Complete genome sequence of the aerobic, heterotroph Marinithermus hydrothermalis type strain (T1T) from a deep-sea hydrothermal vent chimney

Marinithermus hydrothermalis Sako et al. 2003 is the type species of the monotypic genus Marinithermus. M. hydrothermalis T1T was the first isolate within the phylum “Thermus-Deinococcus” to exhibit optimal growth under a salinity equivalent to that of sea water and to have an absolute requirement for NaCl for growth. M. hydrothermalis T1T is of interest because it may provide a new insight into the ecological significance of the aerobic, thermophilic decomposers in the circulation of organic compounds in deep-sea hydrothermal vent ecosystems. This is the first completed genome sequence of a member of the genus Marinithermus and the seventh sequence from the family Thermaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,269,167 bp long genome with its 2,251 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain T1 T (= DSM 14884 = JCM 11576) is the type strain of the species M. hydrothermalis, which is the type species of the monotypic genus Marinithermus [1,2]. The genus name is derived from the Latin word 'marinus' meaning 'of the sea' and the latinized Greek word 'thermos' meaning 'hot', yielding the Neo-Latin word 'Marinithermus' meaning 'an organism living in hot marine places' [1]. The species epithet is derived from the Neo-Latin word 'hydrothermalis' (pertaining to a hydrothermal vent) [1]. Strain T1 T was isolated in November 2000 from the surface zone of a deepsea hydrothermal vent chimney at Suiyo Seamount in the Izu-Bonin Arc, Japan, at a depth of 1,385 m [1]. M. hydrothermalis was the first isolate within the phylum "Thermus-Deinococcus" that grew optimally under a salinity equivalent to that of sea water [1]. Standards in Genomic Sciences The absolute requirement of NaCl for growth distinguishes M. hydrothermalis from members of the genera Thermus and Meiothermus [1,3]. No further isolates have been reported for M. hydrothermalis. Here we present a summary classification and a set of features for M. hydrothermalis T1 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
A representative genomic 16S rRNA sequence of M. hydrothermalis T1 T was compared using NCBI BLAST [4,5] 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 [6] and the relative frequencies of taxa and keywords (reduced to their stem [7]) were determined, weighted by BLAST scores. The most frequently occurring genera were Thermus (91.0%), Oceanithermus (4.9%), Marinithermus (3.3%) and Thermothrix (0.8%) (118 hits in total). Regarding the two hits to sequences from members of the species, the average identity within HSPs was 100.0%, whereas the average coverage by HSPs was 98.0%. Among all other species, the one yielding the highest score was O. profundus (NR_027212), which corresponded to an identity of 91.9% and HSP coverage of 93.3%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highestscoring environmental sequence was EU555123 [8] ('Microbial Sulfide Hydrothermal Vent Field Juan de Fuca Ridge Dudley hydrothermal vent clone 4132B16'), which showed an identity of 91.6% and HSP coverage of 92.1%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'spring' (6.9%), 'hot' (5.3%), 'microbi' (3.7%), 'nation, park, yellowston' (3.2%) and 'skin' (3.0%) (132 hits in total). Environmental samples which yielded hits of a higher score than the highest scoring species were not found. These key words are in accordance with the biotope of the strain T1 T in the original description [1], although 'skin' indicates the possible presence of relatives in a moderate environment. Figure 1 shows the phylogenetic neighborhood of M. hydrothermalis T1 T in a 16S rRNA based tree. The sequences of the three identical 16S rRNA gene copies in the genome differ by two nucleotides from the previously published 16S rRNA sequence (AB079382).
The cells of strain T1 T are Gram-negative, nonmotile, straight rods measuring 7.5 -9.4 µm by 0.9 -1.0 µm during the exponential growth phase [1] ( Figure 2 and Table 1). In the stationary growth phase the cells tend to form filaments [1]. Rotund bodies were not observed from the cells [1]. Cells of strain T1 T have an envelope which consists of a cytoplasmic membrane with a simple outline and a cell wall with an inner, electron-dense thin layer, which presumably represents the peptidoglycan [1]. Colonies are whitish and have 2.5 -3.0 mm of diameter [1]. The organism is an obligate heterotroph and grows only under strictly aerobic culture conditions [1]. Growth was not observed in anaerobic or autotrophic culture conditions [1]. However, it should be noted that according to Mori and colleagues [32] this was tested only in the presence of sulfide. Steinsbu and colleagues [3] argue that it is therefore possible that M. hydrothermalis has the capability of anaerobic growth under unreduced conditions, as has been observed for Rhabdothermus arcticus, Vulcanithermus mediatlanticus, O. profundus and O. desulfurans [3,[32][33][34]. Unlike members of the genus Thermus, reactions were negative for catalase-and cytochrome oxidase and hydrolysis of gelatin, starch or casein was negative [1]. Growth occurs over the temperature range of 50.0 -72.5°C (optimum 67.5°C), pH range 6.25 -7.75 (optimum pH 7.0), and at NaCl concentrations in the range 0.5 -4.5% (optimum 3%) [1]. The generation time under the above listed optimal condition and in medium MJYPV is about 30 minutes [1]. M. hydrothermalis T1 T differs from the members of the genera Oceanithermus by having a higher optimal temperature for growth and a higher oxygen tolerance [3]. Strain T1 T is able to utilize complex organic substrates such as Casamino acids, tryptone and yeast extract as sole energy and carbon sources [1].
Strain T1 T shares with its closest related genomesequenced neighbors, O. profundus [17], Meiothermus silvanus [18] and Thermus thermophilus [16] (Figure 1), the presence of two linked 5S-23S rRNA gene clusters, with two 16S rRNA genes located separately in the genomes, but has one surplus, third 16S rRNA gene copy.  [11]. Rooting was done initially using the midpoint method [12] 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 850 ML bootstrap replicates [13] (left) and from 1,000 maximum-parsimony bootstrap replicates [14] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [15] are labeled with an asterisk, those also listed as 'Complete and Published' with two asterisks [16][17][18][19].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [37], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [38]. The genome project is deposited in the Genomes OnLine Database [15] 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
M. hydrothermalis T1 T , DSM 14884, was grown in DSMZ medium 973 (Marinithermus hydrothermalis medium) [39] 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/DL for cell lysis as described in Wu et al. [38]. DNA is available through the DNA Bank Network [40].

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 [41]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 70 contigs in one scaffold was converted into a phrap [42] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (3,943.0 Mb) was assembled with Velvet [43] and the consensus sequences were shredded into 2.0 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 167.5 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 [42] 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 [41], Dupfinisher [44], 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 97 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 [45]. 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 1,666.5 × coverage of the genome. The final assembly contained 458,684 pyrosequence and 48,027,166 Illumina reads.

Genome annotation
Genes were identified using Prodigal [46] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [47]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant 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 [48].

Genome properties
The genome consists of a 2,269,167 bp long chromosome with a 68.1% GC content ( Figure 3 and Table 3). Of the 2,310 genes predicted, 2,251 were protein-coding genes, and 59 RNAs; 46 pseudogenes were also identified. The majority of the protein-coding genes (75.5%) 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.