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Int J Syst Evol Microbiol 54 (2004), 1987-1990; DOI  10.1099/ijs.0.63058-0
© 2004 International Union of Microbiological Societies

Agromyces ulmi sp. nov., a xylanolytic bacterium isolated from Ulmus nigra in Spain

Raúl Rivas, Martha E. Trujillo, Pedro F. Mateos, Eustoquio Martínez-Molina and Encarna Velázquez

Departamento de Microbiología y Genética, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, Spain

Correspondence
Martha E. Trujillo
mett{at}usal.es


   ABSTRACT
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Two xylan-degrading bacterial strains were isolated from a decayed Ulmus nigra tree in Spain. The isolates were Gram-positive, non-motile, aerobic and formed substrate mycelium which fragmented into irregular rods. 16S rRNA gene sequence analysis indicated that the isolates form a separate branch within the genus Agromyces phylogenetic cluster, with Agromyces mediolanus DSM 20152T being their closest relative (97·7 and 97·6 % sequence similarity). Catalase, nitrate reduction and urease tests differentiated these strains from A. mediolanus. Cell-wall peptidoglycan composition, major menaquinone, predominant fatty acids and phospholipid pattern were typical of the genus Agromyces. The DNA G+C content determined for the type strain XIL01T was 72 mol%. Based on the data presented, a novel species Agromyces ulmi sp. nov. is proposed. The type strain is XIL01T (=LMG 21954T=DSM 15747T).

Abbreviations: DAB, 2,4-diaminobutyric acid

Published online ahead of print on 23 April 2004 as DOI 10.1099/ijs.0.63058-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of strains XIL01T and XIL05 are AY427830 and AY427831, respectively.


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The genus Agromyces proposed by Gledhill & Casida (1969)Go currently harbours ten species, including both non-filamentous and filamentous, microaerophilic to aerobic, catalase- and oxidase-variable actinomycetes (Zgurskaya et al., 1992Go; Suzuki et al., 1996Go; Sasaki et al., 1998Go; Takeuchi & Hatano, 2001Go; Li et al., 2003Go; Ortiz-Martinez et al., 2004Go). During a study of xylanolytic bacteria from decayed stumps of elm trees we isolated two strains designated XIL01T and XIL05. The isolates showed the ability to hydrolyse xylan, a polymer of xylose present in plant cell walls.

The isolation of strains XIL01T and XIL05 was performed on XED agar (7 g xylan, 3 g yeast extract and 25 g agar l–1) as described by Rivas et al. (2003)Go. Cells grew as opaque white colonies on nutrient and XED agar media. Young cultures (15 h) produced branching substrate hyphae that broke into non-motile, diphtheroid and irregular rod-shaped cells (1·3–2·4x0·8–1·0 µm) at later stages. Such cells usually appeared as branching rods when observed by phase-contrast microscopy. Aerial mycelium was not formed. The two strains stained Gram-positive (Doetsch, 1981Go).

Amplification of the 16S rRNA gene and its sequencing was performed according to Rivas et al. (2003)Go. The MEGA2 package (Kumar et al., 2001Go) was used to calculate phylogenetic distances according to the Kimura two-parameter method (Kimura, 1980Go) and the tree was constructed using the neighbour-joining algorithm (Saitou & Nei, 1987Go); bootstrap analyses were based on 1000 resamplings.

Two 16S rRNA gene sequences of 1519 nucleotides were obtained for isolates XIL01T and XIL05; two differences were found between the sequences of the two strains, which formed a separate branch within the genus Agromyces (Fig. 1Go). The sequences of strains XIL01T and XIL05 showed 97·7 and 97·6 % similarity, respectively, with the sequence of Agromyces mediolanus DSM 20152T, which was the closest related species.



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  		Fig. 1. Neighbour-joining tree derived from analysis of 16S rRNA gene sequences (Kimura's two-parameter method) of isolates XIL01T and XIL05 and related Agromyces species. Bootstrap percentages are indicated at branch points. Bar, 1 nucleotide substitution per 100 nucleotides. Microbacterium lacticum IFO 14135T was used as the outgroup.

