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1 Departamento de Microbiología y Genética, Universidad de Salamanca, Spain
2 Departamento de Química-Física, Universidad de Salamanca, Spain
Correspondence
Marta Montero-Barrientos
afta{at}usal.es
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain PPLBT is AY944176.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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on the basis of ribosomal nucleic acid sequences, fatty acids and polar lipid composition. This genus was previously known as Arthrobacter tumescens or Pimelobacter tumescens (Collins et al., 1989). This genus was placed in the family Intrasporangiaceae, together with the genera Intrasporangium (Stackebrandt et al., 1997), Janibacter (Martin et al., 1997), Terracoccus (Prauser et al., 1997), Ornithinicoccus (Groth et al., 1999), Tetrasphaera (Maszenan et al., 2000), Ornithinimicrobium (Groth et al., 2001), Knoellia (Groth et al., 2002), Arsenicicoccus (Olsson & Falsen, 2004) and Serinicoccus (Yi et al., 2004).
Strain PPLBT was isolated under aseptic conditions from a soil sample mixed with Iberian pig hair in the context of a study designed to isolate keratinolytic micro-organisms for biotechnological exploitation. Strain PPLBT was isolated by dilution plating on feather agar (NH4Cl, 0·5 g l–1; NaCl, 0·5 g l–1; K2HPO4, 0·3 g l–1; KH2PO4, 0·4 g l–1; MgCl2.6H2O, 0·1 g l–1; yeast extract, 0·1 g l–1; hammer-milled chicken feathers, 10 g l–1; agar, 20 g l–1) at 32 °C as described Williams et al. (1990) and Kim et al. (2001). Colonies showed transparent ‘haloes' produced by the hydrolysis of keratin from chicken feathers. Strain PPLBT was grown on nutrient agar for 48 h to check for motility by using phase-contrast microscopy (Axioskop 2; Zeiss). On this agar, strain PPLBT was able to grow at temperatures in the range 15–40 °C, but not at 4 or 45 °C. Strain PPLBT was able to grow on nutrient broth supplemented with 2, 5, 7 and 10 % (w/v) NaCl and growth was shown in the presence of 5 % (w/v) NaCl and weakly with 7 % (w/v) NaCl.
Gram-staining was performed as described by Doetsch (1981). Spore staining was carried out by using the Wirtz–Conlin procedure described by Chapin & Murray (1999). Cell morphology was observed under a Zeiss light microscope at x1000 magnification. Strain PPLBT was a Gram-positive, non-motile, non-spore-forming, long-rod-shaped organism (2·0x6·0 µm) and the colonies were yellow on feather agar and nutrient agar. The growth cycle of Terrabacter tumescens includes cocci and irregular rods and the colonies were white or grey. Strain PPLBT is invariably rod-shaped.
Oxidase activity was checked by examining the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride as described Smibert & Krieg (1981); catalase production was demonstrated on a slide by the production of bubbles from a drop of a 3 % hydrogen peroxide solution. The strain was negative for oxidase and positive for catalase. Strain PPLBT was different from Terrabacter tumescens in terms of catalase production. Nitrate reduction, indole and hydrogen sulphide production, the Voges–Proskauer reaction and aesculin hydrolysis were tested as described by Lanyi (1987). DNase activity was determined with DNase test agar (Becton Dickinson). Strain PPLBT differs from Terrabacter tumescens in terms of nitrate reduction and DNase production. Acid production from carbohydrates was detected by the method of Hugh & Leifson (1953). Hydrolysis of casein, gelatin, starch, Tween 80, tyrosine and urea was also determined, as described by Cowan & Steel (1965). The urease activity differed from that of Terrabacter tumescens. Other physiological and biochemical studies were carried out using the API 20NE and API ZYM systems (bioMérieux) according to the manufacturer's instructions. The biochemical, physiological and other differences between strain PPLBT and Terrabacter tumescens are shown in Table 1.
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) was 71 mol%. This value is similar to that of Terrabacter tumescens (73 mol%).
For 16S rRNA gene sequencing, DNA extraction was carried out as previously described by Rivas et al. (2001). The 16S rRNA gene sequence was compared with those deposited in the public databases. Phylogenetic analysis was performed using the software package MEGA (Molecular Evolutionary Genetics Analysis), version 2.1 (Kumar et al., 2001), after multiple alignment of data by using CLUSTAL X (Thompson et al., 1997). The distances were calculated according to the Kimura-2 model (Kimura, 1980) and clustering was determined using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis was based on 1000 resamplings. Fig. 1 shows the phylogenetic placement of strain PPLBT within the family Intrasporangiaceae and the genus Terrabacter. Sequence-similarity calculations performed after a neighbour-joining analysis indicated that the closest relatives of strain PPLBT were Terrabacter tumescens (98·8 %), followed by Terracoccus luteus (96·6 %), Intrasporangium calvum (96·5 %) and Janibacter limosus (96·1 %).
