International Journal of Systematic and Evolutionary Microbiology (2000), 50, 529–536 Printed in Great Britain

Marmoricola aurantiacus gen. nov., sp. nov., a coccoid member of the family isolated from a marble statue

Clara Urzı' ,1 Paola Salamone,1 Peter Schumann2 and Erko Stackebrandt2

Author for correspondence: Clara Urzı' . Tel: j39 090 6765196. Fax: j39 090 392733. e-mail: urzicl!unime.it

1 Institute of Microbiology, A Gram-positive, aerobic bacterium with coccoid cells occurring singly, in pairs Faculty of Science, and in clusters was isolated from the surface of a marble statue. The University of Messina, Salita Sperone 31, I-98166 peptidoglycan contains LL-diaminopimelic acid as diagnostic diamino acid and Villagio S. Agata Messina, a single glycine residue as interpeptide bridge (type A3γ). The major Italy menaquinone is MK-8(H4). The cellular fatty acid pattern consists of straight 2 DSMZ–German Collection chain saturated and monounsaturated components and 10-methyl of Microorganisms and octadecanoic (tuberculostearic) acid as the only branched chain fatty acid. Cell Cultures GmbH, Mascheroder Weg 1b, Phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol occur D-38124 Braunschweig, as characteristic polar lipids. The DNA GMC composition is 72 mol%. According Germany to its phylogenetic position and 16S rDNA signature nucleotides, the organism is a member of the family Nocardioidaceae. The combination of chemotaxonomic characteristics is unique within this family and supports the description of a new genus and new , Marmoricola aurantiacus. The type strain is strain BC 361T (l DSM 12652T).

Keywords: Marmoricola aurantiacus, marble, Nocardioidaceae, chemotaxonomy, phenotypic characteristics

INTRODUCTION namely Nocardioides albus (Prauser, 1976), No- cardioides simplex (O’Donnell et al., 1982), No- with -diaminopimelic acid (- cardioides jensenii (Suzuki & Komagata, 1983; Collins A#pm) cluster into five families belonging to four et al., 1989), Nocardioides luteus (Prauser, 1984), suborders on the basis of characteristic signature Nocardioides plantarum (Collins et al., 1994) and nucleotides of 16S rDNA: (i) Nocardioidaceae (sub- Nocardioides pyridinolyticus (Yoon et al., 1997). order Propionibacterineae), (ii) Propionibacteriaceae Nocardioides fastidiosa proposed by Collins & (suborder Propionibacterineae), (iii) Intrasporangia- Stackebrandt (1989) has been transferred to the genus ceae (suborder Micrococcineae), (iv) Sporychthiacea Aeromicrobium as Aeromicrobium fastidiosum by (suborder Frankineae) and (v) Streptomycetaceae Tamura & Yokota (1994) on the basis of chemo- (suborder Streptomycineae) (Stackebrandt et al., taxonomic and phylogenetic data. Though No- 1997). The genera of these families can readily be cardioides species are heterogeneous with respect to distinguished by their chemotaxonomic characteristics morphology, embracing strains that form mycelium (Schumann et al., 1997; Prauser et al., 1997; Busse which may fragment into irregular rod- and coccus- & Schumann, 1999). The family Nocardioidaceae like cells as well as true rods and cocci, they are (Stackebrandt et al., 1997) comprises the genera coherent in chemotaxonomic properties. The pepti- Nocardioides and Aeromicrobium which differ in their doglycan of all representatives of the genus No- major isoprenoid quinone, cellular fatty acid profile cardioides contains -A#pm and is of the A3γ type and polar lipid pattern. (Schleifer & Kandler, 1972) with a single glycine residue as interpeptide bridge. The genus lacks mycolic Nocardioides The genus includes at present six species, acids and has a tetrahydrogenated menaquinone with

...... eight isoprene units [MK-8(H%)] as predominant iso- prenoid quinone (O’Donnell et al., 1982). Nocardioides Abbreviations: LL-A2pm, LL-diaminopimelic acid; TBSA, 10-methyl octa- decanoic acid (tuberculostearic acid). strains show complex cellular fatty acids including iso- The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of and anteiso-branched, straight-chain saturated and strain BC 361T is Y18629. unsaturated components and 10-methyl octadecanoic

