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Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Correspondence
Akira Yokota
uayoko{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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indicated that the bacterium belonged within the genus Tetrasphaera. A. duodecadis IAM 14868T could be distinguished from other species by the presence of 3-hydroxy meso-diaminopimelic acid in the peptidoglycan and a series of 10-methyl fatty acids. In addition, DNA–DNA relatedness studies indicated that the strain belonged to a genomic species that could be readily distinguished from its nearest neighbours, the type strains of Tetrasphaera elongata, Tetrasphaera japonica and Tetrasphaera australiensis. These features support our proposal to reclassify A. duodecadis in the genus Tetrasphaera, as Tetrasphaera duodecadis comb. nov. The type strain is IAM 14868T (=NBRC 12959T=ATCC 13347T=NCIMB 9222T). An emended description of the genus Tetrasphaera is given.
Tables detailing 16S rRNA gene signature nucleotide patterns of Arthrobacter duodecadis and members of the genus Tetrasphaera and the family Intrasporangiaceae and DNA–DNA relatedness levels between A. duodecadis and related taxa, and a figure showing a TLC chromatogram of purified cell wall peptidoglycan hydrolysates of A. duodecadis strains IAM 14868T and NCIMB 9222T are available as supplementary material in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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as a bacterium isolated from arable soil that indispensably required vitamin B12 for robust growth. In accordance with its marked morphological characteristic, a change from rod to coccus, it was classified in the genus Arthrobacter. Although several chemotaxonomic characteristics of this genus have been reported (Keddie et al., 1966; Skyring & Quadling, 1969; Schleifer & Kandler, 1972; Keddie & Cure, 1977), a full chemotaxonomic analysis has yet to be performed on A. duodecadis.
With respect to the composition of diamino acids in the cell wall peptidoglycan, in particular, Keddie et al. (1966) and Keddie & Cure (1977) reported that the strain contained lysine, which is one of the characteristics that define the genus Arthrobacter. In contrast, Schleifer & Kandler (1972) reported that it contained meso-diaminopimelic acid. The ambiguity of the taxonomic position of this species was pointed out by Keddie et al. (1986). In this study, we carried out chemotaxonomic and genotypic studies on A. duodecadis to clarify its taxonomic position.
The type strains examined in this study, Tetrasphaera australiensis IAM 14901T, Tetrasphaera japonica IAM 14891T, Tetrasphaera elongata IAM 14965T and Janibacter limosus IAM 14889T, were obtained from the IAM culture collection. The culture conditions basically complied with the descriptions in the original reports. With regard to A. duodecadis, for the determination of the composition of diamino acids in the cell wall peptidoglycan, the type strain was obtained from different culture collections: IAM 14868T from the IAM culture collection and NCIMB 9222T from The National Collections of Industrial, Food and Marine Bacteria. The medium and culture conditions used in this study for A. duodecadis were: 1 g polypeptone, 0.2 g yeast extract, 0.1 g glucose, 0.1 g Casamino acids, 0.1 g MgSO4 and 100 µg vitamin B12 in 100 ml distilled water (pH 7.0), at 25 °C (Lochhead, 1958).
The 16S rRNA gene was amplified and sequenced as described by Lin et al. (2004) by using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems), according to the manufacturer's instructions. The partial 16S rRNA gene sequence of A. duodecadis IAM 14868T determined (1485 bases) was aligned using the CLUSTAL W program (Thompson et al., 1994) with similar sequences obtained from the DDBJ database using BLAST (Altschul et al., 1997) and FASTA (Pearson & Lipman, 1988). After ambiguous alignment sites had been removed, a phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987). A. duodecadis IAM 14868T showed high sequence similarity to the genus Tetrasphaera. Sequence similarity values with members of this genus were 97.9 % for T. elongata, 96.8 % for T. australiensis and 97.4 % for T. japonica. By contrast, the sequence similarity with the type strain of Arthrobacter globiformis was 92.3 %. The tree (Fig. 1) showed that A. duodecadis differed from A. globiformis and fell into the cluster of the genus Tetrasphaera with a high bootstrap value (917/1000). This conclusion was also supported by using the maximum-likelihood method with the TREE-PUZZLE program (Schmidt et al., 2002) (data not shown). According to 16S rRNA gene signature nucleotide data (Stackebrandt & Schumann, 2000), the 16S rRNA gene signatures were almost coincident with those of the family Intrasporangiaceae, with some mismatches (see Supplementary Table S1 in IJSEM Online). At positions 1133–1141, those of A. duodecadis and T. elongata were G–C, corresponding to other species of Tetrasphaera (Maszenan et al., 2000) and, for the four species A. duodecadis, T. japonica, T. australiensis and T. elongata, positions 952–1229 (T–A) were exceptions to the rule for the family Intrasporangiaceae.
