculostearic acids (Table 2) highly unsaturated mycolic acids with 68 to 76 carbon atoms with up to seven double bonds. The major products from pyrolysis gas chromatography of methyl mycolates are the straight-chain saturated fatty acids C20:1 and C22:1. Isolated from activated sludge foam, Stoke Bardolph Water Reclamation Works, near Nottingham, UK. Unlike the other isolates, the type strain (N1171 T = DSM 44113T = NCIMB 13947T) did not grow at 10 °C, hydrolyse urea or grow on D (–) arabinose as a sole carbon source. The mean DNA base composition of this isolate was 70% G+C. A complex series of medium-sized peaks corresponding to aldehydes formed by pyrolysis mass spectrometry of purified MAMES prepared from this organism was seen at the following m/e values (main components in bold type): 688 (C48:0), 708 (C50:4), 710 (C50:3), 712 (C50:2), 734 (C52:5), 736 (C52:4), 738 (C52:3), 748 (C53:5), 750 (C53:4), 762 (C54:5), 790 (C56:5) and 792 (C56:4). Fragments corresponding to anhydromycolates, formed by the elimination of water, were found in the higher mass range at m/e 982 (C68:7), 984 (C68:6), 1010 (C70:7), C1012 (C70:6), 1038 (C72:7), 1040 (C72:6) and 1098 (C76:5).

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Acknowledgements J. Chun and S-W. Nam were supported by Overseas Research Student Awards, S.B. Kim and W-Y. Kim by Chevening Scholarships. M. Goodfellow and W-Y. Kim are also grateful for support from UK-KOREA Science & Technology Collaboration Fund (Grant number M1-0027-00-0008). The authors are indebted to Professors R.E. Gordon and R.M. Kroppenstedt and to Drs. L.C. Carlson and A.F. Yassin for the gift of strains (see Table 1).

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