01135 # 2000 IUMS 529 C. Urzı' and others acid (tuberculostearic acid; TBSA). The predominance (Oxoid), 0n4% bacto casamino acids (Difco), 0n07% of iso-C"':! is characteristic for the genus (Yoon et al., (NH%)#HPO%,0n5% NaCl, 0n003% bromocresol purple, and 1997; Park et al., 1998). The polar lipid profiles include the substrates at a final concentration of 1% (Schumann et phosphatidylglycerol, diphosphatidylglycerol and al., 1997). Catalase production was shown by the production two α-glycol positive phospholipids (O’Donnell et al., of bubbles after a suspension of the cells was mixed with a drop of a 3% hydrogen peroxide solution on a slide. DNA 1982). hydrolysis was indicated by clear zones in spot-inoculated The genus Aeromicrobium embraces the two species plates containing DNase agar (Oxoid). The hydrolysis of Aeromicrobium erythreum and A. fastidiosum. Repre- casein and the decomposition of tyrosine, xanthine and sentatives of the genus Aeromicrobium form rod-like hypoxanthine were determined as described previously or coccoid cells. Members of this genus display the (Gordon et al., 1974). same peptidoglycan type A3γ as Nocardioides species Urease activity, nitrate reduction, aesculin hydrolysis, hy- but differ from them in that they contain the tetra- drolysis of starch, hydrolysis of Tween (20, 40, 60, 80), hydrogenated menaquinone with nine isoprenoid units phosphatase production and hydrolysis of gelatin were studied by using previously described methods (Lanyi, 1987). MK-9(H%), exclusively straight-chain saturated and T Strain BC 361 was examined for its ability to grow in monounsaturated fatty acids and phosphatidyl- Czapek Dox modified agar (CZ; Oxoid), potato dextrose ethanolamine (Miller et al., 1991; Tamura & Yokota, agar (PDA; Oxoid), brain-heart infusion agar (BHIA; 1994). Oxoid), 1n5% agar-H#O, YMS-Glc agar (0n5% yeast extract, This paper describes the morphological, physiological 1n5% malt extract, 1% starch, 1% glucose, 1n5% agar), and chemotaxonomic characterization of a coccoid - YCS-Glc agar (0n5% yeast extract, 1n5% casein hydrolysate, 1% starch, 1% glucose, 1 5% agar), Y-Glyc agar (0 5% A pm containing strain isolated from a marble statue n n # yeast extract, 2% glycerol, 1n5% agar), YG-Glyc agar of the Nordfriedhof cemetery in Munich (Germany) (0n5% yeast extract, 1% glucose, 10% glycerol, 1n5% agar) and the determination of its phylogenetic position on (Luedemann & Fonseca, 1989) and in medium M53 Cory- the basis of its 16S rDNA sequence. Because of the nebacterium agar (Collins & Stackebrandt, 1989). unique combination of its characteristics among Cell wall analysis. Purified cell wall preparations were members of the family Nocardioidaceae, a new genus obtained by the method of Schleifer (1985). The amino acids and species, Marmoricola aurantiacus, is proposed. and peptides in cell wall hydrolysates were analysed by two- dimensional ascending TLC on cellulose plates using the METHODS described solvent systems (Schleifer, 1985). Whole-cell sugars were determined as alditol acetates by GC as Bacterial strain. Strain BC 361T was isolated from a marble described (Groth et al., 1996). The molar ratios of amino statue (Wagmu$ ller’s monument), in the old Nordfriedhof acids were determined by GC and GC-MS of N-hepta- cemetery in Munich, Germany. The organism was isolated fluorobutyryl amino acid isobutyl esters (MacKenzie, 1987). by dilution plating on Bunt and Rovira medium (Bunt & The glycolate content of bacterial cell walls was determined Rovira, 1955), modified by addition of 0n5% glucose, 0n5% by the colorimetric method of Uchida & Aida (1984). NaCl and 0 03% Na CO ,pH86 (BRII). For morpho- n # $ n Lipid analysis. Cellular fatty acid methyl esters obtained logical and chemotaxonomic studies, strain BC 361T was from cells grown in bacto tryptic soy broth at 28 C by the cultivated in Luedemann medium (Luedemann, 1968). m method described by Stead et al. (1992) were separated by Staining procedures. Gram staining and acid-fast staining GC (Groth et al., 1996) and identified as described previously were done as described previously (Lanyi, 1987) and (Schumann et al., 1997). Menaquinones were extracted as Luedemann staining was performed as described by described by Collins et al. (1977) and were analysed by Luedemann (1968). HPLC (Groth et al., 1996). Polar lipids extracted by the method of Minnikin et al. (1979) were identified by two- Morphological characteristics. Cell and aggregate mor- dimensional TLC and spraying with specific reagents phology were determined by bright-field microscopy and by (Collins & Jones, 1980). The absence of mycolic acids was phase-contrast microscopy (DML, Leica; oil immersion demonstrated by TLC (Minnikin et al., 1975). objective, magnification i100) and scanning electron micro- scopy (SEM) (Leo Electron Microscopy), from material DNA base composition. DNA was isolated and its GjC obtained from surface growth on agar plates. content was determined by HPLC of deoxyribonucleosides as described previously (Groth et al., 1996). For SEM observations, a 14 d old strain BC 361T culture from Luedemann medium was suspended in PBS (0n13 M 16S rDNA sequence determination and analysis. Extracted NaCl in 0n1MKH#PO%\NaOH buffer, pH 7n4). The cells genomic DNA was used for PCR-mediated amplification of were fixed with 0n5% glutaraldehyde (pH 7n4), washed 16S rDNA (Rainey et al., 1996). The purified PCR product several times and dehydrated in a series of ethanol con- was directly sequenced by using previously described pro- centrations. After the cells were sputter-coated with gold- tocols (Rainey et al., 1996), and the sequence reaction palladium, they were observed under a scanning electron mixtures were electrophoresed with a model 373A automatic microscope (Groth et al., 1997). DNA sequencer. Physiological and biochemical characterization. All physio- The 16S rDNA sequences were manually aligned with the logical tests were performed at 28 mC. Oxidase activity was sequences of members of the order Actinomycetales by using determined by monitoring the oxidation of N,N,Nh,Nh- the ae2 editor (Maidak et al., 1994). Evolutionary distances tetramethyl-p-phenylenediamine on filter paper. Acidifi- for strain BC 361T and for a selection of actinobacterial cation of carbohydrates was verified by a colour change strains (data not shown) were calculated by a previously from blue to yellow in a medium containing 0n5% tryptone described method (Jukes & Cantor, 1969). Phylogenetic

