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Thermobispora bispora (Henssen 1957) Wang et al. 1996 is the type species of the genus Thermobispora. This genus is of great interest because it is strictly thermophilic and because it has been shown for several of its members that the genome contains substantially distinct (6.4% sequence difference) and transcriptionally active 16S rRNA genes. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the second completed genome sequence of a member from the suborder Streptosporangineae and the first genome sequence of a member of the genus Thermobispora. The 4,189,976 bp long genome with its 3,596 protein-coding and 63 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain R51T (= DSM 43833 = ATCC 19993 = JCM 10125) is the type strain of the species Thermobispora bispora, which is the type species of the genus Thermobispora . The generic name of the genus derives from the Greek words ‘thermos’, ‘bis’, and ‘spora’, to indicate high temperature two-spored organisms . Strain R51T was isolated from decaying manure in Berlin (Germany) in 1954 . Other strains were isolated during the same research project from other types of manure in other cities in Germany and in Finland . As deduced from 16S gene sequences, T. bispora was also found in compost in Sweden . Historically, strain R51T was originally classified in 1957 as Thermopolyspora bispora . At the same time, a morphologically similar genus, Microbispora, was described , which has priority and T. bispora was subsequently transferred to the genus Microbispora [5,6]. However, based on thermal preferences [2,7], chemotaxonomic features , and the two-dimensional polyacrylamide gel electrophoresis patterns of the ribosomal protein AT-L30 , Microbispora bispora was subsequently removed from the genus Microbispora to be the type species of the new genus Thermobispora . T. bispora is currently the only species in the genus Thermobispora . In 1997 T. bispora gained interest, as it was described as the first organism to have two distinct (6.4% of total nucleotides) types of transcriptionally active 16S rRNA genes (GenBank accessions U83909 and U83912) . Based on the two copies of the 16S rRNA genes that match best to sequence U83909 the closest related type strain (9% sequence difference ) is Micromonospora pattaloongensis  of the family Micromonosporaceae; based on the two copies of the 16S rRNA genes that match best to sequence U83912 the closest related type strain (8% sequence difference ) is Planotetraspora silvatica  of the family Streptosporangiaceae. Neither fit to the taxonomic position as shown in the List of Procaryotic names with Standing in Nomenclature that shows the genus Thermobispora as a member of the family Pseudonocardiaceae, reflecting the current uncertainty of the taxonomic position of T. bispora . In their recent review of Actinobacteria taxonomy, Zhi et al.  suggested to place Thermobispora in the suborder Streptosporangineae without assignment to a family, which is in accordance with our SSU rRNA tree (Figure 1). 16S rRNA sequences from environmental samples and metagenomic surveys with both 16S rRNA sequences detected phylotypes with approximately 89-92% 16S rRNA gene sequence similarity to both (U83909 and U83912) reference sequences only in a compost metagenome , indicating a very rare occurrence of Thermobispora-spp. in the environment (status March 2010). Here we present a summary classification and a set of features for T. bispora R51T, together with the description of the complete genomic sequencing and annotation.
Figure 1 shows the phylogenetic neighborhood of for T. bispora R51T in a 16S rRNA based tree. The sequences of the four 16S rRNA gene copies in the genome differ from each other by up to 94 nucleotides, and differ by up to 95 nucleotides from the previously published 16S rRNA sequence generated from ATCC 19993 (U58523).
T. bispora cells form substrate mycelia whose hyphae are 0.5 to 0.8 µm in diameter  (Figure 2). The aerial mycelia branch monopodally and bear longitudinal pairs of spores  (not visible in Figure 2). The spore diameters are usually 1.2 to 2.0 µm, but in liquid media spores with a diameter of 3 µm may occur . The aerial mycelia are white, and the substrate mycelia are yellow or yellowish brown on the media used in the respective study (International Streptomyces Project medium 4 agar and IF0328 agar; Institute for Fermentation) . No soluble pigment is produced . T. bispora is an obligately thermophilic organism (Table 1) . Starch is not hydrolyzed; inositol and rhamnose are utilized for growth, but arabinose and glycerol are not utilized . Also, T. bispora is negative for iodinin production and nitrate reduction .
The cell wall of strain R51T contains predominantly the menaquinone MK-9(H0) (75%) and only small amounts of MK-9(H2) and MK-9(H4) . Strain R51T has a type PIV phospholipid pattern, and contains phosphatidylethanolamine but not phosphatidylglycerol and trace amounts of glucosamine-containing phospholipids . The cell wall contains a major amount of meso-diaminopimelic acid, and the whole-cell hydrolysate contains madurose and galactose . The fatty acid composition of strain R51T is dominated by saturated acids, with iso-C16:0 (55%) being the most frequent acid, followed by anteiso-C17:0 (8%), the unsaturated C18:1 (8%), iso-C18:0 (6%) and C16:0 . Also, strain R51T contains minor amounts of 10-methyl-branched chain fatty acids .
This organism was selected for sequencing on the basis of its phylogenetic position , and is part of the Genomic Encyclopedia of Bacteria and Archaea project . The genome project is deposited in the Genome OnLine Database  and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
T. bispora strain R51T, DSM 43833, was grown in DSMZ medium 84 (Rolled oats mineral medium)  at 55°C. DNA was isolated from 1-1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with lysis modification st/FT according to Wu et al. .
The genome of T. bispora was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at http://www.jgi.doe.gov/. 454 pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 4,798 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones . Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 1,181 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. The final assembly consists of 40,290 Sanger and 1.1× pyrosequence based pseudo-reads. Together Sanger reads and pseudo-reads provided 8.19× coverage of the genome.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome an-notation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform .
The genome is 4,189,976 bp long and comprises one main circular chromosome with an overall GC content of 72.4% (Table 3 and Figure 3). Of the 3,659 genes predicted, 3,596 were protein-coding genes, and 63 RNAs; fifty pseudogenes were also identified. The majority of the protein-coding genes (71.9%) were assigned with a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
We would like to gratefully acknowledge the help of Susanne Schneider (DSMZ) for DNA extraction and quality analysis. This work was performed under the auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-1 and SI 1352/1-2.