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. The G+C content of A. duodecadis IAM 14868T was determined by using reversed-phase HPLC (Mesbah et al., 1989) and was found to be 73 mol%, which is consistent with the result obtained using the Tm method (72.5 mol%; Skyring & Quadling, 1969). DNA–DNA hybridization was performed by using the microplate method (Ezaki et al., 1989) with CytoFluor 4000 (Applied Biosystems) being used for the fluorescence intensity measurements. The hybridization steps were carried out at 55 °C for 3 h in 2x SSC, 5x Denhardt's solution, 100 µg denatured salmon DNA, 2.5 % dextran sulfate and 50 % formamide in a final volume of 1 ml, which also contained 50–100 ng biotinylated probe DNA. A. duodecadis IAM 14868T showed less than 20 % DNA–DNA relatedness with each reference strain used: T. japonica IAM 14891T (15 %), T. australiensis IAM 14901T (12 %) and T. elongata IAM 14965T (4 %). Detailed results are available as Supplementary Table S2 in IJSEM Online. Therefore, it is proposed that A. duodecadis represents a separate genomic species (Wayne et al., 1987) in the genus Tetrasphaera.
The cellular fatty acid compositions of A. duodecadis IAM 14868T and members of the genus Tetrasphaera were analysed by using the SHERLOCK Microbial Identification System (MIDI) and the MIDI standard procedure (Sasser, 1990). Analysis of fatty acid methyl esters was carried out according to the standard protocol. Each strain was cultured on brain heart infusion agar (Oxoid) at 31 °C for 45 days. The cellular fatty acid profile of strain IAM 14868T was similar to those of members of the genus Tetrasphaera; however, it contained a series of 10-methyl fatty acids (10-methyl-C16 : 0, 10-methyl-C17 : 0 and 10-methyl-C18 : 0), as shown in Table 1. The dominant fatty acids were i-C15 : 0 (15.8 %), i-C16 : 0 (17.8 %) and 10-methyl-C17 : 0 (20.4 %). Small amounts of 10-methyl-C17 : 0 and 10-methyl-C18 : 0 (tuberculostearic acid) were also present in T. japonica, but no 10-methyl fatty acids were detected in T. elongata or T. australiensis. Thus, the presence of 10-methyl fatty acids is thought to be an indication of the differentiation of A. duodecadis from other closely related species.
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. The amino acid composition was analysed by using HPLC with the Wako PTC-AAA System (Wako Pure Chemical Industries) and diaminopimelic acid isomers were determined by using one- and two-dimensional TLC methods (Harper & Davis, 1979). The acyl types of muramic acids in the peptidoglycan were determined by using the glycolate test (Uchida & Aida, 1984). The HPLC and TLC analyses indicated that each sample contained four amino acids, alanine, glutamic acid, aspartic acid and meso-diaminopimelic acid, in a ratio of 2.9 : 1.0 : 0.8 : 0.8. Interestingly, 50 % or more of the meso-diaminopimelic acid was hydroxylated to 3-hydroxy meso-diaminopimelic acid (Supplementary Fig. S1 in IJSEM Online) and the content increased in accordance with the culture period. These results suggest that meso-diaminopimelic acid is located in the peptide subunit and aspartic acid descends from the interpeptide bridge. These results were consistent with those of Schleifer & Kandler (1972), who identified the peptidoglycan of this bacterium as A4
-type. Other species of the genus have been reported to contain meso-diaminopimelic acid as a major diamino acid in whole-cell hydrolysates (Maszenan et al., 2000; Hanada et al., 2002). The acyl type of muramic acid of A. duodecadis was shown to be acetyl by using the glycolate test.