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, with the modification described by Escara & Hutton (1980) and Huß et al. (1983). DNA was isolated by using the procedure of Cashion et al. (1957). Renaturation rates were computed with the TRANSFER.BAS program of Jahnke (1992). The results of DNA–DNA hybridization showed 36·6 % relatedness between strain PPLBT and Terrabacter tumescens DSM 20308T; this suggests that, on the basis of the species-delineation recommendations of Wayne et al. (1987), strain PPLBT should be classified as a novel species of the genus Terrabacter.
The lipoquinone content of strain PPLBT was determined as described by Komagata & Suzuki (1987). The predominant isoprenoid quinone in strain PPLBT was MK-8(H4); this is characteristic of most species within the family Intrasporangiaceae (Stackebrandt et al., 1997).
The peptidoglycan type was determined as described by Schleifer & Kandler (1972). The peptidoglycan type of strain PPLBT represented the variation A3
, with three glycine residues as the interpeptide bridge. A fourth glycine residue is bound to the 
-carboxyl group of D-glutamic acid in position 2 of the peptide subunit (peptidoglycan type A41.2 according to the Deutsche Sammlung von Mikroorganismen und Zellkulturen murein key). This type is restricted to Terrabacter tumescens, Terracoccus luteus and I. calvum (Schleifer & Kandler, 1972).
Sugar analysis of whole cells and mycolic acid determinations were carried out as described by Staneck & Roberts (1974) and Minnikin et al. (1975), respectively. Mycolic acids were absent (as in Terrabacter tumescens) and the whole-cell sugar profiles contained fucose and large amounts of galactose.
Separation of polar lipids was achieved using TLC as described by Minnikin et al. (1975). The polar lipids detected for strain PPLBT, as well as for Terrabacter tumescens, were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and a phosphoglycolipid of unknown structure. The cellular fatty acid profile was determined by using GLC as described by Stead et al. (1992). The main fatty acids were i-C15 : 0 (34·50 %) and ai-C15 : 0 (18·23 %). Strain PPLBT differs significantly from Terrabacter tumescens (Prauser et al., 1997) in having a large amount of ai-C15 : 0 and a small amount of i-C14 : 0 (2·52 %). Other important fatty acids were ai-C17 : 0 (6·53 %) and i-C16 : 0 (4·59 %). The complete fatty acid profile and the differences with respect to Terrabacter tumescens are shown in Table 2.
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Description of Terrabacter terrae sp. nov.
Terrabacter terrae (ter'rae. L. gen. n. terrae of the earth).
Cells are long irregular rods 2·0x6·0 µm in length. Aerial mycelium is not formed. Gram-positive in young and old cultures, non-motile, non-spore-forming and aerobic. Colonies are glossy and yellow on feather and nutrient agar. Oxidase-negative and catalase-positive. Chemo-organotrophic, with simple nutritional requirements. Metabolism is respiratory; acid is not produced from glucose or other carbohydrates. A wide range of organic compounds are utilized for growth, including glucose, mannitol, citrate and chicken feathers. Does not assimilate arabinose, mannose, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate or phenylacetate. Esterase lipase, lipase, naphthol-AS-BI-phosphohydrolase, keratinase, -galactosidase and valine aminopeptidase are produced. Alkaline phosphatase, esterase, leucine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, acid phosphatase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are not produced. Negative for DNase decomposition, nitrate reduction, the Voges–Proskauer reaction and H2S production. Negative for decomposition of aesculin and Tween 80. Able to hydrolyse urea, arginine, gelatin, starch and casein. The LL-diaminopimelic acid-containing peptidoglycan of group A3 has three glycine residues as an interpeptide bridge. The main menaquinone is MK-8(H4). The fatty acid profile represents the iso, anteiso type, with 13-methyl tetradecanoic acid and 12-methyl tetradecanoic acid predominating. The polar lipids are phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylinositol. Mycolic acids are absent. The DNA G+C content is 71 mol%.
The type strain is PPLBT (=CECT 3379T=LMG 22921T). Isolated from soil mixed with Iberian pig hair.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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