530 International Journal of Systematic and Evolutionary Microbiology 50 Marmoricola aurantiacus gen. nov., sp. nov.

T 80 Strain BC 361 Nocardioides jensenii DSM 20641T (Z78210) 60 T 86 Nocardioides plantarum DSM 11065 (Z78211) Nocardioides pyridinolyticus KCTC 0074 BPT (U61298) 74 Nocardioides simplex DSM 20130T (Z78812) 82 T 100 Nocardioides luteus NCIB 11455 (X53212) Nocardioides albus DSM 43109T (Z78213) T 100 Aeromicrobium erythreum NRRL B-3381 (M37200) Aeromicrobium fastidiosum DSM 10552T (Z78209) T 70 Friedmanniella antarctica DSM 11053 (Z78206) Microlunatus phosphovorus DSM 10555T (Z78207) 74 Propioniferax innocua DSM 8251T (S93388) ...... Luteococcus japonicus DSM 10546T (Z78208) Fig. 1. Phylogenetic dendrogram Propionibacterium freudenreichii DSM 20271T (X53217) based on the results of a 16S rDNA sequence comparison. Bar, 2 nucleo- 2 % tide substitutions per 100 nucleotides. dendrograms were reconstructed by using the treeing in pairs or in tetrads (Fig. 3) and in some cases in algorithm of DeSoete (1983) and the neighbour-joining clusters (old cultures). The temperature range for algorithm contained in the  package (Felsenstein, growth on Luedemann medium was 18–28 mC, no 1993). Bootstrap analyses were used to evaluate the tree growth occurred at 6 and 37 mC. Growth occurred topologies of the neighbour-joining data by performing 500 within a range of pH between 5 1 and 8 7. Addition of resamplings (Felsenstein, 1993). Strain numbers and rDNA n n accession numbers of reference organisms are included in 2% NaCl to Luedemann medium resulted in a Fig. 1. restricted growth, whereas no growth occurred in the presence of 4% NaCl. RESULTS Physiological characteristics Cultural and morphological characteristics The physiological and biochemical characteristics of Growth occurred after 2 weeks in Luedemann medium strain BC 361T are summarized in Table 1. Strain BC T at 28 mC. The colonies were circular (diameter 2–5 361 was negative for oxidase and urease tests. mm), with a smooth surface, shiny and orange Catalase was produced. Nitrate was not reduced coloured (Fig. 2). In old cultures (2–3 months in- to nitrite. No acid was produced from -ribose, cubation), colonies had the tendency to become shaped -arabinose, -xylose, -rhamnose, -glucose, - like craters and were sometimes rough. Cells were mannose, -galactose, maltose, lactose, -cellobiose, spherical (0n5–0n7 µm in diameter) and occurred singly, -trehalose, -raffinose, glycerol, -mannitol and myo- inositol. Casein and gelatin were not hydrolysed. Xanthine and tyrosine were not decomposed, whereas hypoxanthine was decomposed. Aesculin was hy- drolysed, Tween 20, 40, 60 were not hydrolysed, whereas Tween 80 was only weakly hydrolysed. DNA was not hydrolysed. The strain did not grow in the following media: CZ, YMS-Glc, YCS-Glc, YG-Glyc, Y-Glyc, agar-H#O and M53. Growth occurred in PDA and BHIA medium.

