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Complete genome sequence of the thermophilic sulfur-reducer Desulfurobacterium thermolithotrophum type strain (BSAT) from a deep-sea hydrothermal vent
Standards in Genomic Sciences volume 5, pages407–415 (2011)
Desulfurobacterium thermolithotrophum L’Haridon et al. 1998 is the type species of the genus Desulfurobacterium which belongs to the family Desulfurobacteriaceae. The species is of interest because it represents the first thermophilic bacterium that can act as a primary producer in the temperature range of 45–75 °C (optimum 70°C) and is incapable of growing under microaerophilic conditions. Strain BSAT preferentially synthesizes high-melting-point fatty acids (C18 and C20) which is hypothesized to be a strategy to ensure the functionality of the membrane at high growth temperatures. This is the second completed genome sequence of a member of the family Desulfurobacteriaceae and the first sequence from the genus Desulfurobacterium. The 1,541,968 bp long genome harbors 1,543 protein-coding and 51 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain BSAT (= DSM 11699) is the type strain of the species Desulfurobacterium thermolithotrophum, which is the type species of its genus Desulfurobacterium , that currently consists of three validly named species . The genus name is derived from the Latin words ‘de’ meaning ‘from’, ‘sulfur’, and ‘bacterium’ meaning ‘a stick, staff’, yielding the Neo-Latin word ‘Desulfurobacterium’ meaning ‘sulfur-reducing rod-shaped bacterium’ . The species epithet is derived from the latinized Greek word ‘thermê’ meaning ‘heat’, the latinized Greek word ‘lithos’ meaning ‘stone’ and the latinized Greek word ‘trophos’ meaning ‘feeder, rearer, one who feeds’, yielding the Neo-Latin word ‘thermolithotrophum’ meaning ‘referring to its thermophilic way of life and lithotrophic metabolism’ [1,2]. Strain BSAT was collected from the Snake Pit vent field on the mid Atlantic Ridge with the help of the submersible Nautile at a depth of 3,500 m . Although it shares most features with other members of the Aquificales, it is distinct in its inability to grow under microaerophilic conditions . Strain BSAT was the first non-hyperthermophilic primary producer isolated from deep-sea vents . Here we present a summary classification and a set of features for D. thermolithotrophum strain BSAT, together with the description of the complete genomic sequencing and annotation.
Classification and features
A representative genomic 16S rRNA sequence of D. thermolithotrophum BSAT was compared using NCBI BLAST [3,4] under default settings (e.g. considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database  and the relative frequencies of taxa and keywords (reduced to their stem  were determined, weighted by BLAST scores. The most frequently occurring genera were Desulfurobacterium (30.3%), Thermoanaerobacter (18.8%), Thermovibrio (14.2%), Balnearium (11.0%) and Persephonella (4.1%) (80 hits in total). Regarding the two hits to sequences from members of the species, the average identity within HSPs was 98.9%, whereas the average coverage by HSPs was 92.8%. Regarding the single hit to sequences from other members of the genus, the average identity within HSPs was 98.6%, whereas the average coverage by HSPs was 64.4%. Among all other species, the one yielding the highest score was “Desulfurobacterium crinifex” (AJ507320), which corresponded to an identity of 98.6% and HSP coverage of 64.4%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was AF068800 (‘hydrothermal vent clone VC2.1Bac24’), which showed an identity of 99.7% and an HSP coverage of 92.7%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were ‘hydrotherm’ (5.4%), ‘vent’ (4.9%), ‘microbi’ (3.6%), ‘water’ (2.9%) and ‘deep’ (2.0%) (167 hits in total). The most frequently occurring keyword within the labels of those environmental samples which yielded hits of a higher score than the highest scoring species was ‘hydrotherm, vent’ (50.0%) (1 hit in total).
Figure 1 shows the phylogenetic neighborhood of D. thermolithotrophum BSAT in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome differ by two nucleotides from the previously published 16S rRNA sequence (AJ001049).
The cells of strain BSAT are small rods, about 1–2 µm long and 0.4–0.5 µm wide and occur singly or in pairs (Figure 2) . Cells stain Gram-negative and are motile via three polar flagella; spores are not produced . Strain BSAT grows between 40 and 75°C with an optimum around 70°C, while no growth is detected at 37 or 80°C after 48 h incubation . Growth occurs between pH 4.4 and 8, with an optimum around pH 6.25. No growth is detected at pH 3.7 or 8.5 after 48h incubation at 70°C . Growth is observed in sea salts at concentrations ranging from 15 to 70g/l, with an optimum of approximately 35g/l (corresponding to 23 g NaCl/l ). No growth was observed in sea salts at concentrations of 10 and 80 g/l after 48 h incubation at 70°C . Under optimal growth conditions (temperature, pH and NaCl), the doubling time of strain BSAT is around 135 min . Strain BSAT is a strictly anaerobic chemolithotrophic organism that uses sulfur as an electron acceptor in the presence of H+ for growth . It utilizes thiosulfate, sulfite and polysulfides as alternative electron acceptors with H2 as an electron donor. Cysteine, nitrate or nitrite are not utilized and growth on sulfur, thiosulfate, polysulfides or sulfite was accompanied by exponential H2S production . No growth was observed on acetate, formate, methanol, monomethylamine and yeast extract with N2-CO2 or H2 atmosphere in the presence or absence of sulfur . Nitrate, tryptone and yeast extract were used as nitrogen sources . Growth of strain BSAT was inhibited by chloramphenicol, penicillin G and rifampicin at 100 µg/ml but not by streptomycin when added before incubation at the optimum temperature .
The total lipid content of strain BSAT is about 6% of the total dry weight and is characterized by the presence of aminophospholipids and a phospholipid at about 66%, R f 0.7 and 30%, R f 0.5, respectively, as well as minor compounds . Gas chromatographic analysis of fatty acid components of both compounds revealed the presence of saturated and monounsaturated acyl chains . The phosphoinositol contains C16:0 (15%), C18:1 (41%) identified as methyl-oleate, and C18:0 (44%) identified as stearate. The phosphoamino-positive compounds contained C16:0 (14%), C18:1 (43%), C18:0 (31%) and C20:0 (12%), as well as minor compounds .
Genome sequencing and annotation
Genome project history
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 Genomes On Line 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.
Growth conditions and DNA isolation
D. thermolithotrophum strain BSAT, DSM 11699, was grown anaerobically in DSMZ medium 829 (Desulfurobacterium medium)  at 70°C. DNA was isolated from 0.5–1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen 10262) following the standard protocol as recommended by the manufacturer without modifications. DNA is available through the DNA Bank Network .
Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website . Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 96 contigs in one scaffold was converted into a phrap  assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (45.0 Mb) was assembled with Velvet  and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 192.1 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package  was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution , Dupfinisher , or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 101 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI . The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 322.0 × coverage of the genome. The final assembly contained 126,482 pyrosequence and 12,545,740 Illumina reads.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation 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, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform .
The genome consists of one circular chromosome with a total length of 1,541,968 bp and a G+C content of 35.0% (Table 3 and Figure 3). Of the 1,594 genes predicted, 1,543 were protein-coding genes, and 51 RNAs; 34 pseudogenes were also identified. The majority of the protein-coding genes (75.5%) were assigned 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.
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We would like to gratefully acknowledge the help of Thomas Hader (University of Regensburg) for growing D. thermolithotrophum cultures. This work was performed under the auspices of the US Department of Energy’s 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, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, UT-Battelle and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-2.