Complete genome sequence of Oceanithermus profundus type strain (506T)

Oceanithermus profundus Miroshnichenko et al. 2003 is the type species of the genus Oceanithermus, which belongs to the family Thermaceae. The genus currently comprises two species whose members are thermophilic and are able to reduce sulfur compounds and nitrite. The organism is adapted to the salinity of sea water, is able to utilize a broad range of carbohydrates, some proteinaceous substrates, organic acids and alcohols. This is the first completed genome sequence of a member of the genus Oceanithermus and the fourth sequence from the family Thermaceae. The 2,439,291 bp long genome with its 2,391 protein-coding and 54 RNA genes consists of one chromosome and a 135,351 bp long plasmid, and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain 506 T (DSM 14977 = NBRC 100410 = VKM B-2274) is the type strain of Oceanithermus profundus, which is the type species of the genus Oceanithermus [1] of the family Thermaceae [2]. Together with O. desulfurans, there are currently two species placed in the genus [1,3]. The generic name derives from the Latin noun oceanus, meaning ocean and the Neo-Latin masc. substantive (from Gr. adj. thermos) thermus which means hot. Therefore, the name Oceanithermus refers to warmth-loving organisms living in the ocean. The species epithet is derived from the Latin adjective profundus meaning deep, which means pertaining to the abyss, pertaining to the depths of the ocean [1]. Strain 506 T was first isolated from samples of hydrothermal fluids and chimneys collected at the 13ºN hydrothermal vent field on the East Pacific Rise at a depth of 2600 m [1]. There are no further cultivated strains of this species known. The other member of the genus, O. desulfurans, is a thermophilic, sulfur-reducing bacterium isolated from a sulfide chimney in Suiyo Seamount, in the Western Pacific [3]. Here we present a summary classification and a set of features for O. profundus 506 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
A representative genomic 16S rRNA sequence of strain 506 T was compared using NCBI BLAST under default settings (e.g., considering only the highscoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [4] and the relative frequencies, weighted by BLAST scores, of taxa and keywords (reduced to their stem) [5] were determined. The five most frequent genera were Thermus (52.0%), Meiothermus (37.0%), Oceanithermus (7.6%), Marinithermus (2.0%) and Vulcanithermus (1.4%) (156 hits in total). Regarding the four hits to sequences from members of the species, the average identity within HSPs was 99.6%, whereas the average coverage by HSPs was 94.8%. Regarding the two hits to sequences from other members of the genus, the average identity within HSPs was 99.3%, whereas the average coverage by HSPs was 91.0%. Among all other species, the one yielding the highest score was O. desulfurans, which corresponded to an identity of 99.3% and an HSP coverage of 91.0%. The highest-scoring environmental sequence was EU555123 ('Microbial Sulfide Hydrothermal Vent Field Juan de Fuca Ridge Dudley hydrothermal vent clone 4132B16'), which showed an identity of 99.1% and an HSP coverage of 98.0%. The five most frequent keywords within the labels of environmental samples which yielded hits were 'spring' (8.2%), 'hot' (6.2%), 'microbi' (4.5%), 'geochem, nation, park, yellowston' (2.8%) and 'hydrotherm/vent' (2.5%) (94 hits in total). The five most frequent keywords within the labels of environmental samples which yielded hits of a higher score than the highest scoring species were 'hydrotherm/vent' (12.2%), 'field, microbi, ridg' (6.1%), 'fluid' (5.9%), 'dudlei, fuca, juan, sulfid' (3.1%) and 'degre, east, north, ocean, pacif, rise' (3.0%) (3 hits in total). These 16S BLAST results are a confirmation of the kind of environment from which the living strain was isolated and therefore fits the description of the isolate. Figure 1 shows the phylogenetic neighborhood of O. profundus in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome differ by one nucleotide from the previously published 16S rRNA sequence (AJ430586). The cells of O. profundus are described as nonmotile, rod-shaped, 0.5 -0.7 µm in diameter and of various lengths (Figure 2). When grown on proteinaceous substrates, old cultures of O. profundus form filaments and large spheres resembling the 'rotund bodies' typical of aged cells of Thermus species [1,15]. The organism is Gram-negative and non spore-forming (Table 1). O. profundus is microaerophilic, only being able to grow at oxygen concentrations below 6% [1]. No growth has been observed in an atmosphere of air, either in liquid medium or on plates. In an agar tube containing 5 ml of basal medium supplemented with 2 g sucrose and 1 g tryptone per liter with air in the headspace (10 ml), growth occurs in a zone located 20 mm below the agar/air interface [1]. Alternatively, the organism grows anaerobically using nitrate as the electron acceptor. O. profundus grows within a temperature range of 40-68ºC, optimal growth being observed at 60ºC. At 60ºC, it grows between pH 5.5 and 8.4, with an optimum around 7.5 [1]. Strain 506 T grows at NaCl concentrations ranging from 10 to 50 g/l, with an optimum at 30 g/1 [1]. The organism is oxidase-and catalase positive and is able to utilize a wide spectrum of carbohydrates in the presence of either nitrate or oxygen [1]. The highest cell yield is observed in the presence of nitrate with fructose, maltose, sucrose, trehalose, galactose, rhamnose or xylose. Glucose, lactose and starch are utilized, but no growth has been reported