Chemotaxonomic characteristics The peptidoglycan of strain BC 361T contained - A#pm, Ala, Gly and Glu in a molar ratio of 1n0:1n0:0n9:0n7. From these results and from the two- dimensional TLC peptide pattern of partial cell-wall hydrolysates (data not shown), it was concluded that strain BC 361T represented peptidoglycan type A3γ with a single glycine residue as an interpeptide bridge (Schleifer & Kandler, 1972). Glucose and traces of ribose could be detected only as whole-cell sugars. The acyl type was acetyl. The fatty acid profile of strain BC T ...... 361 was characterized by the predominance of hexa- Fig. 2. Single BC 361T colony (after 30 d incubation on decanoic acid (C"':!) and octadecenoic acid (C"):"), Luedemann medium); diameter 4n5 mm. Bar, 1 mm. and by the presence of C"':",C"%:!,C"):! and TBSA as

International Journal of Systematic and Evolutionary Microbiology 50 531 C. Urzı' and others

Table 1. Morphological and biochemical characteristics of strain BC 361T ...... k, Negative; j, positive; w, weakly positive.

Characteristic Strain BC 361T

Colour of colony Orange Cell morphology Coccus, 0n5–0n6 µm, packet, clusters Gram staining j Acid-fast staining k Spore formation k KOH k Catalase j Oxidase k Growth in: k ...... CZ k Fig. 3. Scanning electron micrograph of coccoid cells from 14 d PDA T j old culture of strain BC 361 grown at 28 mC on Luedemann BHIA j medium. Bar, 1 µm. YMS-Glc k YCS-Glc k YG-Glyc k minor components (Table 2). The isoprenoid quinones Y-Glyc k Agar-H O were MK-8(H%), MK-7(H%), MK-8(H#), MK-6(H%); # k the peak area ratio was 73:4:1:1. The polar lipid M53 k pattern consisted of phosphatidylinositol, phosphat- Growth at: idylglycerol, diphosphatidylglycerol and one unknown 6 mC k phospholipid. Mycolic acids were absent. The DNA 18 mC j 22 C GjC composition was 72 mol% (Table 3). m j 25 mC j 28 mC j 16S rDNA sequence analysis 37 mC k Growth at pH: The almost complete 16S rDNA sequence of 1470 nt 5n1 j (95n5% of the Escherichia coli sequence) was deter- 6 T j mined for strain BC 361 and compared to the 7n2 j sequences of the 16S rDNA database of members of 8n7 j the class Actinobacteria (Stackebrandt et al., 1997). Growth in the presence of NaCl: The highest similarity values were found with repre- 0n5% j sentatives of the family Nocardioidaceae (" 93 %), 2% j whereas they were slightly lower with members of the 4% k neighbouring families, i.e. Microlunatus, Propioni- 6% k ferax, Luteococcus, Friedmanniella and Propioni- 10% k bacterium (similarity between 89n6 and 92n6%). Phosphatase k Strain BC 361T shares the highest binary 16S rDNA Nitrate reduction k Degradation tests: similarity with the type strain N. jensenii (96n4%). The two treeing algorithms place these two organisms as a Aesculin j sister lineage to the lineage containing the other species Urea k of Nocardioides [Fig. 1 showing the phylogenetic tree Xanthine k according to the algorithm of DeSoete (1983)], whereas Casein k the Aeromicrobium line of descent branches slightly Hypoxanthine j deeper. The inclusion of the 16S rDNA sequence into Gelatin k the database does not change the branching point of Starch k N. jensenii which has been demonstrated in previous Tween 20 k phylogenetic studies to represent the deepest point Tween 40 k within the genus Nocardioides (Tamura & Yokota, Tween 60 k 1994; Schumann et al., 1997; Yoon et al., 1998a). Tween 80 w Tyrosine k T Although strain BC 361 clusters within the radiation DNA k of Nocardioides species and shares with them the

