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Methanoculleus bourgensis, Methanoculleus olentangyi and Methanoculleus oldenburgensis are subjective synonyms

Susumu Asakawa and Kazunari Nagaoka

Laboratory of Soil Microbiology, Kyushu National Agricultural Experiment Station, Nishigoshi, Kumamoto 861-1192, Japan

Correspondence
Susumu Asakawa
asakawa{at}agr.nagoya-u.ac.jp

	ABSTRACT

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Methanoculleus bourgensis, Methanoculleus olentangyi and Methanoculleus oldenburgensis are subjective synonyms on the basis of phenotypic, genotypic and phylogenetic characteristics. Methanoculleus bourgensis must be the name of the united species because it is the type of the genus Methanoculleus.

The DDBJ/GenBank/EMBL accession number for the 16S rDNA sequence of Methanoculleus oldenburgensis CB-1^T is AB065298.

Present address: Soil Biology and Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.

Present address: National Agricultural Research Center, Tsukuba 305-8666, Japan.

	MAIN TEXT

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The genus Methanoculleus currently includes seven species: Methanoculleus marisnigri, Methanoculleus bourgensis, Methanoculleus thermophilus, Methanoculleus olentangyi, Methanoculleus oldenburgensis, Methanoculleus palmolei and Methanoculleus chikugoensis (Maestrojuán et al., 1990; Romesser et al., 1979; Ollivier et al., 1986; Rivard & Smith, 1982; Corder et al., 1983; Blotevogel et al., 1991; Zellner et al., 1998; Dianou et al., 2001). In the course of the identification of Methanoculleus chikugoensis MG62^T (Dianou et al., 2001), a DNA–DNA hybridization study revealed that Methanoculleus bourgensis MS2^T, Methanoculleus olentangyi RC/ER^T and Methanoculleus oldenburgensis CB-1^T were genomically closely related (67–104 % relatedness to each other), though a similar close relatedness (66 %) between Methanoculleus bourgensis and Methanoculleus olentangyi had already been reported by Xun et al. (1989). We supplemented these results by examining some genotypic and phylogenetic features of the type strains and we now confirm the synonymy of these three species on the basis of these features and phenotypic characteristics already reported.

The 16S rDNA sequence of Methanoculleus oldenburgensis was determined, since it had not previously been available. The type strain, CB-1^T (=DSM 6216^T), was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. Methods for DNA isolation, PCR amplification and sequencing of 16S rDNA were the same as described previously (Dianou et al., 2001). The sequence similarities of strain CB-1^T to Methanoculleus bourgensis MS2^T and Methanoculleus olentangyi RC/ER^T and between strains MS2^T and RC/ER^T were respectively 97·1, 98·7 and 97·7 %, which was consistent with the DNA–DNA hybridization results mentioned above (Dianou et al., 2001).

These three strains seem to have almost the same phenotypic features: the cells are 1–2 µm in diameter, flagellation and motility are not observed (fimbriae-like structures have been reported in the case of Methanoculleus oldenburgensis CB-1^T cells), H₂/CO₂ and formate are used as methanogenic substrates (some secondary alcohols are also used by Methanoculleus bourgensis MS2^T and Methanoculleus olentangyi RC/ER^T), acetate is required for growth and the optimum temperature, pH and NaCl concentration for growth are respectively about 40 °C (37–45 °C), near neutral (6·7–8·0) and about 0·1 M (0·04–0·17 M) (Blotevogel et al., 1991; Ollivier et al., 1986; Corder et al.,1983; Maestrojuán et al., 1990; Zellner et al., 1990; Chong & Boone, 2001; see also Table 2 of Dianou et al., 2001). Some resemblances of whole-cell protein electrophoretic patterns, S-layer structures and polyamine compositions between Methanoculleus bourgensis MS2^T and Methanoculleus olentangyi RC/ER^T were also reported (Maestrojuán et al., 1990; Zellner et al., 1990).

The sole differentiating characteristic is the G+C content of DNA. The G+C contents of Methanoculleus bourgensis MS2^T, Methanoculleus olentangyi RC/ER^T and Methanoculleus oldenburgensis CB-1^T were respectively originally reported to be 59, 54·4 and 48·6 mol%, the first two being determined by the buoyant density method and the latter by thermal denaturation. The range is 10 mol%, which seems too large for members of a single species. Therefore, we measured the G+C contents of DNA from these three strains by the same method (HPLC), as described previously (Dianou et al., 2001), using a Hitachi 683-50 HPLC system. The values for Methanoculleus bourgensis MS2^T, Methanoculleus olentangyi RC/ER^T and Methanoculleus oldenburgensis CB-1^T were respectively 60·5±0·0, 61·1±0·3 and 60·2±0·2 mol% (mean±SD, n=3).

The similarities of phenotypic, genotypic and phylogenetic characteristics described above indicate that Methanoculleus bourgensis (Ollivier et al. 1986) Maestrojuán et al. 1990, Methanoculleus olentangyi (Corder et al. 1988) Maestrojuán et al. 1990 and Methanoculleus oldenburgensis Blotevogel et al. 1998 are subjective synonyms. Rule 15 of the Bacteriological Code (Lapage et al., 1992) indicates that the type of a taxon is permanently associated with that taxon; therefore, since Methanoculleus bourgensis is the type of the genus Methanoculleus Maestrojuán et al. 1990, the name Methanoculleus bourgensis must be retained for the united species.

The physiological variance of strain CB-1, such as its slightly higher temperature for optimum growth, might indicate its affiliation to a subspecies within the united species. However, further information concerning the phenotypic characters of strain CB-1 and the other strains will be needed for the taxonomic description of strain CB-1.

Emended description of Methanoculleus bourgensis (Ollivier et al. 1986) Maestrojuán et al. 1990
The descriptions of the genus Methanoculleus and the species Methanoculleus bourgensis have been given by Maestrojuán et al. (1990). Strains RC/ER and CB-1, previously the type strains of Methanoculleus olentangyi and Methanoculleus oldenburgensis, respectively, are reference strains of the species Methanoculleus bourgensis. The type strain is strain MS2^T (=ATCC 43281^T=DSM 3045^T).

	ACKNOWLEDGEMENTS

 
We thank Y. Koga of the University of Occupational and Environmental Health, Japan, Y. Kamagata of the National Institute of Advanced Industrial Sciences and Technology and T. Ito of the Japan Collection of Microorganisms, RIKEN, for their helpful advice and Y. Hamahira of Hitachi Instruments Service Co., Ltd for his help in operating the HPLC apparatus.

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