with ribose, galactose, arabinose, dextrin or cellobiose [1]. Acetate and propionate are produced during growth with sucrose as a growth substrate and nitrate as the electron acceptor. Nitrite is the only product of denitrification [1]. O. profundus grows well with complex proteinaceous substrates such as beef extract, tryptone or papaic digest of soybean (1-1.5 g/l). However, growth is strongly inhibited by higher concentrations of these substrates [1]. The isolate does not grow with Casamino acids or yeast extract as sole sources of carbon and energy, though 100 mg/l yeast extract is required for growth [1]. O. profundus is able to utilize acetate, pyruvate and propionate as growth substrates. It also grows with methanol, ethanol and mannitol, though the cell yield is lower [1]. O. profundus is able to grow lithoheterotrophically using molecular hydrogen as the energy source, yeast extract as the carbon source and nitrate as the electron acceptor. Other electron acceptors (sulfate, elemental sulfur, thiosulfate and nitrite) do not support growth, regardless of growth substrate [1]. Detailed studies on the metabolism of maltose, acetate, pyruvate, and hydrogen have been undertaken by Fedosov et al. [26]. Standards in Genomic Sciences profundus relative to the other type strains within the family Thermaceae. The tree was inferred from 1,420 aligned characters [6,7] of the 16S rRNA gene sequence under the maximum likelihood criterion [8]. Rooting was initially done using the midpoint method [9] and then checked for its accordance with the current taxonomy (see Table 1) and rooted accordingly. The branches are scaled in terms of the expected number of substitutions per site. Numbers to the right of bifurcations are support values from 1,000 bootstrap replicates [10] if larger than 60%. Lineages with type strain genome sequencing projects that are registered in GOLD [11] but remain unpublished are labeled with one asterisk, published genomes with two asterisks [12][13][14].  Phylum "Deinococcus-Thermus" TAS [18,19] Class Deinococci TAS [20,21] Order Thermales TAS [21,22] Family Thermaceae TAS [21,23] Genus , 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 [25] If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Chemotaxonomy
The polar lipid pattern of strain 506 T comprises three phospholipids, whereas glycolipids have not been detected [1]. This differentiates the organism from members of the genera Vulcanithermus, Rhabdothermus, Thermus and Meiothermus, where phospholipids and glycolipids have both been detected [27,28]. It should be noted that the major phospholipid detected in O. profundus has the same Rf and staining behavior as the 2′-O-(1, 2diacyl-sn-glycero-3-phospho)-3′-O-(α-N-acetylglucosaminyl)-N-glyceroyl alkylamine reported to occur in members of the genera Meiothermus and Thermus [29]. On the basis of Rf value and staining behavior this lipid also appears to be present in members of the genera Vulcanithermus and Rhabdothermus, which also synthesize glycolipids [30,31] Although members of the genus Deinococcus may also produce glycolipids in addition to a novel series of phosphoglycolipids [32,33] the latter are absent in members of the genera Thermus and Meiothermus. The absence of glycolipids was one of the arguments for Miroshnichenko et al. for placing strain 506 T in a new genus [1].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [38] and is part of the Genomic Encyclopedia of Bacteria and Archaea project [39]. The genome project is deposited in the Genome On Line Database [11] and the complete genome sequence is deposited in Gen-Bank. 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
O. profundus strain 506 T , DSM 14977, was grown anaerobically in DSMZ medium 975 (Oceanithermus profundus medium) [40] at 60°C. DNA was isolated from 0.5-1 g of cell paste using Jetflex Genomic DNA Purification Kit following the standard protocol as recommended by the manufacturer, but with an additional proteinase K (20 µl) digestion for 45 min at 58°C. DNA is available through the DNA Bank Network [41].

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 [42]. Pyrosequencing reads were assembled using the Newbler assembler version 2.3-PreRelease-8-23-2009 (Roche). The initial Newbler assembly, consisting of nine contigs in four scaffolds, was converted into a phrap assembly by [43] making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (208 Mb) was assembled with Velvet [44] 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 306.1 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 [43] 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 gap-Resolution [42], Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [45]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 177 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 [46]. 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 282.8 × coverage of the genome. The final assembly contained 1,258,374 pyrosequence and 5,792,221 Illumina reads.

Genome annotation
Genes were identified using Prodigal [47] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [48]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uni-Prot, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) [49].

Genome properties
The genome consists of a 2,303,940 bp long chromosome with a G+C content of 70% and a 135,351 bp plasmid with a G+C content of 66% (Table 3 and Figure 3). Of the 2,445 genes predicted, 2,391 were protein-coding genes, and 54 RNAs; 18 pseudogenes were also identified. The majority of the protein-coding genes (69.9%) were assigned with 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.