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Description of Methanobrevibacter Gottschalkii Sp. Nov International Journal of Systematic and Evolutionary Microbiology (2002), 52, 819–822 DOI: 10.1099/ijs.0.02022-0 Description of Methanobrevibacter gottschalkii NOTE sp. nov., Methanobrevibacter thaueri sp. nov., Methanobrevibacter woesei sp. nov. and Methanobrevibacter wolinii sp. nov. Wadsworth Center, New Terry L. Miller and Chuzhao Lin York State Department of Health, Albany, NY 12201-0509, USA Author for correspondence: Terry L. Miller. Tel: j1 518 474 0015. Fax: j1 518 474 8590. e-mail: terry.miller!wadsworth.org Formal nomenclature is proposed for five methanogens, isolated from horse, pig, cow, goose and sheep faeces, that represent four novel species of the genus Methanobrevibacter. The four species, Methanobrevibacter gottschalkii sp. nov., Methanobrevibacter thaueri sp. nov., Methanobrevibacter woesei sp. nov. and Methanobrevibacter wolinii sp. nov., are distinguished from each other by a lack of genomic DNA reassociation and from previously described members of the genus on the basis of differences in the sequences of the 16S rRNA genes. Keywords: Methanobrevibacter, faecal methanogens Six of the seven species of Methanobrevibacter were (Conway de Macario et al., 1987; Ko$ nig, 1986; Miller isolated from gastrointestinal ecosystems (Miller, et al., 1986). 2001). Methanobrevibacter ruminantium, the type spe- The coccobacilli isolated from animals are distingui- cies, was isolated from the bovine rumen. Methano- shed from M. arboriphilicus, M. cuticularis, M. curva- brevibacter smithii is the predominant CO -reducing # tus and M. filiformis on the basis of differences in methanogen isolated from the human colon (Miller et morphology and physiology (Table 1). However, the al., 1986; Lin & Miller, 1998). The type strain of M. coccobacilli species of the genus Methanobrevibacter smithii was originally isolated from a municipal sewage and the animal isolates are not easily distinguishable digestor and was probably present in human faeces on the basis of biochemical and physiological charac- entering the treatment facility. Ferrari et al. (1994) iso- teristics (Table 1). lated Methanobrevibacter oralis from the human oral cavity. Three different species of Methanobrevibacter Lin & Miller (1998) showed that the isolates from were isolated from termite hindguts: Methanobrevi- horse, pig, cow, goose and sheep faeces probably bacter cuticularis, Methanobrevibacter curvatus and represented four novel species on the basis of (i) Methanobrevibacter filiformis (Leadbetter & Breznak, genomic DNA reassociation studies and (ii) com- 1996; Leadbetter et al., 1998). The type strain of parison of 16S rRNA gene sequences with the type the seventh species, Methanobrevibacter arboriphilicus strains of the species of the genus available for analysis [corrected to Methanobrevibacter arboriphilus in Miller at that time. Subsequent comparison of almost com- (2001); this correction has not yet been validly plete 16S rRNA gene sequences deposited in GenBank published], was isolated from decaying cottonwood showed that the animal faecal isolates shared only trees, and four other strains were isolated from sewage 91–93% sequence similarity with M. cuticularis, M. digestors (Miller, 2001). curvatus and M. filiformis (data not shown). However, the relationship of the animal isolates to M. oralis was et al Miller . (1986) isolated and described methanogens not known because a 16S rRNA gene sequence was from various animal faeces. The morphology, physi- not available for phylogenetic analysis. Recently, ology, cell-wall chemistry, GjC content of the geno- Kulik et al. (2001) included the animal faecal methano- mic DNA and immunology of the isolates are con- gen sequences and a partial sequence for M. oralis in a Methanobrevibacter sistent with those of the genus phylogenetic comparison of methanogen sequences ................................................................................................................................................. amplified from human dental plaque. Their analysis of Published online ahead of print on 3 December 2001 as DOI 10.1099/ partial 16S rRNA gene sequences (599 bp) showed that ijs.0.02022-0. the animal isolates were phylogenetically distinct from 02022 # 2002 IUMS Printed in Great Britain 819 T. L. Miller and C. Lin Table 1. Some phenotypic traits of the species of the genus Methanobrevibacter ................................................................................................................................................................................................................................................................................................................. Taxa are indicated as: 1, M. ruminantium M1T;2,M. arboriphilicus