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DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Stefan Spring
ssp{at}dsmz.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DSM 2832T is AJ862839.
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. Later, Asakawa & Nagaoka (2003) united Methanoculleus bourgensis, Methanoculleus olentangyi and Methanoculleus oldenburgensis in one species under the name Methanoculleus bourgensis. All described species of the genus Methanogenium, with the exception of [Methanogenium] frittonii, are psychrophilic to mesophilic and require acetate for growth. In contrast, the original description of [Methanogenium] frittonii by Harris et al. (1984) reports that this species is thermophilic and does not require acetate as a carbon source for growth. However, because of the lack of a 16S rRNA gene sequence, the phylogenetic relationship of [Methanogenium] frittonii to other members of the genus Methanogenium remained unclear. To clarify the taxonomic position of [Methanogenium] frittonii, we determined the almost complete 16S rRNA gene sequence of the type strain DSM 2832T. The strains [Methanogenium] frittonii DSM 2832T and Methanoculleus thermophilus DSM 2373T were grown under the conditions indicated in the DSMZ catalogue of strains (www.dsmz.de). For the isolation of DNA, each strain was cultured in about 10 l of the respective DSMZ medium. After incubation for 48 h at 55 °C, cells were harvested by centrifugation. It turned out that both strains were very sensitive to lysis during resuspension of the cell pellet, which was done under oxic conditions. However, the loss of DNA could be efficiently prevented by resuspending the pellet in 80 % (v/v) 2-propanol (aqueous solution), which stabilized the nucleic acids.
Genomic DNA extraction and amplification of the 16S rRNA gene of strain DSM 2832T was carried out as reported by Rainey et al. (1996). Purified PCR products were sequenced with the CEQTM DTCS Quick Start kit (Beckman Coulter), as described in the manufacturer's protocol. The CEQTM 8000 Genetic Analysis System was used for the electrophoresis of the sequence reaction products. An almost complete 16S rRNA gene sequence of 1451 nucleotides was determined, and was phylogenetically analysed using the ARB software package (Ludwig et al., 2004). The phylogenetically most closely related species of [Methanogenium] frittonii turned out to be Methanoculleus thermophilus. The 16S rRNA gene sequences of both type strains were almost identical (99·9 % similarity), and had similarity values around 96 % with sequences of other type strains of the genus Methanoculleus, which suggested the affiliation of strain DSM 2832T to this genus. In contrast, [Methanogenium] frittonii was only distantly related to representatives of the genus Methanogenium, as indicated by similarity values of below 92 % among 16S rRNA gene sequences.
Genomic DNA for the determination of DNA base composition and DNA–DNA hybridization experiments was isolated by using a French pressure cell, and was purified by chromatography on hydroxyapatite, as described by Cashion et al. (1977). DNA–DNA hybridization experiments between both strains were carried out in 2xSSC buffer containing 5 % (v/v) formamide at 68 °C, according to the method of De Ley et al. (1970). DNA renaturation rates were measured by using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with an in situ temperature probe (Varian). The estimated level of DNA–DNA binding between the strains DSM 2832T and DSM 2373T was 86 %. According to Wayne et al. (1987), reassociation values above 70 % indicate that the strains belong to the same species.
The affiliation of both strains to one species is supported by several morphological and physiological traits. The sole important physiological difference is the requirement for acetate as a carbon source by Methanoculleus thermophilus, whereas [Methanogenium] frittonii can grow autotrophically. However, Romesser et al. (1979) reported that Methanoculleus marisnigri, a species closely related to Methanoculleus thermophilus, can also grow without acetate as a carbon source. Consequently, within the genus Methanoculleus the requirement for acetate seems to be variable among strains.
A further differentiating characteristic between both type strains is the DNA G+C content. In the original species descriptions of Methanoculleus thermophilus and [Methanogenium] frittonii, G+C contents of 59 mol% (buoyant density method; Rivard & Smith, 1982) and 49 mol% (buoyant density method; Harris et al., 1984), respectively, were reported. Generally, the variability of G+C content among strains within the same species does not exceed 5 mol% (Rosselló-Mora & Amann, 2001). To check this discrepancy, we repeated the determination of the DNA G+C content of both strains by using reversed-phase HPLC of nucleosides, according to Mesbah et al. (1989). The G+C content of Methanoculleus thermophilus DSM 2373T and [Methanogenium] frittonii DSM 2832T was found to be 59·1 and 56·1 mol%, respectively. While the reported value for strain DSM 2373T could be confirmed by our results, the obtained value for strain DSM 2832T was inconsistent with the data reported by Harris et al. (1984), but was in good agreement with values obtained by Zabel et al. (1985) and Widdel et al. (1988) by using the thermal denaturation method for three other strains affiliated to the species Methanoculleus thermophilus (55–57 mol%). Thus, we conclude that the originally reported DNA G+C content of 49 mol% for strain DSM 2832T was too low, due to experimental error.
Based on the presented results, it is proposed to unite the species [Methanogenium] frittonii and Methanoculleus thermophilus. According to rules 38, 42 and 24b(2) of the Bacteriological Code (Lapage et al., 1992), the validly published name Methanoculleus thermophilus has priority and hence should be used for the unified taxon.
In addition, the presented data and a critical review of the relevant literature permit an emendation of the genus Methanogenium, thereby enabling a more distinct differentiation from the related genus Methanoculleus (Table 1).
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and Zabel et al. (1985), with the following exceptions. Secondary alcohols might be used as electron donor by single strains (Widdel et al., 1988). Most strains require acetate as a carbon source for growth, but single strains might be autotrophic. Trypticase peptone or yeast extract stimulates growth in all strains. The DNA G+C content is 55–59 mol%; the type strain has a G+C content of 59 mol%. The type strain is DSM 2373T (=OCM 174T).
Emended description of the genus Methanogenium Romesser et al. 1981 emend. Maestrojuán et al. 1990
The description of this genus is that given by Romesser et al. (1979), emended by Maestrojuán et al. (1990), except that optimal temperatures for growth are between 15 and 35 °C and optimal NaCl concentrations range from 0·35 to 1·25 M.
The type species is Methanogenium cariaci.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). A rapid method for the base ratio determination of bacterial DNA. Anal Biochem 81, 461–466.[CrossRef][Medline]
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Ludwig, W., Strunk, O., Westram, R. & 29 other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.
Maestrojuán, G. M., Boone, D. R., Xun, L., Mah, R. A. & Zhang, L. (1990). Transfer of Methanogenium bourgense, Methanogenium marisnigri, Methanogenium olentangyi, and Methanogenium thermophilicum to the genus Methanoculleus gen. nov., emendation of Methanoculleus marisnigri and Methanogenium, and description of new strains of Methanoculleus bourgense and Methanoculleus marisnigri. Int J Syst Bacteriol 40, 117–122.
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.
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