Complete genome sequence of Vulcanisaeta distributa type strain (IC-017T)

Vulcanisaeta distributa Itoh et al. 2002 belongs to the family Thermoproteaceae in the phylum Crenarchaeota. The genus Vulcanisaeta is characterized by a global distribution in hot and acidic springs. This is the first genome sequence from a member of the genus Vulcanisaeta and seventh genome sequence in the family Thermoproteaceae. The 2,374,137 bp long genome with its 2,544 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain IC-017 T (= DSM 14429 = JCM 11212) is the type strain of the species Vulcanisaeta distributa, which is the type species of its genus Vulcanisaeta [1]. The only other species in the genus is V. souniana [1,2]. The genus name derives from the Latin words 'vulcanicus' meaning volcanic, and 'saeta' meaning stiff hair, to indicate a rigid rod inhabiting volcanic hot springs [1]. The species epithet derives from the Latin 'distributa', referring to the wide distribution of strains belonging to this species [1]. The type strain IC-017 T was isolated from a hot spring in Ohwakudani, Kanagawa, Japan [1] [1]. At the time of the species description, the terminus 'distributa' referred simply to a wide distribution within Japan [1]. However, 16S rRNA sequences which probably belong to the genus Vulcanisaeta (≥95% sequence similarity to V. distributa) have been obtained from 117°C hot deep-sea hydrothermal fluid in the south Mariana area [3]. Clone sequences that are highly similar to the 16S rRNA gene sequence of strain IC-017 T were obtained from an acidic hot spring water at the Tatung Volcano area in Northern Taiwan (99%, FJ797325), the hot Sylvan Spring in Yellowstone National Park (=YNP, USA, 98%, DQ243774), at the Cistern Hot Spring at Norris Geyser Basin in YNP (98%, DQ924709) and also at other springs in YNP (98%, DQ833773). Metagenomic sequences from uncultured clones in YNP (94%, ADKH01000984) also support these observations. The 16S rRNA gene similarity values to non-hot-spring metage-nomes, e.g., from marine, soil, or human gut, were all below 83%, indicating that Vulcanisaeta is probably not found in these habitats (status July 2010). Although it is not the case for the type strain IC-017 T , V. distributa recently received further interest, as it was found that strain IC-065 contained a 691 bp large intron within its 16S rRNA sequence [4]. Novel 16S rRNA introns have been found in several members of the family Thermoproteaceae [4]. Here we present a summary classification and a set of features for V. distributa strain IC-017 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
The cells of strain IC-017 T are rigid, straight to slightly curved rods ( Figure 1 and Table 1) [4]. Occasionally, they bend, branch out, or bear spherical bodies at the terminae (not seen in Figure 1), which have been termed as 'golf clubs'. Most cells are 0.4-0.6 µm in width and 3-7 µm long [4]. Pili have been observed to rise terminally or laterally; motility has not been observed [4]. Usually, strain IC-017 T grows anaerobically. However, when cultured in media in which sulfur is replaced by sodium thiosulfate (1.0 g/l), strain IC-017 T showed weak growth in a low-oxygen atmosphere (1%), but not in air [4]. In contrast to Thermocladium or Caldivirga strains, V. distributa grows well even in the absence of a vitamin mixture or archaeal cell-extract solution in the medium [4]. All seven tested strains of V. distributa were shown to be resistant to chloramphenicol, kanamycin, oleandomycin, streptomycin and vancomycin, but sensitive to erythromycin, novobiocin and rifampicin (all at 100 µg per ml) [4]. V. distributa needs acidic conditions to grow (pH 3.5 to 5.6). Under optimal growth conditions, the doubling time is 5.5 to 6.5 hours [4]. Sulfur or thiosulfate is required as an electron acceptor. Strain IC-017 T does not utilize D-arabinose, D-fructose, lactose, sucrose, D-xylose, acetate, butyrate, formate, fumarate, propionate, pyruvate, succinate, methanol, formamide, methylamine or trimethylamine as carbon sources and does not utilize fumarate, malate or nitrate as electron acceptors [4]. Figure 2 shows the phylogenetic neighborhood of V. distributa IC-017 T in a 16S rRNA based tree. The sequence of the single 16S rRNA gene copy in the genome of strain IC-017 T does not differ from the previously published 16S rRNA sequence (AB063630).

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [26], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [27]. The genome project is deposited in the Genome OnLine Database [20] 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
V. distributa IC-017 T , DSM 14429, was grown anaerobically in DSMZ medium 88 (Sulfolobus medium) [28] at 90°C. DNA was isolated from 0.5-1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the standard protocol as recommended by the manufacturer.

Genome sequencing and assembly
The genome was sequenced using a 454 sequencing platform. All general aspects of library construction and sequencing can be found at the JGI website. Pyrosequencing reads were assembled using the Newbler assembler version 2.0.0-PostRelease-09/05/2008 (Roche). The initial Newbler assembly consisted of 147 contigs in 13 scaffolds and was converted into a phrap assembly by making fake reads from the consensus, and collecting the read pairs in the 454 paired end library. Draft assemblies were based on 252.4 Mb 454 draft 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 following finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible misassemblies were corrected with gapResolution, Dupfinisher [29], or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [30].
Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 97 additional reactions were necessary to close gaps 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 contains 0.8 million pyrosequencing reads that provide 106.3× coverage of the genome.

Figure 2.
Phylogenetic tree highlighting the position of V. distributa IC-017 T relative to the other type strains within the genus Vulcanisaeta and the type strains of the other genera within Thermoproteales. The tree was inferred from 1,356 aligned characters [16,17] of the 16S rRNA gene sequence under the maximum likelihood criterion [18] and rooted with the type strains of the genera of Desulfurococcales and Acidilobales. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 150 bootstrap replicates [19] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [20] are shown in blue, published genomes [21][22][23][24] and INSDC accessions CP000504 and CP00852 in bold.  Phylum Crenarchaeota TAS [7,8] Class Thermoprotei TAS [8,9] Order Thermoproteales TAS [10][11][12][13] Family Thermoproteaceae TAS [10,12,13] 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 of the Gene Ontology project [15]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

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) nonredundant data-base, UniProt, TIGRFam, 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 a 2,374,137 bp long chromosome with a 45.4% GC content (Table 3 and Figure 3). Of the 2,593 genes predicted, 2,544 were protein-coding genes, and 49 RNAs; fifty one pseudogenes were also identified. The majority of the protein-coding genes (57.2%) 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.