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1 Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone 31, I-98166 Messina, Italy
2 DSMZ – German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
3 GBF – Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Erko Stackebrandt
erko{at}dsmz.de
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ABSTRACT |
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Published online ahead of print on 29 August 2003 as DOI 10.1099/ijs.0.02745-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains BC512, BC444T, BC517, BC521 and BC412 are AJ316570–AJ316574.
Detailed fatty acid compositions of B. aggregatus DSM 4725T and nine novel strains are available as supplementary material in IJSEM Online.
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), Modestobacter (Mevs et al., 2000) and Blastococcus (Ahrens & Moll, 1970; Urzì et al., 2001). The result of a detailed taxonomic investigation of some of the isolates that cluster with Blastococcus aggregans is reported.
Isolation and cultivation
Ten strains were isolated from various sites (Table 1) by dilution plating on Bunt & Rovira medium (Bunt & Rovira, 1955), modified by addition of 0·5 % glucose, 0·5 % NaCl and 0·03 % Na2CO3, pH 8·6 (medium BRII), and maintained in Luedemann medium (LM; Luedemann, 1968). Growth was tested on LM agar plates at 6, 10, 20, 32, 37 and 45 °C. NaCl tolerance was checked at concentrations of 0·5, 3, 5, 6 and 10 % (w/v). Growth at different pH values was tested at pH 4, 5, 6·8, 8·6, 10 and 12.
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) (no growth for strain BC521). Growth was not supported on potato dextrose agar (Oxoid). Growth was observed at 20 and 37 °C, with an optimum at 32 °C. Strains BC412 and BC518 also grew at 15 °C. No growth occurred below 10 °C or at 45 °C. Growth in LM occurred at pH 5–8·6 with an optimum at pH 6·8; except for strains BC412, BC512 and BC521, which grew at up to pH 8·6, all strains grew at up to pH 10·5. No growth was observed at or above 3 % NaCl, except for strain BC448, which grew at 5 % NaCl.
Colony and cell morphology
Cell and aggregate morphology were determined by bright-field microscopy and by phase-contrast microscopy (DML; Leica) (oil-immersion objective, magnification x100). For scanning electron microscopy analysis, bacterial samples were dehydrated with a graded series of acetone, critical-point-dried with CO2 and sputter-coated with gold before examination in a Zeiss field emission scanning electron microscope DSM982 Gemini at 5 kV using the Everhart-Thornley SE-detector and the in-lens SE-detector in a 50 : 50 ratio. Morphology of colonies was studied with a stereomicroscope (Wild M10; Leica). Colonies of all strains were pink- to orange-pigmented, circular (diameter 2–3 mm) with a smooth or rough surface. Cell morphology was characterized by coccoid cells (0·5–1·7 µm in diameter) occurring singly, in pairs or in tetrads, often tending to form aggregates (Fig. 1a, b). Under the conditions used, bud formation was observed only for strains BC412, BC509, BC517, BC518 and BC521, and small motile zoospores were produced by BC509, BC412 and BC521. Strains BC412 and BC521 formed elongated cells, produce germ tubes and long filaments.
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; Schumann et al., 1997) of B. aggregatus DSM 4725T and the BC strains were characterized by the predominance of iso-C16 : 0 and iso-C16 : 1 acids. Other fatty acids, some occurring in major amounts (C17 : 1
8c), differed among strains and are indicated in the species description. The fatty acid profiles are available as supplementary material in IJSEM Online. The major respiratory quinones, determined as described previously (Collins et al., 1977; Groth et al., 1996), were MK-9(H4) and MK-9; homologues of the H4 series (7, 8) as well as the MK-9 series (H2, H6) may occur in minor amounts. The polar lipid pattern (Minnikin et al., 1979; Collins & Jones, 1980) of strain BC444T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine. B. aggregatus DSM 4725T and strain BC412 also contained two unknown phospholipids. The cell walls of strains BC444T, BC521 and BC412 and of B. aggregatus DSM 4725T contained meso-diaminopimelic acid (meso-A2pm), confirming the observation stated to be characteristic of members of the family Geodermatophilaceae (Normand et al., 1996). The DNA G+C content (Mesbah et al., 1989) of B. aggregatus DSM 4725T was 73·9 mol%.
