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1 Departamento de Microbiología y Genética, Edificio Departamental, Lab 209, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany
Correspondence
Martha E. Trujillo
mett{at}usal.es
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Published online ahead of print on 5 November 2004 as DOI 10.1099/ijs.0.63361-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain WA201T is AJ626950.
Cellular fatty acid profiles and an extended phylogenetic dendrogram are available as supplementary material in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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) are reported to inhabit soil, water, marine environments and sediments (Lüdemann & Brodsky, 1963; Kawamoto, 1989). Micromonospora endolithica, isolated from an extreme Antarctic sandstone environment, has been described recently (Hirsch et al., 2004), showing that this genus is distributed in very different environments. Kasai et al. (2000) redefined and reduced the genus to 14 species based on DNA–DNA relatedness, 16S rRNA and gyrB phylogenetic data. We report the classification of an organism representing a novel Micromonospora species.
Isolate WA201T was recovered from a water sample of a pond, located on a former uranium mine (Ciudad Rodrigo, Spain) where radon is known to be dissolved in water (Lozano et al., 2002). The isolation procedure using soil extract agar pH 6·5 at 28 °C was described previously by Trujillo et al. (2004). Long-term maintenance of strain WA201T was accomplished by storage in glycerol suspension (20 %, w/v) at –80 °C. The isolate was cultured on Bennett's agar, glucose-yeast extract agar and nutrient agar to check for growth rate. Growth was slow on these media and therefore a basal medium (SA1) was designed to improve it. SA1 contained glucose, 10 g; yeast extract (Difco), 3 g; tryptone (Difco), 5 g; tryptose (Difco), 2 g; starch (Fluka), 2 g; CaCO3, 100 mg; CoCl2, traces; ferric citrate, traces; Bacto-agar (Difco), 18 g; and distilled water, 1 l. The pure culture was then routinely grown on SA1 agar.
Extraction of genomic DNA, PCR amplification of the 16S rRNA gene and sequencing of the purified PCR products were described previously (Rivas et al., 2003). The sequence of isolate WA201T was manually aligned and compared with representative sequences of members of the order Actinomycetales obtained from GenBank/EMBL. Phylogenetic distances were calculated with the Kimura two-parameter model and tree topologies were inferred using the least-squares (De Soete, 1983), maximum-parsimony (Fitch, 1972) and neighbour-joining methods (Saitou & Nei, 1987). One thousand bootstrap replications were performed using the MEGA program as described by Kumar et al. (2001).
Morphological features were studied on glucose-yeast extract agar, nutrient agar and SA1 agar at 28 °C. Cell morphology and motility were observed by phase-contrast microscopy using 5-day-old cultures. Spore production was examined on 3-week-old cultures on SA1 agar using a scanning electron microscope (Zeiss, DSM 940). Agar plugs were fixed overnight in phosphate buffer (pH 7·0) which contained 2 % paraformaldehyde and 0·2 % glutaraldehyde, dehydrated through a graded ethanol series, critical-point dried and sputter-coated with gold. The Gram and acid-fast stains (Doetsch, 1981) were performed using 3-day-old cultures.
The ability of isolate WA201T to grow on a range of sole carbon sources at 1 % (w/v) was determined according to Williams et al. (1983) with agar and a nitrogen base without amino acids (Difco). NaCl tolerance and temperature (4–45 °C) growth ranges were determined on SA1 medium.
Isolate WA201T and Micromonospora carbonacea DSM 43168T were also characterized using the API 20NE and API ZYM systems (bioMérieux) according to the manufacturer's instructions and incubation of the strips for 48 h at 28 °C. Catalase and oxidase activity were recorded as described previously (Rivas et al., 2003). Hydrolysis of casein (1 % skimmed milk), aesculin, arbutin, starch (1 % w/v), tyrosine (0·5 %) and xylan (0·4 %) were tested on SA1 as the basal medium.