The menaquinone fraction was prepared from lyophilized cells of A. duodecadis IAM 14868T (Collins et al., 1977). The composition and structures were analysed by HPLC (Tamaoka et al., 1983) and mass spectrometry (GCMS-QP 5050A; Shimadzu). MK-8(H4) was shown to be the major isoprenoid quinone (>95 %).
Detailed morphological and physiological properties of A. duodecadis have been published previously (Lochhead, 1958; Owens & Keddie, 1969; Keddie et al., 1986). In this study, we re-examined the bacterium's phenotypic properties using commercial kits. Aminopeptidase and O/F tests were carried out using a Bactident aminopeptidase kit (Merk KgaA) and Pourmedia OF medium (Eiken Chemical Co.), respectively. Acid production, decomposition and utilization of substrates and other biochemical reactions were carried out using the Biotest kit (Eiken Chemical Co.). The results are given in the description. We also observed morphological changes during culture using scanning electron microscopy and confirmed that a morphological change from rod to coccus occurred. In contrast, T. australiensis and T. japonica have been reported as not showing any significant change in cell shape. On the other hand, T. elongata, which has the highest 16S rRNA gene sequence similarity to A. duodecadis, was reported to produce L-shaped clumps of cells in culture (Hanada et al., 2002). A comparison of the characteristics of A. duodecadis and Tetrasphaera species is shown in Table 2.
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Emended description of the genus Tetrasphaera Maszenan et al. 2000
The emended description is based on data from Maszenan et al. (2000), Hanada et al. (2002) and this study.
Cells are cocci, occurring singly or in pairs but predominantly as tetrads or clusters, or exhibit morphological change from rod to coccus and V- and T-shaped formations. Aerobic, non-motile, non-endospore-forming and Gram-positive to Gram-variable. Catalase-positive but negative for indole production. The major cell wall diamino acid is meso-diaminopimelic acid or 3-hydroxy meso-diaminopimelic acid. The major menaquinone is MK-8(H4). The major cellular fatty acids are branched fatty acids; 10-methyl branched fatty acids are present in some species. The G+C content is 68–73 mol%. All species have G–C at positions 1133–1141 and T–A at positions 952–1229 as specific 16S rRNA gene signature nucleotides. The type species is Tetrasphaera japonica.
Description of Tetrasphaera duodecadis comb. nov.
Tetrasphaera duodecadis (du.o.de'ca.dis. L. n. duodecas -adis the number twelve; L. gen. n. duodecadis of twelve, referring to the requirement of the organism for vitamin B12).
Basonym: Arthrobacter duodecadis Lochhead 1958.
Morphological and basic growth descriptions are taken from the original study by Lochhead (1958) and the description of peptidoglycan structure is based on data from Schleifer & Kandler (1972). Cells are Gram-variable and exhibit rod to coccus morphological change; rods vary in length from 1.5 to 4 µm and are 0.5–0.6 µm wide. Many V- and T-shaped formations occur; coccoid cells are 0.4–0.6 µm in diameter. Exhibits slow growth. For regrowth from an L-drying ampoule on the medium of Lochhead (1958), visible colonies may not appear for a week or more. Vitamin B12 and thiamine are required for growth. Grows well at 20–32 °C; growth occurs at 10 °C but not at 37 °C. Colonies on agar are circular, entire and convex, with a smooth surface, and cream-coloured to pale-brown and somewhat translucent, and may reach a diameter of 1.5 to 2 mm. Aminopeptidase, oxidase, indole test, Voges–Proskauer test and production of H2S are negative, but catalase reaction, nitrate reduction and 
-galactosidase are positive. O/F test is positive. Fructose, glucose, mannitol and sucrose are assimilated, but not xylose, mannose, arabinose, maltose or rhamnose. Gelatin, acetoamide and aesculin are hydrolysed, but not urea or arginine. Citrate and malonic acid are not utilized. The G+C content is 73 mol%. The cell wall peptidoglycan contains alanine, glutamic acid, aspartic acid and meso-diaminopimelic acid. A large amount of 3-hydroxy meso-diaminopimelic acid is present instead of meso-diaminopimelic acid. Peptidoglycan structure is A4-type. The acyl type of the cell wall is acetyl. The predominant cellular fatty acids are 10-methyl-C17 : 0, iso-C16 : 0 and iso-C15 : 0. In addition, 10-methyl-C18 : 0 (tuberculostearic acid) and 10-methyl-C16 : 0 are present.
