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1 DSMZ – German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
2 Centre for Agricultural Landscape and Land Use Research (ZALF), Institute of Primary Production and Microbial Ecology, Gutshof 7, D-14641 Paulinenaue, Germany
3 Centre for Agricultural Landscape and Land Use Research (ZALF), Institute of Primary Production and Microbial Ecology, Eberswalder Straße 84, D-15374 Müncheberg, Germany
4 Biological Resource Centre, Biotechnology Centre, National Institute of Technology and Evaluation (NITE), Kazusa Academia Park, Kisarazu, Chiba 292-0812, Japan
Correspondence
Peter Schumann
psc{at}dsmz.de
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and Agreia bicolorata Evtushenko et al. 2001. The two species were found to share genus-specific chemotaxonomic characteristics such as the occurrence of D-ornithine and L-2,4-diaminobutyric acid in the peptidoglycan and the profile of cellular fatty acids and 1,1-dimethoxy-alkanes. DNA–DNA relatedness of only 47·8 % and differences in phenotypic features such as the menaquinone profile and oxidase and Voges–Proskauer reactions confirmed the distinct species status of S. pratensis and A. bicolorata. On the basis of the data from phylogenetic and phenotypic analyses, the reclassification of S. pratensis as Agreia pratensis comb. nov. is proposed. As a result of this reclassification, the two genera are coherent, in that the cell wall composition and 1,1-dimethoxy-alkane spectrum are significant genus-specific characteristics.
Published online ahead of print on 6 June 2003 as DOI 10.1099/ijs.0.02664-0.
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Without being aware of the similarity of their research subjects, two research groups isolated strains of coryneform bacteria that contained a hitherto novel B-type peptidoglycan containing ornithine and 2,4-diaminobutyric acid (DAB). One group decided in favour of proposing the new genus Agreia to accommodate the isolates (Evtushenko et al., 2001), whereas the other submitted a proposal for a novel species of the phylogenetically closest genus Subtercola, Subtercola pratensis (Behrendt et al., 2002), during the review process of the description of Agreia. For obvious reasons, the type strains of Agreia bicolorata and S. pratensis could not be compared at that time. The taxonomic status of A. bicolorata and S. pratensis had to be reconsidered after valid publication of both names. The type strains A. bicolorata DSM 14575T and S. pratensis DSM 14246T were therefore subjected to supplementary chemotaxonomic studies, detailed phylogenetic analyses and DNA–DNA hybridization.
To clarify whether S. pratensis and A. bicolorata indeed display the same type of peptidoglycan, enantiomeric diamino acid isomers were determined according to Sasaki et al. (1998). The type strain of A. bicolorata and the type strain of S. pratensis contained both D-orn and L-DAB in almost equal amounts in their cell walls and thus can be clearly differentiated from Subtercola boreus and Subtercola frigoramans, which contain only DAB in their peptidoglycan. S. pratensis and A. bicolorata display identical two-dimensional TLC patterns of peptides and amino acids resulting from partial hydrolysis (Schleifer, 1985) of the peptidoglycan (data not shown). Dinitrophenylation (Schleifer, 1985) of peptidoglycan samples of S. pratensis and A. bicolorata revealed free amino groups at Orn and DAB. Since position 3 of the peptide subunit of all known B-type peptidoglycan structures contains an L-isomeric amino acid (Schleifer & Kandler, 1972; DSMZ, 2001), it may be concluded that L-dab occupies position 3 of the peptide subunit and D-Orn is involved in the interpeptide bridge. Muramic acid residues of the peptidoglycans of both S. pratensis and A. bicolorata are acetylated, as determined according to Uchida et al. (1999). These results suggest that S. pratensis and A. bicolorata display the same peptidoglycan structure.
A further chemotaxonomic feature, the profiles of fatty acid methyl esters and 1,1-dimethoxy-alkanes of A. bicolorata and S. pratensis type strains, was analysed by GC and GC/MS according to Schumann et al. (1997). As shown in Table 1, similar patterns were found for the two strains. Supplementary to the report of Evtushenko et al. (2001), it was found that A. bicolorata also contained 1,1-dimethoxy-anteiso-pentadecane (a-15 : 0 DMA). Comparison of profiles with those of S. boreus and S. pratensis (Männistö et al., 2000) revealed a similar spectrum for iso- and anteiso-branched fatty acids, but differences were found in the composition of 1,1-dimethyl-alkanes. In addition to a-15 : 0 DMA, 1,1-dimethoxy-iso-hexadecane (i-16 : 0 DMA) and 1,1-dimethoxy-anteiso-heptadecane (a-17 : 0 DMA) constitute the predominant 1,1-dimethyl-alkanes of S. boreus and S. frigoramans (Table 1). Accordingly, the 1,1-dimethoxy-alkane profile is an additional chemotaxonomic feature useful for discrimination of the genera Agreia and Subtercola.