Susceptibility to various antibiotics was examined for the isolate and M. carbonacea DSM 43168T using amikacin (30 µg), amoxicillin/clavulanic acid (20/10 µg), ampicillin (2 µg), cefaclor (30 µg), cefazolin (30 µg), ciprofloxacin (5 µg), cloxacillin (1 µg), cefuroxime (30 µg), erythromycin (2 and 15 µg), gentamicin (10 µg), neomycin (5 µg), netilmicin (30 µg), oxacillin (1 µg), oxytetracycline (30 µg), penicillin G (10 U), piperacillin (100 µg), polymyxin B (300 IU), tetracycline (30 µg) and vancomycin (30 µg) disks (Oxoid), and SA1 as the basal medium. Readings were taken at 3, 5 and 10 days.
Isomers of diaminopimelic acid (DAP) in whole-cell hydrolysates were determined by TLC on cellulose (modified method of Hasegawa et al., 1983; Rhuland et al., 1955). Whole-cell sugars were analysed according to Staneck & Roberts (1974). Menaquinones were extracted and purified by the method of Minnikin et al. (1984) and analysed by HPLC (Hewlett Packard 1100). Methyl esters of cellular fatty acids were prepared from cells grown for 24 h on trypticase soy agar cultures (28 °C) and analysed by GLC (Schröder et al., 1997). Polar lipids were extracted and identified by two-dimensional TLC (Minnikin et al., 1984). The DNA G+C content was determined using the thermal melting method (Mandel & Marmur, 1968).
DNA–DNA relatedness was measured spectrophotometrically between isolate WA201T and M. carbonacea DSM 43168T following the method of De Ley et al. (1970) with the modification of Huß et al. (1983). DNA was purified on hydroxyapatite as described by Cashion et al. (1977). Renaturation rates were calculated using the TRANSFER.BAS program of Jahnke (1992).
The 16S rRNA gene sequence of isolate WA201T (1517 nt) showed a close relationship with members of the family Micromonosporaceae and fell within the genus Micromonospora clade. The highest sequence similarities found were with M. carbonacea DSM 43168T (98·5 %) and Micromonospora matsumotoense IFO 14550T (98·1 %), which correspond to 22 and 28 nucleotide differences (1473 nucleotides compared), respectively. The various tree-making algorithms yielded similar tree topologies although some of the branches varied slightly, but isolate WA201T was always recovered in the same branch as M. carbonacea DSM 43168T. The closest phylogenetic relatives of strain WA201T are represented on the tree corresponding to the Kimura two-parameter method and the least-squares method (Fig. 1). An extended phylogenetic dendrogram is provided as Supplementary Fig. A in IJSEM Online.
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Strain WA201T grew at between 20 and 37 °C, but no growth was recorded at 4, 10, 15 or 45 °C after 3 weeks. Tolerates NaCl up to 3 %; no growth occurred at 5 %. Nitrate was not reduced. The isolate hydrolysed aesculin, arbutin, casein, gelatin, starch and xylan.
WA201T and M. carbonacea DSM 43168T produced the following enzymes (API ZYM kit): alkaline phosphatase, esterase lipase (C8), lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase.
The above strains produced identical antibiotic profiles, which showed resistance to ampicillin (2 µg) and susceptibility to amikacin (30 µg), amoxicillin/clavulanic acid (20/10 µg), cefaclor (30 µg), cefuroxime (30 µg), cefazolin (30 µg), ciprofloxacin (5 µg), cloxacillin (1 µg), erythromycin (2 and 15 µg), gentamicin (10 µg), neomycin (5 µg), netilmicin (30 µg), oxytetracycline (30 µg), penicillin G (10 U), piperacillin (100 µg), polymyxin B (300 IU), oxacillin (1 µg), tetracycline (30 µg) and vancomycin (30 µg).
