Complete genome sequence of the thermophilic sulfate-reducing ocean bacterium Thermodesulfatator indicus type strain (CIR29812T)

Thermodesulfatator indicus Moussard et al. 2004 is a member of the Thermodesulfobacteriaceae, a family in the phylum Thermodesulfobacteria that is currently poorly characterized at the genome level. Members of this phylum are of interest because they represent a distinct, deep-branching, Gram-negative lineage. T. indicus is an anaerobic, thermophilic, chemolithoautotrophic sulfate reducer isolated from a deep-sea hydrothermal vent. Here we describe the features of this organism, together with the complete genome sequence, and annotation. The 2,322,224 bp long chromosome with its 2,233 protein-coding and 58 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
The genus Thermodesulfatator currently contains two species, both of which are anaerobic, thermophilic, chemolithoautotrophic sulfate reducers isolated from deep-sea hydrothermal vents [1,2]. Strain CIR29812 T (= DSM 15286 = JCM 11887) is the type strain of the species Thermodesulfatator indicus [1]. The strain was isolated from a chimney fragment taken from a black smoker in the Kairai vent field, Central Indian Ridge [1]. The genus name was derived from a combination of the Greek term thermos, hot, and the Neo-Latin desulfatator, sulfatereducer, meaning the thermophilic sulfate-reducer [1]; the species epithet was derived from the Latin adjective indicus, referring to the Indian Ocean, from where the strain was isolated [1]. The other species in this genus is T. atlanticus, which was isolated from the wall of a chimney at the Standards in Genomic Sciences Rainbow vent field on the Mid-Atlantic Ridge [2]. The major difference between the two Thermodesulfatator species is that T. indicus is strictly chemolithoautotrophic, while T. atlanticus is able to utilize organic carbon sources [2]. Here we present a summary classification and a set of features for T. indicus CIR29812 T , together with the description of the genomic sequencing and annotation.

Classification and features
A representative genomic 16S rRNA sequence of T. indicus CIR29812 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 Desulfovibrio (22.5%), Thermodesulfatator (22.0%), Thermodesulfobacterium (16.9%), Methylococcus (10.9%) and Thermodesulforhabdus (5.7%) (38 hits in total). Regarding the two hits to sequences from members of the species, the average identity within HSPs was 99.9%, whereas the average coverage by HSPs was 95.8%. Among all other species, the one yielding the highest score was "Geothermobacterium ferrireducens" (AF411013), which corresponded to an identity of 90.1% and an HSP coverage of 64.7%. (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 AJ874315 ('continuous enrichment hydrothermal black chimney clone 850'), which showed an identity of 96.7% and an HSP coverage of 93.9%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'spring' (6.2%), 'microbi' (4.8%), 'hot' (4.2%), 'nation, park' (2.7%) and 'yellowston' (2.6%) (212 hits in total). These keywords fit reasonably well to the habitat of a thermophilic sulfate-reducer. Environmental samples which yielded hits of a higher score than the highest scoring species were not found. Figure 1 shows the phylogenetic neighborhood of T. indicus in a 16S rRNA based tree. The sequences of the two 16S rRNA gene copies in the genome differ from each other by two nucleotides, and differ by up to four nucleotides from the previously published 16S rRNA sequence (AF393376).
T. indicus cells are Gram-negative rods with a length of 0.8-1.0 μm and a width of 0.4-0.5 μm [1]. An electron micrograph of T. indicus is shown in Figure 2. Cells are motile with a single polar flagellum and can be found separately or in groups of two or three cells [1]. The temperature range for growth is 55-80°C with an optimum at 70°C [1]. The salinity range is 10-35 g/L NaCl, with an optimum of 25 g/L NaCl [1]. The pH range is 6.0-6.7 with 6.25 as the optimum [1]. T. indicus is strictly anaerobic and strictly chemolithoautotrophic, growing with H2 as electron donor, sulfate as electron acceptor, and CO 2 as the carbon source [1]. Some organic compounds stimulated growth [1]. Ammonium, nitrate, peptone and tryptone could serve as nitrogen sources [1].

Chemotaxonomy
The major respiratory quinone found in T. indicus is menaquinone with seven isoprene subunits (MK-7) [1]. The major phospholipids are phosphatidylinositol and phosphatidylethanolamine. Phosphatidylglycerol and three unidentified phospholipids are present in lesser amounts [1]. The major fatty acids are C 18:0 and C 18:1 , and hydroxylated fatty acids are also present [1]. T. indicus was found to be sensitive to tetracycline, ampicillin, chloramphenicol, and rifampicin, and resistant to penicillin, kanamycin, and streptomycin [1].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [23], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [24]. 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.  [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 maximumparsimony 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.

Current classification
Domain Bacteria TAS [15] Phylum Thermodesulfobacteria TAS [16] Class Thermodesulfobacteria TAS [17,18] Order Thermodesulfobacteriales TAS [17,19] Family Thermodesulfobacteriaceae TAS [17,20] Genus Evidence codes -IDA: Inferred from Direct Assay (first time in publication); 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 the Gene Ontology project. If the evidence code is IDA, then the property was directly observed for a living isolate by one of the authors or an expert mentioned in the acknowledgements [22].

Growth conditions and DNA isolation
T. indicus strain CIR29812 T , DSM 15286, was grown anaerobically in DSMZ medium 383 (Desulfobacterium medium) [25] at 70°C. DNA was isolated from 0.5-1 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with modification st/LALM for cell lysis as described in Wu et al. 2009 [24]. DNA is available through the DNA Bank Network [26].

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 [27]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 49 contigs in one scaffold was converted into a phrap [28] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (427.0 Mb) was assembled with Velvet [29] 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 298.3 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 [28] 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 (C. Han, unpublished), Dupfinisher [30], 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 95 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 (A. Lapidus, unpublished). 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 310.6 × coverage of the genome. The final assembly contained 759,221 pyrosequence and 11,861,111 Illumina reads.

Genome annotation
Genes were identified using Prodigal [31] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [32]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [33], RNAMMer [34], Rfam [35], TMHMM [36], and SignalP [37].

Genome properties
The genome consists of a 2,322,224 bp long circular chromosome with a 42.4% G+C content (Table 3 and Figure 3). Of the 2,291 genes predicted, 2,233 were protein-coding genes, and 58 RNAs; 38 pseudogenes were also identified. The majority of the protein-coding genes (73.2%) 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. a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.  a) The percentage is based on the total number of protein coding genes in the annotated genome.