Genome analysis of Elusimicrobium minutum, the first cultivated representative of the Elusimicrobia phylum (formerly Termite Group 1) Page: 4 of 37
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42 discussed in light of physiological data, and potential functions indicated by the genome
43 annotation are compared to requirements imposed by the intestinal environment. Using the
44 concatenated sequences of 22 single-copy marker genes of E. minutum and of the uncultivated
45 "Endomicrobium" strain Rs-D17, an endosymbiont of termite gut flagellates (22), we also
46 investigated the phylogenetic position of Elusimicrobia relative to other bacterial phyla.
Material and Methods
47 DNA preparation. A 400-ml culture of Elusimicrobium minutum strain Peil91T grown on
48 glucose (14) was harvested by centrifugation. Cells were resuspended in 500 pl TE buffer (10
49 mM Tris-HCl, 1 mM EDTA, pH 8.0), and 30 pl of 10% SDS and 3 pl of proteinase K (20
50 mg/ml) were added. The mixture was incubated at 37 C for 1 h. The lysate was extracted
51 three times with an equal volume of phenol-chloroform-isoamyl alcohol (49:49:1, by vol)
52 using Phase Lock Gel tubes (Eppendorf). The supernatant was transferred to a fresh tube, and
53 the DNA was precipitated with 0.6 volumes of isopropanol, washed with ice-cold 80%
54 (vol/vol) ethanol, and air-dried. Quality and quantity were checked by agarose gel
55 electrophoresis.
56 Genome sequencing, assembly, and gap closure. The genome of E. minutum was sequenced
57 at the Joint Genome Institute (JGI) using a combination of 8-kb and 40-kb Sanger libraries
58 and 454 pyrosequencing. All general aspects of library construction and sequencing performed
59 at the JGI can be found at http://www.jgi.doe.gov/. 454 pyrosequencing reads were assembled
60 using the Newbler assembler (Roche). Large Newbler contigs were chopped into 1871
61 overlapping fragments of 1000 bp and entered into the assembly as pseudo-reads. TheHerlemann et al.
4
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Herlemann, D. P. R.; Geissinger, O.; Ikeda-Ohtsubo, W.; Kunin, V.; Sun, H.; Lapidus, A. et al. Genome analysis of Elusimicrobium minutum, the first cultivated representative of the Elusimicrobia phylum (formerly Termite Group 1), article, February 1, 2009; Berkeley, California. (https://digital.library.unt.edu/ark:/67531/metadc928715/m1/4/: accessed April 23, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.