Phylogenetic analyses
Of the isolates for which partial 16S rRNA gene sequences (5550 nucleotides) have been published previously (Urzì et al., 2001), six strains were selected for determination of almost complete sequences. These strains represented three different lineages, branching adjacent to that of B. aggregatus DSM 4725T. Sequencing methods and analyses of phylogenetic relatedness followed described procedures (Rainey et al., 1996). The phylogenetic analysis supported analyses based on partial sequences: three lineages emerged, defined by strains BC444T/BC448/BC512/BC517, sharing >99·9 % similarity, BC412 and BC521; sequences of members of these lineages were 97·6–98·2 % similar among themselves. B. aggregatus DSM 4725T was about equidistantly related to the BC521 and BC412 lineages (99·2 and 98·6 % similar) and slightly less closely related to strain BC444T and related strains (98·0 %). Based upon partial sequences, the BC444T lineage contained several additional isolates that were >99·9 % similar to strain BC444T (Urzì et al., 2001). Similarity values were transformed into phylogenetic distance values that compensate for multiple substitutions at any given site in the sequence (Jukes & Cantor, 1969). A distance matrix dendrogram (DeSoete, 1983) is shown in Fig. 2.
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; Stackebrandt et al., 2002) using the restriction enzymes PvuII and PstI. The patterns were in accord with 16S rRNA gene sequence analysis in that members of the BC444T cluster were homogeneous, while members of the other lineages were defined by individual patterns (Fig. 3).
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; Huß et al., 1983; Jahnke, 1992) revealed that strain BC444T shared only 39 % similarity with strain BC412 and 50 % similarity with strain BC521. All three isolates were clearly unrelated to B. aggregatus DSM 4725T (25–37 % similarity). Because of the low sequence similarity (98·2 %), the binary DNA–DNA reassociation value was not determined for strains BC521 and BC412.
Physiology
Physiological tests were performed at 28 °C using Biolog GN, API ZYM and API 20 NE substrate panels. Catalase and oxidase activities were determined by the formation of bubbles in a 3 % hydrogen peroxide solution; oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine was done on filter paper. Properties are compiled in Table 2. The metabolic reactions of members of the three novel Blastococcus lineages showed significant differences from that of B. aggregatus DSM 4725T as well as among themselves.
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studied 65 isolates from natural stones and monuments attributed to the family Geodermatophilaceae and showed that, on rock surfaces, members of the genera Geodermatophilus, Modestobacter and Blastococcus had different distributions. In fact, strains belonging phylogenetically to Geodermatophilus were very rare, while strains belonging to Modestobacter were very common and were distributed on stone surfaces located in different climates. Strains taxonomically affiliated to the only species of the genus Blastococcus, B. aggregatus, isolated from the Baltic Sea (Ahrens & Moll, 1970), were found on rock surfaces located in the Mediterranean basin and, in particular, they were isolated from monuments and rocks in Greece, Italy and Malta. The ten strains share a few characteristics with B. aggregatus (Table 2
) such as the formation of pink/orange colonies, aerobic metabolism and the presence of MK-9(H4) as the predominant menaquinone. On the other hand, differences were observed in the composition of fatty acids and physiological properties. Compared with the stone surface isolates, B. aggregatus DSM 4725T is characterized by a rather low metabolic profile. Based on phylogenetic, genomic and phenotypic grounds, strains of the BC444T cluster also differ from strains BC521 and BC412 and can therefore be described as representatives of a novel species of the genus Blastococcus, for which the name Blastococcus saxobsidens sp. nov. is proposed.
As a consequence of newly reported properties not included in the original description of strain DSM 4725T (Ahrens & Moll, 1970), the description of the genus Blastococcus must be emended. At this stage, we refrain from describing strains BC521 and BC412, though, based upon genomic and phenetic evidence, these single-strain taxa would merit formal description. The rationale for not doing so is based on the finding that the novel species B. saxobsidens shows a significant number of variable and weak physiological reactions. This indicates that the intraspecies metabolic diversity is higher than is expressed by the type strain.
Emended description of the genus Blastococcus Ahrens and Moll 1970
Blastococcus (Blas.to.coc'cus. Gr. n. blastos sprout, bud; Gr. n. coccus a grain, berry; N.L. masc. n. Blastococcus the budding coccus).
A formal description has never been provided. The description is based on the morphological observations of Ahrens & Moll (1970), supplemented with data reported in this study.
Gram-positive. Coccoid units, often reproducing by budding and multiple fission, giving rise to a variety of cell forms and aggregates. Single cells may be motile rods and vibrioid or non-motile cocci that tend to form aggregates. Formation of buds is common but not universal. One to four membranous bodies per cell may be present, which are linked to the cytoplasmic membrane. Aerobic; some cells may be microaerophilic. Strains from surfaces of marble and limestone utilize a broader spectrum of organic compounds than the only known marine strain. Predominant fatty acids are iso-C16 : 0, iso-C16 : 1, C18 : 19c and, in some strains, C17 : 18c, iso-C15 : 0 and C17 : 0. Peptidoglycan contains meso-A2pm as diagnostic diamino acid. Predominant menaquinone is MK-9(H4); MK-9 may also occur in large amounts. Polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine; two unknown phospholipids may occur. DNA G+C content is 74 mol%. Phylogenetically, a member of the family Geodermatophilaceae.
