Genomic Analysis of Vulcanisaeta thermophila Type Strain CBA1501T Isolated from Solfataric Soil

Hyperthermophilic archaea have been isolated from high-temperature environments such as geothermally heated soils, sulfur-rich hot springs, and submarine volcanic habitats; optimal growth of these organisms occurs above 80°C (Stetter, 1999, 2006, 2013). The genus Vulcanisaeta belongs to the family Thermoproteaceae, order Thermoproteales, phylum Crenarchaeota, and was first proposed by Itoh et al. (2002). It currently includes 3 validly named species, that is, Vulcanisaeta distributa (Itoh et al., 2002), V. souniana (Itoh et al., 2002), and V. thermophila (Yim et al., 2015), as per the List of Prokaryotic Names with Standing in Nomenclature database (Parte, 2014). Members of the genus Vulcanisaeta are rod-shaped, anaerobic, hyperthermophilic, and acidophilic (Itoh et al., 2002). To date, 15 genomes, including two complete genomes, V. distributa and “Vulcanisaeta moutnovskia” (Mavromatis et al., 2010; Gumerov et al., 2011), have been reported for the genus Vulcanisaeta, as per the NCBI genome database (http://www.ncbi.nlm.nih.gov/genome/). 
 
Hyperthermophilic enzymes are stable and active at high temperatures of >70°C (Vieille et al., 1996). These enzymes can be studied using model systems to elucidate enzyme mechanisms and evolution of proteins stable at high temperatures and to determine the higher temperature limit for enzyme stability (Vieille and Zeikus, 2001). In a previous study, V. thermophila CBA1501T (= ATCC BAA-2415T = JCM 17228T) was isolated from solfataric soil in the Republic of the Philippines (Yim et al., 2015). It was found to grow at 75–90°C, pH 4.0–6.0, and 0–1.0% (w/v) NaCl, with optimal growth at 85°C, pH 5.0, and 0% (w/v) NaCl. Here, a genome sequence of V. thermophila CBA1501T has been reported and information of hyperthermophilic enzymes of high biotechnological value has been provided.

Hyperthermophilic enzymes are stable and active at high temperatures of >70 • C (Vieille et al., 1996). These enzymes can be studied using model systems to elucidate enzyme mechanisms and evolution of proteins stable at high temperatures and to determine the higher temperature limit for enzyme stability (Vieille and Zeikus, 2001). In a previous study, V. thermophila CBA1501 T (= ATCC BAA-2415 T = JCM 17228 T ) was isolated from solfataric soil in the Republic of the Philippines (Yim et al., 2015). It was found to grow at 75-90 • C, pH 4.0-6.0, and 0-1.0% (w/v) NaCl, with optimal growth at 85 • C, pH 5.0, and 0% (w/v) NaCl. Here, a genome sequence of V. thermophila CBA1501 T has been reported and information of hyperthermophilic enzymes of high biotechnological value has been provided.

Culture Conditions and DNA Extraction
In a previous study, we isolated V. thermophila CBA1501 T from the solfataric soil of the Mayon volcano in the Republic of the Philippines (Yim et al., 2015) and cultivated it on modified JCM medium no. 236 (M236) (containing per liter salt base solution: 2.94 g trisodium citrate dihydrate, 0.5 g yeast extract, 10.0 ml trace vitamins, 1.0 mg resazurin, 0.5 g Na 2 S·9H 2 O, and 20 mM thiosulfate). For DNA extraction, the strain was enriched at 80 • C in M236 medium, using a serum bottle. Its genomic DNA was extracted using the G-spin total DNA extraction kit (iNtRON Biotechnology, Korea) and QuickGene DNA tissue kit S (Kurabo, Japan).

General Genomic Features of V. thermophila CBA1501 T
The draft genome sequence of V. thermophila CBA1501 T was 2,022,594 bp in length, with a G+C content of 49.1 mol % in 10 contigs. The largest contig was 791,731 bp long, and the N50 value was 634,758 bp. The genome was found to contain 2170 CDSs, one 16S-23S-5S rRNA gene operon, and 41 tRNA genes. Genomic features are shown in Figure 1. On the basis of information from the EggNOG v. 4.1 database, 1927 genes were categorized into Clusters of Orthologous Groups of proteins (COGs) functional groups. The most abundant COG category was "Function unknown" (S; 729 genes), followed by "Energy production and conversion" (C; 178 genes), "Amino acid transport and metabolism" (E; 168 genes), "Translation, ribosomal structure and biogenesis" (J; 159 genes), "Carbohydrate transport and metabolism" (G; 96 genes), and "Coenzyme transport and metabolism" (H; 87 genes). Among the SEED subsystem categories, "Carbohydrates" (181 genes), "Amino Acids and Derivatives" (171 genes), "Protein Metabolism" (142 genes) and "Cofactors, Vitamins, Prosthetic Groups, Pigments" (116 genes) were the most dominant categories (>10% of a total of 1,094 matched SEED subsystem categories).

Comparative Genomics Data
V. thermophila CBA1501 T had lesser than 73% orthoANI values with all of the related strains in the genus Vulcanisaeta (Supplementary Table 1). In the orthoANI values-based dendrogram, the strain CBA1501 T was located as an outgroup to the other related strains in Vulcanisaeta (Supplementary Figure 1B). These results indicate that V. thermophila CBA1501 T is evolutionarily distinct from other related strains. The pan-genome analysis showed that 10 genomes in the genus Vulcanisaeta have the core genome, comprised of 979 POGs. In contrast, only the genome of strain CBA1501 T had 211 POGs as a singleton. Among these singletons, various enzymes, including arylformamidase, shikimate kinase, formyl-CoA transferase, xanthine dehydrogenase, hydrogensulfite reductase, and amidase, were detected.
In conclusion, the information provided here is useful as the genome of V. thermophila CBA1501 T will provide insights into the metabolism of hyperthermophilic archaea and aid in identifying opportunities for biotechnological applications of novel hyperthermophilic enzymes.
FIGURE 1 | Graphic circular map of the Vulcanisaeta thermophila CBA1501 T genome. Outer circle shows genes on the sense and antisense strands (colored according to COG categories), and RNA genes (red, tRNA; blue, rRNA) are shown from the outside of the circle to the center. Inner circles show the GC skew, with yellow and blue indicating positive and negative values, respectively; the GC content is indicated in red and green. This genome map was visualized using CLgenomics 1.52 (Chun Lab Inc.).

AUTHOR CONTRIBUTIONS
SWR designed and coordinated all the experiments. KJY performed cultivation, DNA extraction and purification. JYK, HSS, YBK, D-GL, JK, and K-SO performed the sequencing, genome assembly, gene prediction, gene annotation and comparative genomic analysis. JYK, KJY, and SWR wrote manuscript. All authors have read the manuscript and approved.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.01639