532 International Journal of Systematic and Evolutionary Microbiology 50 Marmoricola aurantiacus gen. nov., sp. nov.

Table 2. Cellular fatty acid composition of strain BC 361T and of type strains of Aeromicrobium and Nocardioides species ...... Fatty acid: 1, i-C"%:!;2,C"%:!;3,i-C"&:!; 4, ai-C"&:!;5,C"&:!;6,i-C"':!;7,i-C"':";8,C"':!;9,C"':"; 10, i-C"(:!; 11, ai-C"(:!; 12, ai-C"(:"; 13, C"(:!; 14, C"(:"; 15, i-C"):!; 16, C"):!; 17, C"):"; 18, TBSA (10-methyl octadecanoic acid). i-C"&:",C"&:",i-C"(:" and C"):# were not observed. Values less than 1% are not shown; i, iso; ai, anteiso.

Taxon Fatty acid composition (%)*

123456789101112131415161718

BC 361T† 3n441n414n92n433n72n8 N. jensenii 1n716n71n328n62n84n32n39n61n03n96n51n51n610n32n4 N. albus 2n31n71n570n83n01n92n12n16n02n12n61n9 N. simplex 4n62n641n81n13n85n01n95n213n15n2 N. plantarum 3n41n026n02n91n05n416n86n14n01n815n71n9 N. pyridinolyticus‡ 5n11n447n34n21n21n714n22n11n21n71n02n03n4 A. erythreum§ 3n13n147n05n22n42n13n018n95n0 A. fastidiosum¶ 1n11n420n65n42n720n215n8 * Data (except for strain BC 361T and N. pyridinolyticus) from Schumann et al. (1997). † Strain contained additionally 1n4% 2-hydroxy hexadecanoic acid. ‡ Data from Yoon et al. (1997). Strain contained additionally 5n5% 10-methyl heptadecanoic acid and 5n0% 10-methyl hexadecanoic acid. § Strain contained additionally 4n6% 2-hydroxy hexadecanoic acid. ¶ Strain contained additionally 20n3% 2-hydroxy hexadecanoic acid.

Table 3. Differential characteristics of strain BC 361T and members of the family Nocardioidaceae ...... Abbreviations: DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PG-OH, PG containing 2-hydroxy fatty acids; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PL, unknown phospholipid(s); menaquinones e.g. MK-9(H%), menaquinone with two of nine isoprene units hydrogenated.

Taxon Cell Major Polar lipids GjC Reference morphology menaquinone (mol%)

T BC 361 Cocci in packets MK-8(H%) PI, PG, DPG, PL 72 Aeromicrobium Rods MK-9(H%) PE, PG 71–73 Miller et al. (1991); Tamura & Yokota (1994) N. albus Hyphae MK-8(H%) PG, DPG, PL, 66n5–68n6 Prauser (1986); Collins et al. (1989); PG-OH O’Donnell et al. (1982) N. simplex Rods\cocci MK-8(H%) PG, DPG, PL, 71n7 Collins et al. (1989); O’Donnell et al. PG-OH (1982) family-specific 16S rDNA signature nucleotides, strain that a great variety of Actinobacteria could be con- BC 361T exhibits several secondary-structure-forming sidered as the major colonizer of stone surfaces nucleotides that are not present in the 16S rDNA exposed to the outdoor environment. Among them, sequences of any other member of the family. These strains of Micrococcus, Geodermatophilus and Micro- nucleotides are found at positions (numbered accord- monospora (Urzı' & Realini, 1998) were very often ing to E. coli) 183–194 (G–C), 987–1218 (A–U), isolated and representatives of novel taxa might be 1002–1138 (A–U), 1012–1017 (G–C) 1089–1216 expected (unpublished data). Tests based upon mor- (G–C), 1422–1478 (U–G) and 1423–1479 (U–A). phological as well as biochemical and physiological characteristics turned out to be insufficient to identify DISCUSSION all isolated strains at the genus level. In the case of strain BC 361T, the first attempt carried out using Rock and monument surfaces support a high variety classical identification methods misplaced it in the of microflora that often have been only poorly studied. genus Micrococcus. Later on, -A#pm was found to In the last few years, however, it has become evident be the characteristic diamino acid of the cell wall. This