The type species is Blastococcus aggregatus.
Emended description of Blastococcus aggregatus Ahrens and Moll 1970
The emendation is based on the description of Ahrens & Moll (1970), supplemented with data reported in this study.
Gram-positive, highly irregular vibrioid or rod-shaped cells occurring singly or in three-dimensional coccoid aggregates. The manifestation of one of these stages is influenced by environmental conditions. Low salt concentrations, temperatures of 5–10 °C and microaerophilic conditions favour the motile rod stage. Single cells are either motile vibrioid (0·3–1·5x0·4–3·0 µm) or rods or ellipsoid (1·2–1·5x1·5–3·0 µm). Rods are often separated by disc-like septa. These motile or non-motile rods carry one to six vibrioform buds, attached to the mother cell by a tapered pole. Increased temperatures and salt concentrations induce the formation of non-motile coccoid aggregates (1·2–2·5 µm in diameter), appearing as linear, band-like or column-like three-dimensional forms. Larger aggregates (1 mm diameter) are separated by rectangular and/or radial partitions. Cell types of different size occur mostly side by side. After 5 days on peptone/yeast extract agar at 20 °C, colonies are pink, turbid, round and convex with a shiny surface. In liquid media, thin turbidity and formation of pink sediment is observed. Catalase-positive, oxidase-negative. No growth in mineral medium; weak growth in the presence of ethanol, but not with glucose, acetate, citrate, methanol, ethylamine or paraffin. Good growth in mineral medium plus peptone (0·5 %) and yeast extract (0·1 %); in these media, addition of 1 % of either glucose, acetate, citrate, ethylamine or paraffin reduces growth. Growth is enhanced in peptone/yeast extract medium by the addition of 1 % NaCl. No acid or gas from glucose, galactose, fructose, xylose, sucrose, maltose, lactose or glycerin. Methyl-red and Voges–Proskauer reactions are negative. No hydrolysis of starch, chitin, alginate or cellulose. Reactions towards substrates provided by the API ZYM, API 20 NE and Biolog GP2 panels are indicated in Table 2. Major fatty acids are 14-methyl pentadecanoic acid (iso-C16 : 0) and 14-methyl pentadecenoic acid (iso-C16 : 1). Diamino acid of peptidoglycan, menaquinone and polar lipids are as described for the genus. Two unknown phospholipids may occur. Extinction maxima of carotenoids at 470 and 500 nm. The DNA G+C content is 73·9 mol%.
The type strain, DSM 4725T (=ATCC 25902T), was isolated from the Baltic Sea.
Description of Blastococcus saxobsidens sp. nov.
Blastococcus saxobsidens (sax.ob'si.dens. L. n. saxum rock; L. v. obsideo to occupy; N.L. part. adj. saxobsidens rock-occupying).
Aerobic, Gram-positive. Motile and non-motile cells (1·0–1·7 µm in diameter) are coccoid, occurring in tetrads with a tendency to remain aggregated. Sparse growth on LM, malt agar and YCS-Glyc agar. No growth on potato dextrose agar. Temperature range for growth is 20–37 °C, with an optimum at 32 °C; growth at pH 5–8·6, with an optimum at pH 6·8. No growth at 3 % NaCl, except strain BC448, which grows at 5 % NaCl. Colonies are pink/orange-pigmented, irregular, convex with a smooth to rough surface (2–3 mm in diameter). Catalase- and oxidase-positive. Nitrate is not reduced to nitrite. Carbon sources utilized for growth are D-glucose, D-fructose, L-arabinose, ribose, myo-inositol and lactose. No acid production from D-fructose, myo-inositol or lactose. Casein, gelatin and starch are not hydrolysed. DNA, tyrosine, xanthine and hypoxanthine are not decomposed. Tweens 20, 40, 60 and 80 are not hydrolysed. Major fatty acids are 14-methyl pentadecanoic acid (iso-C16 : 0), cis-9-heptadecenoic acid (C17 : 18c) and 13-methyl tetradecanoic acid (iso-C15 : 0). Diamino acid of peptidoglycan, major menaquinone and polar lipids are as described for the genus.
The type strain, BC444T (=DSM 44509T=NRRL 24246T), was isolated from limestone sampled in Malta. Other strains, BC448, BC509, BC512, BC517, BC518, BC519 and BC525, were isolated from calcarenite and marble from Italy and Greece. Phenotypic differences from the type strain are shown in Table 2.
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ACKNOWLEDGEMENTS |
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-glucosidase, assimilation of mannitol, N-acetylglucosamine, maltose, caproate, citrate and phenylacetate, lipase, 
-galactosidase,