 
Biochemical and physiological properties of the two isolates investigated included carbon source assimilation, temperature growth rates and degradation of organic compounds (Rivas et al., 2003Go). Amylase, caseinase, catalase, oxidase and cellulase activities were detected as described by Rivas et al. (2003)Go. The two strains grew between 15 and 37 °C, were negative for oxidase reaction and showed very similar results for the other characteristics studied; however, strain XIL05 could be differentiated from XIL01T on the basis of assimilation of malate and arbutin degradation. Differential phenotypic characteristics between strains XIL01Tand XIL05 and their phylogenetically closest relatives are presented in Table 1Go. Other results are reported under the species description.


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  		Table 1. Characteristics that differentiate Agromyces ulmi sp. nov. from other phylogenetically close Agromyces species

Data are from this study and Takeuchi & Hatano (2001)Go, Li et al. (2003)Go and Dorofeeva et al. (2003)Go. +, Positive; –, negative; W, weak; ND, not determined.

 
Isolates XIL01T and XIL05 were grown on trypticase soy broth (Becton Dickinson, BBL) for 4 days at 28 °C for chemotaxonomic characterization, which included analyses of peptidoglycan composition, cell-wall sugars, cellular fatty acids, menaquinones and polar lipids. Studies were carried out according to the methods described by Schleifer & Kandler (1972)Go, Staneck & Roberts (1974)Go, Schleifer (1985)Go and Zimmermann et al. (1998)Go.

Both strains contained 2,4-diaminobutyric acid (DAB), glycine and glutamic acid in their cell walls, but alanine was not found. This composition is consistent with the B2{gamma}-type peptidoglycan, which has been reported for all strains currently representing the genus Agromyces. Cell-wall sugars found in strains XIL01T and XIL05 were rhamnose, fucose and glucose. Rhamnose and glucose have been reported for most species of the genus Agromyces, whereas fucose has been found only in Agromyces fucosus (Zgurskaya et al., 1992Go; Ortiz-Martinez et al., 2004Go).

The major fatty acids found for strains XIL01T and XIL05 were anteiso-C15 : 0 (46·2 and 45·6 %), iso-C16 : 0 (25·3 and 24·3 %) and anteiso-C17 : 0 (19·6 % and 22·0 %). This result is in agreement with other Agromyces species described (Suzuki et al., 1996Go; Takeuchi & Hatano, 2001Go; Li et al., 2003Go).

HPLC analysis of the menaquinones revealed major amounts of MK-12 and MK-11 for both strains, a result that is also reported for many Agromyces species (Takeuchi & Hatano, 2001Go; Dorofeeva et al., 2003Go). Isolate XIL01T contained the menaquinones MK-12, -11, -10, -13 and -9 (50 : 35 : 11 : 2 : 2), whereas XIL05 contained MK-12, -11, -13, -9 and -10 (61 : 23 : 7 : 4 : 3). Polar lipids were only studied for strain XIL01T and were composed of diphosphatidylglycerol, phosphatidylglycerol and glycolipid.

Determination of DNA base composition and DNA–DNA hybridization analyses were carried out as previously described by Rivas et al. (2003)Go. The G+C contents of strains XIL01T and XIL05 were 72·0 and 71·7 mol%, respectively. DNA–DNA relatedness values between strains XIL01T and XIL05, A. mediolanus DSM 20152T and Agromyces luteolus DSM 14595T were 100, 48·3 and 53·4 %, respectively, which clearly indicates the strains to be members of a separate genomic species (Wayne et al., 1987Go)

Thus, the results presented in this paper show that strains XIL01T and XIL05 should be classified as a novel species within the genus Agromyces, for which the name Agromyces ulmi sp. nov. is proposed.

Description of Agromyces ulmi sp. nov.
Agromyces ulmi (ul'mi. L. fem. gen. n. ulmi of the elm tree, referring to the isolation source of this micro-organism).

