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Int J Syst Evol Microbiol 57 (2007), 2191-2193; DOI  10.1099/ijs.0.65051-0
© 2007 International Union of Microbiological Societies

Thermoanaerobacter pseudethanolicus sp. nov., a thermophilic heterotrophic anaerobe from Yellowstone National Park

Rob U. Onyenwoke1, Vadim V. Kevbrin2, Anatolly. M. Lysenko2 and Juergen Wiegel1

1 Department of Microbiology, The University of Georgia, Athens, GA 30602-2605, USA
2 Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/2, 117 312 Moscow, Russia

Correspondence
Juergen Wiegel
jwiegel{at}uga.edu


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Strain 39ET, originally characterized as Clostridium thermohydrosulfuricum strain 39E and later renamed as Thermoanaerobacter ethanolicus strain 39E, shows less than 97 % 16S rRNA gene sequence similarity with the type strain of the type species of the genus Thermoanaerobacter, T. ethanolicus strain JW 200T. On the basis of a polyphasic analysis that included DNA–DNA hybridization studies with the subspecies of Thermoanaerobacter brockii, its closest phylogenetic relatives, strain 39ET represents a novel species of the genus Thermoanaerobacter, for which the name Thermoanaerobacter pseudethanolicus sp. nov. is proposed. The type strain is 39ET (=DSM 2355T=ATCC 33223T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 39ET is L09164.

The results of DNA–DNA hybridizations between strain 39ET and three Thermoanaerobacter brockii subspecies are presented in a supplementary table available with the online version of this paper.


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Strain 39ET was isolated and characterized by Zeikus et al. (1980)Go as a strain of Clostridium thermohydrosulfuricum (Hollaus & Sleytr, 1972Go) and was later described as a Thermoanaerobacter ethanolicus strain, 39E (Lee et al., 1993Go).

As first reported by Bateson et al. (1989)Go and Rainey et al. (1993)Go, the 16S rRNA gene sequence data clearly places strain 39ET closer phylogenetically (>98 % similarity) to the subspecies of Thermoanaerobacter brockii (Zeikus et al., 1979Go), i.e. T. brockii subsp. brockii DSM 1457T (Zeikus et al., 1979Go; Lee et al., 1993Go; Cayol et al., 1995Go), T. brockii subsp. finnii DSM 3389T (Schmid et al., 1986Go; Cayol et al., 1995Go) and T. brockii subsp. lactiethylicus DSM 9801T (Cayol et al., 1995Go), than to T. ethanolicus strain JW 200T, the type strain of the type species of the genus Thermoanaerobacter (Wiegel & Ljungdahl, 1981Go; Fig. 1Go). To clarify the relationship between strain 39ET and the subspecies of T. brockii, DNA–DNA hybridization experiments were carried out.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequence data with maximum-likelihood correction for synonymous changes, showing the estimated relationships between strain 39ET and related taxa. The 16S rRNA gene sequence data used represents Escherichia coli DSM 30083T nucleotide positions 23–1450. Numbers at nodes indicate bootstrap percentages (based on 1000 replicates). The GenBank accession numbers for each reference strain are shown in parentheses. Bar, 0.02 nucleotide substitutions per site.

