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Departamento de Microbiología y Genética, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain
Correspondence
Martha E. Trujillo
mett{at}usal.es
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. The G+C content determined was 69 mol%. Based on the present data, it is proposed that strain XIL02T (=LMG 20991T=CECT 5976T) be classified as the type strain of a novel Microbacterium species, for which the name Microbacterium ulmi sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of isolate XIL02T is AY062021.
A 16S rDNA-based neighbour-joining tree including all species of Microbacterium is available as supplementary material in IJSEM Online.
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-1,4-xylanases, arabinofuranosidases, -glucuronidases and -xylosidases, which are produced by a wide range of micro-organisms, many of them belonging to the high-G+C Gram-positive bacteria (Beg et al., 2000
; Busch & Stutzenberger, 1997; Chamberlain & Crawford, 2000; Rivas et al., 2003; Ruiz-Arribas et al., 1995; Mayorga-Reyes et al., 2002; Wang et al., 1998).
A study was undertaken to investigate the bacterial diversity in decayed trees of Ulmus nigra with the aim of isolating xylanolytic strains. A series of coryneform bacteria were isolated from various samples; one of these strains produced significant xylan hydrolysis activity and was chosen for further study. Chemotaxonomic, morphological, physiological and genetic characterization of this strain suggested that it belongs to the emended genus Microbacterium (Takeuchi & Hatano, 1998), which accommodated 33 species with validly published names at the time of writing. Members of the genus Microbacterium contain the unusual type-B peptidoglycan, and strains representing these species have been isolated from various sources (Zlamala et al., 2002). The data presented in this paper indicate that this strain represents a novel species of the genus Microbacterium, for which the name Microbacterium ulmi sp. nov. is proposed.
A sample of sawdust from a decayed tree of U. nigra was collected under aseptic conditions and 1 g was suspended in 100 ml sterile water and stirred for 30 min. From this suspension, 100 µl was spread on XED medium (0·7 % xylan, 0·3 % yeast extract and 2·5 % agar) and incubated at 28 °C. A bacterial strain that produced a conspicuous clearing zone around the area of growth was isolated after 10 days incubation and a pure culture was maintained as a glycerol suspension (25 %, v/v) at -80 °C.
Isolate XIL02T was observed by phase-contrast microscopy using 48-h-old cultures grown on nutrient broth to check for cell shape and motility. Cells were also Gram-stained as described by Doetsch (1981).
DNA extraction, PCR amplification of the 16S rRNA gene and sequencing of the PCR products were performed as described previously (Rivas et al., 2003). An almost complete 16S rRNA gene sequence was obtained and compared with those from the public databases. Sequences were aligned using the CLUSTAL X software (Thompson et al., 1997). An evolutionary-distance matrix was calculated using the algorithm of Jukes & Cantor (1969). The phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) and its topology was compared to that of a tree obtained using the maximum-parsimony method (Fitch, 1971). Bootstrap analyses were based on 1000 resamplings. The MEGA2 package (Kumar et al., 2001) was used for all analyses.
Strain XIL02T was cultivated in TSB (Becton Dickinson) for 4 days at 28 °C in a rotary shaker (90 r.p.m.) for cell wall and menaquinone analyses. The same medium amended with 1·5 % agar was used to cultivate the strain for fatty acid composition analysis. Menaquinone and cellular fatty acid compositions were determined as described by Zimmermann et al. (1998). Determination of the peptidoglycan type was carried out as described by Schleifer (1985) and Schleifer & Kandler (1972). Sugar analyses were performed according to described procedures (Staneck & Roberts, 1974).
The ability to solubilize phosphate was detected in YED-P plates (3 g yeast extract, 7 g glucose, 15 g agar and 3 g dibasic calcium phosphate l-1) as described previously (Peix et al., 2001). Amylase, catalase and oxidase activities were detected as described by Rivas et al. (2003). Casein hydrolysis activity was detected on skimmed milk agar. Cellulases were detected after 7 days incubation on plates containing 0·5 % carboxymethylcellulose as the carbon source, 0·3 % yeast extract and 1·5 % agar. Plates were stained with a 1 % aqueous Congo red solution. Other physiological and biochemical tests were done using API 20NE and API 50CH strips (bioMérieux) following the manufacturer's instructions.
DNA for base composition analysis was prepared according to Chun & Goodfellow (1995). The G+C content of DNA was determined using the thermal denaturation method (Mandel & Marmur, 1968).
DNA–DNA relatedness was tested [in 2x SSC plus 10 % (v/v) DMSO at 68 °C] between strain XIL02T and strains Microbacterium arborescens DSM 20754T and Microbacterium imperiale DSM 20530T. DNA was isolated using the procedure of Cashion et al. (1977) and DNA–DNA hybridization was carried out as described by De Ley et al. (1970) with the modification of Huß et al. (1983) and Escara & Hutton (1980). Renaturation rates were calculated using the TRANSFER.BAS program (Jahnke, 1992).
Isolate XIL02T was a Gram-positive, aerobic, non-motile, non-spore-forming and rod-shaped organism. Colonies on nutrient agar or XED medium showed a typical coryneform morphology. They were convex, smooth, white, opaque and 1–3 mm in diameter within 7 days at 28 °C.
