The strains and their corresponding

The strains and their corresponding learn more GenBank accession numbers for 16S rRNA genes are (type=T): Methanobrevibacter gottschalkii … Figure 2 Transmission electron microscopy of negatively stained Methanobrevibacter sp. AbM4 cells. Genome sequencing and annotation Genome project history Methanobrevibacter sp. AbM4 was selected for genome sequencing on the basis of its phylogenetic position as a representative of organisms whose nearest relative is M. wolinii. AbM4 was isolated from a sample of sheep abomasal contents, whereas the type strain of M. wolinii SH was isolated from enrichment cultures of sheep feces [15]. AbM4 grows readily in broth cultures making it amenable to experimentation in the laboratory. A summary of the genome project information is shown in Tables 1 and and22.

Table 1 Classification and general features of Methanobrevibacter sp. AbM4 Table 2 Project information Growth conditions and DNA isolation AbM4 was grown in BY medium [26] with added SL10 Trace Elements solution (1 ml added l-1 ) [27], selenite/tungstate solution (final conc. of selenite and tungstate are 3 and 4 ��g l-1 respectively) [28]; and Vitamin 10 solution (0.1 ml added to 10 ml culture before inoculation) [2]. H2 was supplied as the energy source by pumping the culture vessels to 180 kPa over pressure with an 80:20 mixture of H2:CO2. Genomic DNA was extracted from freshly grown cells using a modified version of a liquid N2 and grinding method [29]. Briefly, AbM4 cultures were harvested by centrifugation at 20,000 �� g for 20 min at 4 oC and cell pellets combined into 40 ml Oakridge centrifuge tubes and frozen at -80 oC.

The frozen cell pellets were placed in a sterile, pre-cooled (-85 oC) mortar and ground to a powder with periodic addition of liquid N2. Buffer B1 (5 ml Qiagen Genomic-Tip 500 Maxi kit, Qiagen, Hilden, Germany) containing RNase (2 ��g ml-1 final concentration) was added to the powdered cell pellet to create a slurry which was then removed. An additional 6 ml of B1 buffer was used to rinse the remaining material from the mortar and pestle and combined with the cell slurry, which was then treated following the Qiagen Genomic-Tip 500/G Maxi kit instructions. Finally, the genomic DNA was precipitated by addition of 0.7 vol isopropanol, and collected by centrifugation at 12,000 �� g for 10 min at room temperature and re-dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 7.

5). Genome sequencing and assembly The complete genome sequence of AbM4 was determined using pyrosequencing of 3kb mate paired-end sequence libraries using a 454 GS FLX platform with titanium chemistry (Macrogen, Korea). Pyrosequencing reads provided 234�� coverage of the genome and were assembled using the Newbler assembler version 2.7 (Roche Cilengitide 454 Life Sciences, USA). The Newbler assembly resulted in 30 contigs across 4 scaffolds.

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