 
DNA–DNA hybridization experiments were performed spectrophotometrically as described by De Ley et al. (1970)Go and modified by Huß et al. (1983)Go. Chromosomal DNA for DNA–DNA hybridizations was isolated according to Marmur (1961)Go. The results of DNA–DNA hybridizations between strain 39ET and T. brockii subsp. brockii DSM 1457T, T. brockii subsp. finnii DSM 3389T and T. brockii subsp. lactiethylicus DSM 9801T gave reassociation values of 56, 51 and 45 %, respectively (see Supplementary Table S1 available in IJSEM Online). The fact that all of the values were less than 70 % indicates that strain 39ET is not related to any of the T. brockii subspecies at species level (Wayne et al., 1987Go). This result was corroborated by the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany (P. Schumann, personal communication), where a DNA–DNA reassociation value of 23–34 % was obtained (using the spectrophotometric method of De Ley et al., 1970Go) between strain 39ET and T. brockii subsp. brockii DSM 1457T. Our DNA–DNA hybridizations between the known subspecies of T. brockii (T. brockii subsp. brockii DSM 1457T to T. brockii subsp. finnii DSM 3389T and T. brockii subsp. lactiethylicus DSM 9801T) gave values of 65 and 67 %, as compared with reported values of 89–97 % and 76–85 %, respectively. Our value for DNA–DNA hybridization between T. brockii subsp. finnii DSM 3389T and T. brockii subsp. lactiethylicus DSM 9801T was 62 %, while the reported value is 76–85 %. All of the values obtained in this study were significantly lower than the results obtained by Cayol et al. (1995)Go (see Supplementary Table S1). However, these aberrations might be due to the fact that Cayol et al. (1995)Go employed the tritium-labelled nucleotide method for determining DNA–DNA relatedness, whereas the results obtained in this work relied upon the spectrophotometric protocol of De Ley et al. (1970)Go. Since our DNA–DNA hybridization results between the various T. brockii subspecies are not substantially below 70 % (see Supplementary Table S1), no changes in the status of the subspecies T. brockii subsp. brockii DSM 1457T, T. brockii subsp. finnii DSM 3389T and T. brockii subsp. lactiethylicus DSM 9801T (Cayol et al., 1995Go) are proposed, although the data raise some doubts as to the validity of these subspecies, especially T. brockii subsp. lactiethylicus (Fig. 1Go). However, on the basis of the fact that the DNA–DNA hybridization values obtained for strain 39ET and the T. brockii subspecies are significantly below 70 %, strain 39ET does not represent a novel subspecies of T. brockii but instead represents a novel species of the genus Thermoanaerobacter, for which the name Thermoanaerobacter pseudethanolicus sp. nov. is proposed.

The classification of strain 39ET as a novel species is mainly based on previously published physiological properties (Table 1Go), 16S rRNA gene sequence analysis (Fig. 1Go) and DNA–DNA hybridization results. The name was chosen because strain 39ET (the proposed type strain of T. pseudethanolicus sp. nov.) produces fermentation products in proportions similar to those of strain JW 200T (the type strain of T. ethanolicus), with high levels of ethanol being formed per mole of glucose utilized.


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  		Table 1. Differential phenotypic characteristics for some species of the genus Thermoanaerobacter

Strains: 1, T. brockii subsp. brockii DSM 1457T; 2, T. brockii subsp. finnii DSM 3389T; 3, T. brockii subsp. lactiethylicus DSM 9801T; 4, T. pseudethanolicus 39ET; 5, T. ethanolicus JW 200T. Data for reference strains were taken from Cayol et al. (1995)Go. +, Positive; –, negative; V, variable result.

 
Description of Thermoanaerobacter pseudethanolicus sp. nov.
Thermoanaerobacter pseudethanolicus [pseud'e.tha.no'li.cus. Gr. adj. pseudes false; N.L. adj. ethanolicus a bacteria-specific epithet; N.L. masc. adj. pseudethanolicus a false (Thermoanaerobacter) ethanolicus].

Other names include Thermoanaerobacter ethanolicus strain 39E (Lee et al., 1993Go) and Clostridium thermohydrosulfuricum strain 39E (Zeikus et al., 1980Go).

The description is based mainly on those given by Zeikus et al. (1980)Go and Lee et al. (1993)Go for strain 39ET. Cells are rod-shaped and form round, terminal, mother-cell-distending (drumstick-shaped) spores during growth on xylose-containing medium. Gram-stain reaction is variable, but the cell wall is Gram-type positive (Wiegel, 1981Go). No polymyxin B–lipopolysaccharide interaction is found (Wiegel & Quandt, 1982Go). Cells are motile and reduce thiosulfate to H2S. Fermented carbohydrates include xylose, cellobiose, starch, glucose, maltose and sucrose. No growth is observed using CO2/H2. The temperature optimum is 65 °C. The doubling time at 65 °C is 75 min. The DNA G+C content of the type strain is 34.4±0.3 mol% (Tm).

The type strain, 39ET (=DSM 2355T=ATCC 33223T), was isolated from the Octopus Spring algal–bacterial mat in Yellowstone National Park, WY, USA, using modified Trypticase-yeast extract-glucose medium (containing 5 % xylose instead of glucose) at 65 °C.