International Journal of Systematic and Evolutionary Microbiology 50 533 C. Urzı' and others

finding was in conflict with the affiliation of strain BC of chemotaxonomic and phylogenetic characteristics. 361T to the genus Micrococcus or related genera Since it could readily be distinguished from repre- (Stackebrandt et al., 1995) and initiated a study of sentatives of all phylogenetically related genera, it is chemotaxonomic characteristics and phylogenetic re- concluded that the organism should be assigned to a latedness of the strain in question. new genus and species for which the name Marm- oricola aurantiacus is proposed. The combination of the chemotaxonomic character- istics, peptidoglycan structure, menaquinone profiles and fatty acid composition is very helpful to dif- Description of Marmoricola gen. nov. ferentiate genera of the order Actinomycetales and Marmoricola. (Mar.mo.ri co.la. L. neutr. n. marmor especially to distinguish strains containing -A pm in h # marble; L. masc. suffix -cola inhabitant of; Mar- their cell wall (Schumann et al., 1997; Prauser et al., moricola inhabitant of marble). 1997). Strain BC 361T showed the peptidoglycan type A3γ with a single glycine residue as interpeptide bridge, Cells are Gram-positive, not acid-fast, spherical, oc- MK-8(H%) as predominant menaquinone and only curring singly, in pairs, tetrads and small clusters. straight-chain saturated and unsaturated fatty acids Non-motile and non-sporulating. No rod\coccus life and TBSA (Tables 2 and 3). Peptidoglycan structure cycle. Colonies are circular, convex, entire, orange- and the major menaquinone of strain BC 361T are pigmented, shiny and smooth. They can become rough consistent with the genus description of the genus and shaped like craters in old cultures. Aerobic. Nocardioides, but the complete absence of iso- and Catalase-positive, oxidase-negative. The cell wall pep- anteiso-branched chain fatty acids and the presence of tidoglycan contains -A#pm as characteristic diamino phosphatidylinositol clearly separates strain BC 361T acid and a single glycine residue as interpeptide bridge from representatives of this genus. Exclusively (type A3γ). The acyl type is acetyl. Phospholipids are straight-chain saturated and unsaturated fatty acids phosphatidylinositol, phosphatidylglycerol and di- and TBSA in combination with peptidoglycan type phosphatidylglycerol. Mycolic acids are absent. The A3γ with a single glycine residue as interpeptide bridge cellular fatty acid profile consists of straight-chain are in agreement with the genus description of saturated and monounsaturated components and Aeromicrobium, but strain BC 361T differs from all TBSA as the only branched chain fatty acid. The strains of this genus in containing MK-8(H%) as major major menaquinone is MK-8(H%). The DNA GjC menaquinone and by the absence of phosphatidyl- content is 72 mol%. Phylogenetically, this genus is a ethanolamine. In regard to its chemotaxonomic char- member of the family Nocardioidaceae (Stackebrandt acteristics, strain BC 361T seemed to occupy an et al., 1997). The type species is Marmoricola auran- ‘intermediate position’ between the genera No- tiacus. cardioides and Aeromicrobium. The phylogenetic analysis based on 16S rDNA se- Description of Marmoricola aurantiacus sp. nov. quence comparison placed strain BC 361T between the Marmoricola aurantiacus (au.ran.ti a.cus. M.L. adj. genera Nocardioides and Aeromicrobium and revealed h aurantiacus orange-coloured). N. jensenii (96n4% similarity) as its closest phylogenetic neighbour (Fig. 1). As published by Yoon et al. (1998a, Cells are spherical occurring singly, in pairs, tetrads b), N. jensenii shows the highest 16S rDNA similarity and small clusters (0n5–0n7 µm in diameter). Non- (95n0%) and the highest 16S–23S internally tran- motile and non-sporulating. No rod\coccus life cycle. scribed spacer sequence similarity (73n1%) of all Colonies are circular, convex, entire, orange-pig- validly published Nocardioides species to Aero- mented, shiny and smooth (2–5 mm in diameter). They microbium erythreum, the type species of the genus can become rough and shaped like craters in old Aeromicrobium. Despite its phylogenetic proximity to cultures. Aerobic. Catalase-positive, oxidase-negative. the genus Aeromicrobium, the fatty acid profile and Nitrate is not reduced to nitrite. No acid production is all other chemotaxonomic features characterize N. observed from -ribose, -arabinose, -xylose, - jensenii as a true member of the genus Nocardioides rhamnose, -glucose, -mannose, -galactose, malt- (Schumann et al., 1997; Suzuki & Komagata, 1983; ose, lactose, -cellobiose, -trehalose, -raffinose, Collins et al., 1989). Strain BC 361T differs from its glycerol, -mannitol or myo-inositol. Casein and closest phylogenetic neighbour N. jensenii not only by gelatin are not hydrolysed. Xanthine and tyrosine chemotaxonomic characteristics but also by sequences are not decomposed; hypoxanthine is decomposed. of the PCR primer designed for rapid identification of Aesculin is hydrolysed, Tween 80 is only weakly N. jensenii by multiplex PCR (Park et al., 1998) and of hydrolysed. DNA is not hydrolysed. The strain does 16S rDNA of strain BC 361T. The nucleotide pair G-G not grow in the following media: CZ, YMS-Glc, YCS- at position 445 and 446 in N. jensenii is replaced by the Glc, YG-Glyc, Y-Glyc, agar-H#O and M53. Growth pair C-A in the sequence of strain BC 361T. In addition, occurs in Luedemann, PDA and BHIA media. The cell T strain BC 361 exhibits several secondary-structure- wall peptidoglycan contains -A#pm as characteristic forming nucleotides that are not present in the 16S diamino acid and a single glycine residue as inter- rDNA sequences of any other member of the family. peptide bridge (type A3γ). The acyl type is acetyl. Thus, strain BC 361T possesses a unique combination Phospholipids are phosphatidylinositol, phosphatidyl-