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. S. pratensis clustered with A. bicolorata with both the neighbour-joining and maximum-likelihood methods, displaying the high sequence similarity of 99·6 % (Figs 1 and 2). This branch was supported by a high bootstrap value, as was the clustering of both with S. boreus and S. frigoramans. However, the originally described species S. boreus and S. frigoramans (Männistö et al., 2000) did not form a monophyletic branch. The similarity of S. boreus to S. pratensis and A. bicolorata was up to 1 % higher than its similarity to S. frigoramans. Moreover, clustering of S. frigoramans and S. boreus, as shown in Fig. 1, was not supported by bootstrap analysis and could not be confirmed by either the maximum-likelihood method (Fig. 2) or the consensus tree, according to Felsenstein (1993). These results show that the genera Subtercola and Agreia cannot be differentiated unambiguously on the basis of phylogenetic analyses. A comprehensive phylogenetic comparison of the two genera could not be performed by Evtushenko et al. (2001) since the publication of the genus Subtercola and the review process of the description of the genus Agreia coincided.
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; Huß et al., 1983; Jahnke, 1992). The DNA–DNA relatedness of the type strains DSM 14246T and DSM 14575T was found to be 47·8 %. The finding that the two type strains represent different genomospecies according to Wayne et al. (1987) is supported by several differentiating phenotypic features. In contrast to S. pratensis, cells of A. bicolorata are motile and positive for the oxidase and Voges–Proskauer reactions. Furthermore, the isoprenoid quinones of A. bicolorata are dominated by MK-10, whereas those of S. pratensis predominantly comprise MK-10 and MK-11.
The polyphasic approach to the definition of a new genus is based on consideration of both phylogenetic relationships and phenotypic properties. When 16S rDNA sequence comparison and chemotaxonomic analyses provide discordant results, selection of the decisive dataset requires taxonomic expertise. The unification of the genera Microbacterium (containing Lys in the peptidoglycan) and Aureobacterium (containing Orn in the peptidoglycan) by Takeuchi & Hatano (1998) is an example of attributing priority to analyses of partial 16S rDNA sequence similarity for genus allocation. On the other hand, the recent re-evaluation of the genus status of Oerskovia (unified earlier with the phylogenetically related genus Cellulomonas) by Stackebrandt et al. (2002) showed that genus-specific chemotaxonomic features can also be of greater concern when defining a genus. Thus, the close phylogenetic relationship but low DNA–DNA relatedness of S. pratensis and A. bicolorata and the clear differentiation of both from the original psychrophilic species of Subtercola on the basis of chemotaxonomic features support the separate genus status of Agreia and, hence, we propose the reclassification of S. pratensis as Agreia pratensis comb. nov.
Description of Agreia pratensis (Behrendt et al. 2002) comb. nov.
Agreia pratensis (pra.ten'sis. L. fem. adj. pratensis pertaining to meadows/grassland).
Basonym: Subtercola pratensis Behrendt et al. 2002.
The description is as given by Behrendt et al. (2002). The type strain is DSM 14246T (=P 229/10T=LMG 21000T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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DSMZ (2001). Catalogue of Strains, 7th edn. Braunschweig: Deutsche Sammlung von Mikroorganismen und Zellkulturen.
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Felsenstein, J. (1993). PHYLIP (Phylogeny Interference Package), version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA.
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.
Jahnke, K. D. (1992). Basic computer program for evaluation of spectroscopic DNA renaturation data from GILFORD System 2600 spectrometer on a PC/XT/AT type personal computer. J Microbiol Methods 15, 61–73.
Männistö, M. K., Schumann, P., Rainey, F. A., Kämpfer, P., Tsitko, I., Tiirola, M. A. & Salkinoja-Salonen, M. S. (2000). Subtercola boreus gen. nov., sp. nov. and Subtercola frigoramans sp. nov., two new psychrophilic actinobacteria isolated from boreal groundwater. Int J Syst Evol Microbiol 50, 1731–1739.
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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.[CrossRef][Medline]
Schleifer, K. H. (1985). Analysis of the chemical composition and primary structure of murein. Methods Microbiol 18, 123–156.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Schumann, P., Prauser, H., Rainey, F. A., Stackebrandt, E. & Hirsch, P. (1997). Friedmanniella antarctica gen. nov., sp. nov, an LL-diaminopimelic acid-containing actinomycete from Antarctic sandstone. Int J Syst Bacteriol 47, 278–283.[CrossRef][Medline]
Stackebrandt, E., Breymann, S., Steiner, U., Prauser, H., Weiss, N. & Schumann, P. (2002). Re-evaluation of the status of the genus Oerskovia, reclassification of Promicromonospora enterophila (Jáger et al. 1983) as Oerskovia enterophila comb. nov. and description of Oerskovia jenensis sp. nov. and Oerskovia paurometabola sp. nov. Int J Syst Evol Microbiol 52, 1105–1111.[Abstract]
Takeuchi, M. & Hatano, K. (1998). Union of the genera Microbacterium Orla-Jensen and Aureobacterium Collins et al. in a redefined genus Microbacterium. Int J Syst Bacteriol 48, 739–747.[CrossRef][Medline]
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Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef]
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