The genome sequence for strain 39ET is presently available under the name T. ethanolicus 39E at http://genome.ornl.gov/microbial/teth_39e/.


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Bateson, M. M., Weigel, J. & Ward, D. M. (1989). Comparative analysis of 16S ribosomal RNA sequences of thermophilic fermentative bacteria isolated from hot spring cyanobacterial mats. Syst Appl Microbiol 12, 1–7.[Medline]

Cayol, J. L., Ollivier, B., Patel, B. K. C., Ravot, G., Magot, M., Ageron, E., Grimont, P. A. D. & Garcia, J. L. (1995). Description of Thermoanaerobacter brockii subsp. lactiethylicus subsp. nov., isolated from a deep subsurface French oil well, a proposal to reclassify Thermoanaerobacter finnii as Thermoanaerobacter brockii subsp. finnii comb. nov., and an emended description of Thermoanaerobacter brockii. Int J Syst Bacteriol 45, 783–789.[Abstract/Free Full Text]

Cook, G. M., Janssen, P. H. & Morgan, H. W. (1991). Endospore formation by Thermoanaerobium brockii HTD4. Syst Appl Microbiol 14, 240–244.

De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]

Hollaus, F. & Sleytr, U. (1972). On the taxonomy and fine structure of some hyperthermophilic saccharolytic clostridia. Arch Microbiol 86, 129–146.

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.

Lee, Y. E., Jain, M. K., Lee, C., Lowe, S. E. & Zeikus, J. G. (1993). Taxonomic distinction of saccharolytic thermophilic anaerobes: description of Thermoanaerobacterium xylanolyticum gen. nov., sp. nov., and Thermoanaerobacterium saccharolyticum gen. nov., sp. nov.; reclassification of Thermoanaerobium brockii, Clostridium thermosulfurogenes, and Clostridium thermohydrosulfuricum E100-69 as Thermoanaerobacter brockii comb. nov., Thermoanaerobacterium thermosulfurigenes comb. nov., and Thermoanaerobacter thermohydrosulfuricus comb. nov., respectively; and transfer of Clostridium thermohydrosulfuricum 39E to Thermoanaerobacter ethanolicus. Int J Syst Bacteriol 43, 41–51.[Abstract/Free Full Text]

Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.

Onyenwoke, R. U., Brill, J. A., Farahi, K. & Wiegel, J. (2004). Sporulation genes in members of the low G+C Gram-type positive phylogenetic branch (Firmicutes). Arch Microbiol 182, 182–192.[Medline]

Rainey, F. A., Ward, N. L., Morgan, H. W., Toalster, R. & Stackebrandt, E. (1993). Phylogenetic analysis of anaerobic thermophilic bacteria: aid for their reclassification. J Bacteriol 175, 4772–4779.[Abstract/Free Full Text]

Schmid, U., Giesel, H., Schoberth, S. M. & Sahm, H. (1986). Thermoanaerobacter finnii spec. nov., a new ethanologenic sporogenous bacterium. Syst Appl Microbiol 8, 80–85.

Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & 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.[Free Full Text]

Wiegel, J. (1981). Distinction between the Gram reaction and the Gram type of bacteria. Int J Syst Bacteriol 31, 88[Abstract/Free Full Text]

Wiegel, J. & Ljungdahl, L. J. (1981). Thermoanaerobacter ethanolicus gen. nov., spec. nov., a new, extreme thermophilic, anaerobic bacterium. Arch Microbiol 128, 343–348.[CrossRef]

Wiegel, J. & Quandt, L. (1982). Determination of the Gram type using the reaction between polymyxin B and lipopolysaccharides of the outer cell wall of whole bacteria. J Gen Microbiol 128, 2261–2270.[Abstract/Free Full Text]

Zeikus, J. G., Hegge, P. W. & Anderson, M. A. (1979). Thermoanaerobacter brockii gen. nov. and sp. nov., a new chemoorganotrophic, caldoactive, anaerobic bacterium. Arch Microbiol 122, 41–48.[CrossRef]

Zeikus, J. G., Ben-Bassat, A. & Hegge, P. (1980). Microbiology of methanogenesis in thermal, volcanic environments. J Bacteriol 143, 432–440.[Abstract/Free Full Text]




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