The type strain is IAM 14868T (=ATCC 13347T=NCIMB 9222T=NBRC 12959T), which was isolated from soil.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Hanada, S., Liu, W.-T., Shintani, T., Kamagata, Y. & Nakamura, K. (2002). Tetrasphaera elongata sp. nov., a polyphosphate-accumulating bacterium isolated from activated sludge. Int J Syst Evol Microbiol 52, 883–887.[Abstract]
Harper, J. J. & Davis, G. H. G. (1979). Two-dimensional thin-layer chromatography for amino acid analysis of bacterial cell walls. Int J Syst Bacteriol 29, 56–58.
Keddie, R. M. & Cure, G. L. (1977). The cell wall composition and distribution of free mycolic acids in named strains of coryneform bacteria and in isolates from various natural sources. J Appl Bacteriol 42, 229–252.[Medline]
Keddie, R. M., Leask, B. G. S. & Grainger, J. M. (1966). A comparison of coryneform bacteria from soil and herbage: cell wall composition and nutrition. J Appl Bacteriol 29, 17–43.
Keddie, R. M., Collins, M. D. & Jones, D. (1986). Genus Arthrobacter Conn and Dimmick 1947, 300AL. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1288–1301. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Lin, Y.-C., Uemori, K., de Briel, D. A., Arunpairojana, V. & Yokota, A. (2004). Zimmermannella helvola gen. nov., sp. nov., Zimmermannella alba sp. nov., Zimmermannella bifida sp. nov., Zimmermannella faecalis sp. nov. and Leucobacter albus sp. nov., novel members of the family Microbacteriaceae. Int J Syst Evol Microbiol 54, 1669–1676.
Lochhead, A. G. (1958). Two new species of Arthrobacter requiring respectively vitamin B12 and the terregens factor. Arch Mikrobiol 31, 163–170.[CrossRef]
Maszenan, A. M., Seviour, R. J., Patel, B. K. C., Schumann, P., Burghardt, J., Tokiwa, Y. & Stratton, H. M. (2000). Three isolates of novel polyphosphate-accumulating Gram-positive cocci, obtained from activated sludge, belong to a new genus, Tetrasphaera gen. nov., and description of two new species, Tetrasphaera japonica sp. nov. and Tetrasphaera australiensis sp. nov. Int J Syst Evol Microbiol 50, 593–603.[Abstract]
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Owens, J. D. & Keddie, R. M. (1969). The nitrogen nutrition of soil and herbage coryneform bacteria. J Appl Bacteriol 32, 338–347.[Medline]
Pearson, W. R. & Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 2444–2448.
Saito, H. & Miura, K. I. (1963). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochem Biophys Acta 72, 619–629.[Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sasser, M. (1990). Identification of bacteria through fatty acid analysis. In Methods in Phytobacteriology, pp. 119–204. Edited by Z. Klement, K. Rudolph & D. C. Sands. Budapest: Akadèmiai Kiadó.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan type of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Schmidt, H. A., Strimmer, K., Vingron, M. & von Haeseler, A. (2002). TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18, 502–504.
Skyring, G. W. & Quadling, C. (1969). Soil bacteria: a principal component analysis and guanine-cytosine contents of some arthrobacter-coryneform soil isolates and of some named cultures. Can J Microbiol 16, 95–106.
Stackebrandt, E. & Schumann, P. (2000). Description of Bogoriellaceae fam. nov., Dermacoccaceae fam. nov., Rarobacteraceae fam. nov. and Sanguibacteraceae fam. nov. and emendation of some families of the suborder Micrococcineae. Int J Syst Evol Microbiol 50, 1279–1285.[Abstract]
Tamaoka, J., Katayama-Fujimura, Y. & Kuraishi, H. (1983). Analysis of bacterial menaquinone mixtures by high performance liquid chromatography. J Appl Bacteriol 54, 31–36.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Uchida, K. & Aida, K. (1984). An improved method for the glycolate test for simple identification of acyl type of bacterial cell walls. J Gen Appl Microbiol 30, 131–134.
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.
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