Gram-positive and non-spore-forming bacterium. Young cultures produce branching substrate hyphae that break up into non-motile, diphtheroid and irregular-shaped cells after 15 h. Aerial mycelium absent. Colonies on XED and nutrient agar are circular convex, white, opaque and usually 1–2 mm in diameter within 7 days at 28 °C. Aerobic; oxidase- and catalase-negative. Optimal growth temperature is 28 °C. Chemo-organotrophic. The following substrates are used as carbon sources: L-arabinose, cellulose, mannose, N-acetylglucosamine, gentiobiose, maltose, starch and xylan. By contrast, adipate, caproate, citrate, malate, mannitol and phenylacetate are not used. Produces acid from glucose, glycerol, D-xylose, galactose, fructose, mannose, L-sorbose, rhamnose, methyl {alpha}-D-mannoside, methyl {alpha}-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, lactose, maltose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, glycogen, xylitol, {beta}-gentiobiose and D-lyxose. Aesculin is hydrolysed; caseinase, {beta}-galactosidase and gelatinase are produced. Arginine dehydrolase, indole, tryptophan deaminase and urease are not produced. Nitrate is not reduced to nitrite. The peptidoglycan type is B2{gamma}. Other chemotaxonomic properties are described in the text and in Table 1Go.

The type strain, XIL01T (=LMG 21954T=DSM 15747T), was isolated from the decayed stump of an elm tree, Ulmus nigra.


   ACKNOWLEDGEMENTS
 
This work was supported by the CAICYT-DGES and JCyL (Spanish government). We thank M. Sánchez for the 16S rRNA gene sequences. We also acknowledge the DSMZ staff for their help with chemotaxonomic and DNA–DNA hybridization analyses.


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Dorofeeva, L. V., Krausova, V. I., Evtushenko, L. I. & Tiedje, J. M. (2003). Agromyces albus sp. nov., isolated from a plant (Androsace sp.). Int J Syst Evol Microbiol 53, 1435–1438.[Abstract/Free Full Text]

Gledhill, W. E. & Casida, L. E., Jr (1969). Predominant catalase-negative soil bacteria. III. Agromyces, gen. n., microorganisms intermediary to Actinomyces and Nocardia. Appl Microbiol 18, 340–349.

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]

Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). Molecular Evolutionary Genetics Analysis Software. Tempe, AZ: Arizona State University.

Li, W.-J., Zhang, L.-P., Xu, P., Cui, X.-L., Xu, L.-H., Zhang, Z., Schumann, P., Stackebrandt, E. & Jiang, C.-L. (2003). Agromyces aurantiacus sp. nov., isolated from a Chinese primeval forest. Int J Syst Evol Microbiol 53, 303–307.[Abstract/Free Full Text]

Ortiz-Martinez, A., Gonzalez, J. M., Evtushenko, L. I., Jurado, V., Laiz, L., Groth, I. & Saiz-Jimenez, C. (2004). Reclassification of Agromyces fucosus subsp. hippuratus as Agromyces hippuratus sp. nov., comb. nov. and emended description of Agromyces fucosus. Int J Syst Evol Microbiol 54, 1553–1556.[Abstract/Free Full Text]

Rivas, R., Sánchez, M., Trujillo, M. E., Zurdo-Piñeiro, J. L., Mateos, P. F., Martínez-Molina, E. & Velázquez, E. (2003). Xylanimonas cellulosilytica gen. nov., sp. nov., a xylanolytic bacterium isolated from a decayed tree (Ulmus nigra). Int J Syst Evol Microbiol 53, 99–103.[Abstract/Free Full Text]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Sasaki, J., Chijimatsu, M. & Suzuki, K. (1998). Taxonomic significance of 2,4-diaminobutyric acid isomers in the cell wall peptidoglycan of actinomycetes and reclassification of Clavibacter toxicus as Rathayibacter toxicus comb. nov. Int J Syst Bacteriol 48, 403–410.[Abstract/Free Full Text]

Schleifer, K. H. (1985). Analysis of the chemical composition and primary structure of murein. Methods Microbiol 18, 123–156.

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Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to identification of aerobic actinomycetes by thin layer chromatography. Appl Microbiol 28, 226–231.[Medline]

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