Complete genome sequence of the thermophilic sulfur-reducer Desulfurobacterium thermolithotrophum type strain (BSAT) from a deep-sea hydrothermal vent

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.


Introduction
Strain BSA T (= DSM 11699) is the type strain of the species Desulfurobacterium thermolithotrophum, which is the type species of its genus Desulfurobacterium [1], that currently consists of three validly named species [19]. 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' [1]. 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 BSA T 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 [1]. Although it shares most features with other members of the Aquificales, it is distinct in its inability to grow under microaerophilic conditions [1]. Strain BSA T was the first non-hyperthermophilic primary producer isolated from deep-sea vents [1]. Here we present a summary classification and a set of features for D. thermolithotrophum strain BSA T , together with the description of the complete genomic sequencing and annotation.

Classification and features
A representative genomic 16S rRNA sequence of D. thermolithotrophum BSA T 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 [5] and the relative frequencies of taxa and keywords (reduced to their stem [6] 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 BSA T 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 BSA T are small rods, about 1-2 µm long and 0.4-0-5 µm wide and occur singly or in pairs ( Figure 2) [1]. Cells stain Gram-negative and are motile via three polar flagella; spores are not produced [1]. Strain BSA T 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 [1]. 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 [1]. 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 [1]). No growth was observed in sea salts at concentrations of 10 and 80 g/l after 48 h incubation at 70°C [1]. Under optimal growth conditions (temperature, pH and NaCl), the doubling time of strain BSA T is around 135 min [1]. Strain BSA T is a strictly anaerobic chemolithotrophic organism that uses sulfur as an electron acceptor in the presence of H + for growth [1]. 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 [1]. 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 [1]. Nitrate, tryptone and yeast extract were used as nitrogen sources [1]. Growth of strain BSA T was inhibited by chloramphenicol, penicillin G and rifampicin at 100 µg/ml but not by streptomycin when added before incubation at the optimum temperature [1].

Figure 1
Phylogenetic tree highlighting the position of D. thermolithotrophum relative to the type strains of the other species within the order Aquificales. The tree was inferred from 1,422 aligned characters [7,8] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [9]. Rooting was done initially using the midpoint method [10] and then checked for its agreement with the current classification ( Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 1,000 ML bootstrap replicates [11] (left) and from 1,000 maximum parsimony bootstrap replicates [12] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [13] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks (referenced in [14][15][16][17] and CP002444).   [18] and the NamesforLife database [19].

Current classification
Domain Bacteria TAS [20] Phylum 'Aquificae' TAS [22] Class Aquificae TAS [23,24] Order Aquificales TAS [23,25,26] Family Desulfurobacteriaceae TAS [25] Genus Evidence codes -TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [30].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [31], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [32]. The genome project is deposited in the Genomes On Line Database [13] 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.

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 [36]. 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 [37] 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 [38] 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.

Genome annotation
Genes were identified using Prodigal [41] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [42]. 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 [33].

Genome properties
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.