An almost complete 16S rRNA gene sequence was obtained for isolate XIL02T and this indicated that the organism was phylogenetically related to members of the genus Microbacterium within the family Microbacteriaceae. Fig. 1 shows the relationship of strain XIL02T with its nearest phylogenetic relatives based on the neighbour-joining method. The closest relatives were M. imperiale (97·8 % similarity) and M. arborescens (97·4 %). Similar results were obtained using the maximum-parsimony method (data not shown). A fuller phylogenetic tree that includes all Microbacterium species with validly published names can be found as supplementary material in IJSEM Online.
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; Chabot et al., 1998; Rodríguez & Fraga, 1999), and their use as plant-growth-promoting rhizobacteria may be an important contribution to biofertilization of agricultural crops. Most strains studied for this purpose belong to the 
-proteobacteria, especially members of the genera Mesorhizobium and Rhizobium (Peix et al., 2001). During a screening programme for phosphate-solubilizing strains, we isolated strain XIL02T; to our knowledge, this is the first report of a Microbacterium strain that solubilizes inorganic phosphate. It is worth mentioning that a laboratory bacterial contaminant characterized and identified as Friedmanniella spumicola (BUTO1) also produced significant phosphate-clearing zones when it was incubated on a medium that contained dibasic calcium phosphate as the only phosphorus source (M. E. Trujillo, unpublished data). These data suggest that the biodiversity of phosphate-solubilizing bacteria is still poorly understood and that isolation of these micro-organisms is not restricted to the soil. On the other hand, Gram-positive bacteria with high G+C content have been observed as dominant members of phosphate-removing activated-sludge communities (Bond et al., 1995; Maszenan et al., 1999) and are believed to play a major role in phosphorus turnover. It cannot be concluded whether phosphate removal is linked to the solubilization of rock phosphate; however, a better knowledge of these processes will improve our understanding of the diversity and interactions of these micro-organisms.
The presence of the D-diamino acid D-ornithine in the cell wall indicated that strain XIL02T contained the unusual peptidoglycan type B2 (Schleifer & Kandler, 1972), which has been reported for 67 % of strains that currently belong to the genus Microbacterium (Behrendt et al., 2001). Other species, including M. arborescens and M. imperiale, contain L-lysine as the cell-wall diamino acid (Behrendt et al., 2001; Takeuchi & Hatano, 1998). The cell-wall sugars detected for strain XIL02T were galactose, fucose, xylose and rhamnose, while mannose was not found. Galactose and rhamnose have been found in cell walls of many other Microbacterium species, while fucose has only been reported for Microbacterium aurantiacum and Microbacterium aurum and xylose has been detected in Microbacterium chocolatum and Microbacterium laevaniformans. The cellular fatty acid pattern of strain XIL02T was composed of iso- and anteiso-branched fatty acids. The main fatty acids detected were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. These results are in accordance with those reported for the genus Microbacterium. The major isoprenoid quinones detected for this strain were MK-12 and MK-13, with minor amounts of MK-11 and MK-14 and traces of MK-10. This pattern has been reported for other Microbacterium species, including Microbacterium arabinogalactonolyticum, Microbacterium esteraromaticum, Microbacterium terregens, Microbacterium trichothecenolyticum and Microbacterium keratanolyticum (Behrendt et al., 2001). The major polar lipids detected for strain XIL02T were phosphatidylglycerol, diphosphatidylglycerol (cardiolipin) and an unknown glycolipid. Chemotaxonomic differences found between strain XIL02T and its closest phylogenetic relatives are shown in Table 1
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The G+C content of strain XIL02T was 69 mol%. This value is similar to those obtained in species from the genus Microbacterium.
DNA–DNA reassociation studies were used to confirm the species status of the novel isolate in relation to its closest phylogenetic neighbours. The results of DNA–DNA hybridization showed that strain XIL02T presented 43·9 and 32·6 % relatedness, respectively, with M. arborescens DSM 20754T and M. imperiale DSM 20530T. These results indicate that isolate XIL02T does not belong to either of these species when the recommendation of a threshold value of 70 % DNA–DNA similarity for species definition is considered (Wayne et al., 1987).
Therefore, on the basis of phylogenetic, chemotaxonomic and phenotypic data, we propose that isolate XIL02T should be classified as the type strain a novel species, for which the name Microbacterium ulmi sp. nov. is proposed.
Description of Microbacterium ulmi sp. nov.
Microbacterium ulmi (ul'mi. L. gen. fem. n. ulmi of the elm tree).
Gram-positive, aerobic or facultatively anaerobic, non-motile, non-spore-forming rods. It grows between 15 and 37 °C. The pH range for growth is 5–8. Catalase- and oxidase-negative. Utilizes D-arabinose, carboxymethylcellulose, cellobiose, D-fructose, gentiobiose, maltose, mannitol, D-mannose, phenylacetate, starch and xylan as carbon sources. Does not use L-arabinose, malate, N-acetyl glucosamine or citrate as carbon sources. Aesculin, casein and gelatin are hydrolysed; does not reduce nitrate. Arginine dihydrolase, ornithine decarboxylase, lysine decarboxylase, tryptophan deaminase and urease are not produced. Chemotaxonomic properties are listed in Table 1.
The type strain is strain XIL02T (=LMG 20991T=CECT 5976T), isolated from sawdust of a decayed tree of Ulmus nigra.
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ACKNOWLEDGEMENTS |
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