534 International Journal of Systematic and Evolutionary Microbiology 50 Marmoricola aurantiacus gen. nov., sp. nov. glycerol and diphosphatidylglycerol. Mycolic acids are actinomycetes with diaminobutyric acid in the cell wall. Int J absent. The cellular fatty acid profile consists of Syst Bacteriol 46, 234–239. straight-chain saturated and monounsaturated com- Groth, I., Schumann, P., Rainey, F. A., Martin, K., Schuetze, B. & ponents and TBSA as the only branched chain fatty Augsten, K. (1997). Bogoriella caseilytica gen. nov., sp. nov., a acid. The major menaquinone is MK-8(H%). The DNA new alkaliphilic actinomycete from a soda lake in Africa. Int J GjC content is 72 mol%. Phylogenetically, it is a Syst Bacteriol 47, 788–794. member of the family Nocardioidaceae. The only Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein known isolation site is a Carrara marble statue molecules. In Mammalian Protein Metabolism, vol. 3, pp. (Wagmu$ ller’s monument) located in the Nordfriedhof 21–132. Edited by H. N. Munro. New York: Academic Press. Cemetery in Munich, Germany. The type strain is Lanyi, B. (1987). Classical and rapid identification methods for strain BC 361T (bacterial collection of the Institute of medically important . Methods Microbiol 19, 1–67. Microbiology, Messina). It has been deposited in the Luedemann, G. M. (1968). Geodermatophilus, a new genus of the DSMZ – German Collection of Microorganisms and Dermatophilaceae (Actinomycetales). J Bacteriol 96, 1848–1858. T Cell Cultures GmbH as strain DSM 12652 . Luedemann, G. M. & Fonseca, A. F. (1989). Genus Geoder- matophilus Luedemann 1968, 1857AL.InBergey’s Manual of Systematic Bacteriology, vol. 4, pp. 2406–2409. Edited by S. T. ACKNOWLEDGEMENTS Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams & This study was supported by the financial contribution of Wilkins. CNR (PF 97.00720. 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