DH1T;3,M. curvatus RFM-2T;4,M. cuticularis RFM-1T;5, M. filiformis RFM-3T;6,M. oralis ZRT;7,M. smithii PST;8,M. gottschalkii HOT;9,M. thaueri CWT; 10, M. woesei GST; 11, M. wolinii SHT. Abbreviations: , not known; AA, amino acid mixture; AcH, acetate; Bvits, B vitamin mixture; , coccobacillus; CoM, coenzyme M; DTT, dithiothreitol (cysteine and H#S inhibit growth); FaecX, faecal extract; 2-MB, 2- methylbutyric acid; NutB, nutrient broth; RF, rumen fluid; Trp, Trypticase; , strain-dependent; VFA, volatile fatty acid mixture; YE, yeast extract. All strains use H#jCO# for growth; no substrate other than formate and H#jCO# is used for growth by any of the strains shown. Trait 1 2 3 4 5 6 7 8 9 10 11 Cell shape Short to long rods Curved rods Short rods Rods in in aggregates with polar fibres filaments DNA GjC content (mol%) 31 26 28 30 29 38 30 33 Optimum temperature for 38 30–37 30 37 30 37 38 37 37 37 37 growth (mC) Optimum pH 7n27n5–7n87n27n77n26n9–7n46n9–7n47777 Growth with bile kk jj jkj k Formate used for growth j k j kkj kkj k Medium additions for good AcH, AA, CoM, Bvits, CO# NutB, RF AA, RF, YE DTT, YE FaecX AcH, Bvits AcH, AcH, AcH, AcH, growth NH%,2-MB TrpjYE TrpjYE TrpjYE CoMjVFA M. oralis as well as from the methanogen sequences antibody probes raised against M. ruminantium (strain they amplified from dental plaque (Kulik et al., 2001). M1T), M. smithii (strains PST, ALI), M. arboriphilicus (strains DH1T, AZ, DC) or any other methanogen All of the available molecular phylogenetic evidence antisera in the antibody probe bank (Conway de supports the conclusion that the animal isolates Macario et al., 1987). The DNA G C content is 29 represent novel species of the genus Methanobrevi- j mol% (T ). bacter. We propose a formal nomenclature for the m animal isolates in this communication. Strain HOT was isolated from enrichments of horse faeces. Strain PG was isolated from enrichments of pig Description of Methanobrevibacter gottschalkii sp. faeces. The two species share " 99% 16S rRNA gene nov. sequence similarity and their genomic DNA reas- sociates at 73–108%, indicating that they are strains of Methanobrevibacter gottschalkii (gott.schalhki.i. N.L. the same species (Lin & Miller, 1998). gen. n. gottschalkii of Gottschalk, named in honour of T T The type strain is strain HO (l DSM 11977 l Gerhard Gottschalk for his notable contributions to T the understanding of the biochemistry of methano- OCM 813 ). The GenBank accession number of its genesis). 16S rRNA sequence is U55238. Strain PG (l DSM 11978 l OCM 816) is a reference strain. The GenBank Coccobacillus with rounded ends, about 0n7 µmin accession number of its 16S rRNA sequence is U55239. width and 0n9 µm in length, occurring in pairs or short chains. Gram-positive reaction. Cell walls are com- posed of pseudomurein (Ko$ nig, 1986). The peptide Description of Methanobrevibacter thaueri sp. nov. moiety contains glutamate, alanine and lysine (strains T T HO and PG) and may contain ornithine (strain HO ). Methanobrevibacter thaueri (thauher.i. N.L. gen. n. Galactosamine may be present at very low concentra- thaueri of Thauer, named in honour of Rolf K. Thauer tions or absent from cell-wall hydrolysates (Ko$ nig, for his fundamental contributions to the delineation of 1986). Cells are resistant to lysis by SDS. Optimum the biochemistry of methanogenesis). temperature 37 mC. Optimum pH 7. Coccobacillus, with slightly tapered ends, about 0n5 Strict anaerobe. Cells grown in rumen fluid medium µm in width and 0n6–1n2 µm in length, occurring in are catalase-negative. Grows and produces methane pairs and short chains. Some chains may have elongat- from H# and CO# (Miller et al., 1986). Does not grow ed cells. Gram-positive reaction. Cell walls are com- or produce methane from formate, acetate, methanol, posed of pseudomurein (Ko$ nig, 1986). The peptide trimethylamines or methanol with H#. Requires either moiety contains glutamate, alanine and lysine. acetate and\or one or more components of trypticase Galactosamine and glucosamine may be present at or yeast extract for growth. Does not require coenzyme very low concentrations in cell-wall hydrolysates M or branched-chain fatty acids for growth. Grows in (Ko$ nig, 1986). Cells are resistant to lysis by SDS. medium with salt concentrations similar to sea water Optimum temperature 37 mC. Optimum pH 7. (medium 3 of Balch et al., 1979). Growth is not inhibited by bile salts (Miller et al., 1986). Neither Strict anaerobe. Cells grown in rumen fluid medium strain HOT nor strain PG reacts with polyvalent are catalase-negative. Grows and produces methane 820 International Journal of Systematic and Evolutionary Microbiology 52 Animal isolates of Methanobrevibacter T T from H# and CO# (Miller et al., 1986). Does not grow The type strain is strain GS (l DSM 11979
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