Various physiological and biochemical results useful to differentiate between isolate WA201T and its closest phylogenetic neighbours M. carbonacea DSM 43168T and M. matsumotoense IFO 14550T are given in Table 1. Other results are presented in the species description.
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(Schleifer & Kandler, 1972). Glucose in large amounts, galactose, mannose and xylose were found as whole-cell sugars. The fatty acid pattern for the isolate was composed of saturated and unsaturated iso/anteiso-branched fatty acids. A significant amount of 10-methyl-branched 17 : 0 (5·24 %) was found in addition. Straight-chain saturated and unsaturated fatty acids were present only in minor amounts (Supplementary Table A). The main menaquinones were MK-10(H4) (77 %) and MK-10(H6) (23 %). A small amount of MK-9(H4) (5 %) was also found. MK-10(H2) and MK-9(H6) occurred only as traces. This menaquinone profile differs from that reported for M. carbonacea DSM 43168T, which contains MK-9(H4) in large amounts (Kawamoto, 1989), while M. matsumotoense IFO 14550T contains MK-10(H6), MK-10(H8) and MK-10(H4) in a ratio of 1·9 : 1·8 : 1 (Lee et al., 1999). The menaquinone composition within the genus Micromonospora has been reported to be complex and heterogeneous in comparison to the menaquinone composition found in other actinomycete genera, which show similar high levels of intrageneric relatedness (Koch et al., 1996). The polar lipid pattern was mainly composed of phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and the diagnostic phosphatidylethanolamine. Some glycolipids of unknown structure were also found. This pattern correlates with phospholipid pattern II sensu Lechevalier et al. (1977). The G+C content of the DNA was 68·6 mol%. Most chemotaxonomic markers support the placement of isolate WA201T in the genus Micromonospora.
The DNA–DNA relatedness value (36·5 %) clearly indicated that isolate WA201T does not belong to the species M. carbonacea, as this value is well below the threshold value of 70 % for definition of bacterial species according to Wayne et al. (1987).
The chemical, morphological and phylogenetic data suggest that strain WA201T represents a novel species when compared with the type strains of species with validly published names within the genus Micromonospora. Thus, on the basis of this polyphasic taxonomic study, strain WA201T merits classification as a novel species within the genus Micromonospora and the name Micromonospora mirobrigensis sp. nov. is proposed.
Description of Micromonospora mirobrigensis sp. nov.
Micromonospora mirobrigensis (mi.ro.bri.gen'sis. N.L. fem. adj. mirobrigensis pertaining to Mirobriga, the region in Spain where the type strain was isolated).
Gram-positive, chemo-organotrophic and strictly aerobic. Colonies on SA1 agar are 2–3 mm in diameter after 2 weeks. Colonies are raised, folded and orange, turning brown to black on sporulation. Well-developed substrate hyphae bearing single slightly warty spores; aerial mycelium is not produced. Optimum pH and temperature for growth are 7 and 28 °C, respectively. Oxidase- and catalase-positive. Nitrate is not reduced; urease-negative. Growth is not observed in the presence of crystal violet 0·001 % or sodium azide 0·01 %. Resistant to ampicillin (2 µg). Acid is produced from glucose. Carbon sources assimilated are L-arabinose, D-cellobiose, D-galactose, D-glucose, D-maltose, D-mannose, D-raffinose, D-sucrose and D-trehalose. Adipate, caprate, citrate, malate, D-mannitol, D-melezitose, phenyl-acetate, L-rhamnose, D-sorbitol, L-sorbose and xylitol are not assimilated. Contains meso-DAP in its cell wall; major menaquinone is MK-10(H4). Major fatty acids are iso-15 : 0, iso-16 : 0, iso-17 : 1 and anteiso-17 : 0.
The type strain, WA201T (=DSM 44830T=LMG 22229T), was isolated from the region of Mirobriga (Ciudad Rodrigo, Spain).
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ACKNOWLEDGEMENTS |
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