Complete genome sequence of Thermaerobacter marianensis type strain (7p75aT)

Thermaerobacter marianensis Takai et al. 1999 is the type species of the genus Thermaerobacter, which belongs to the Clostridiales family Incertae Sedis XVII. The species is of special interest because T. marianensis is an aerobic, thermophilic marine bacterium, originally isolated from the deepest part in the western Pacific Ocean (Mariana Trench) at the depth of 10.897m. Interestingly, the taxonomic status of the genus has not been clarified until now. The genus Thermaerobacter may represent a very deep group within the Firmicutes or potentially a novel phylum. The 2,844,696 bp long genome with its 2,375 protein-coding and 60 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain 7p75a T (= DSM 12885 = ATCC 700841 = JCM 10246) is the type strain of T. marianensis which is the type species of the genus Thermaerobacter [1,2]. Currently, there are five species placed in the genus Thermaerobacter [1,3]. The generic name derives from the Greek words 'thermos' meaning 'hot', 'aeros'(air) and the Neo-Latin word 'bacter' meaning 'a rod', which altogether means able to grow at high temperatures in the presence of air [2]. The species epithet is derived from the Neo-Latin word 'marianensis' pertaining to the Mariana Trench, the location from which the strain was isolated from [2]. T. marianensis strain 7p75a T was isolated from a mud sample of the Challenger Deep in the Mariana Trench at the depth of 10,897 m [2]. No further isolates have been obtained for T. marianensis. Other members of the genus Thermaerobacter were isolated from mud of the bottom of the Challenger Deep [2], shallow marine hydrothermal vent, Japan [4], water sediment slurries of the runoff channel of New Lorne Bore, Australia [5], a coastal hydrothermal beach, Japan [6] and from food sludge compost, Japan [7]. Here we present a summary classification and a set of features for T. marianensis 7p75a T , together with the description of the complete genomic sequencing and annotation.

Classification and features
The 16S rRNA gene sequences of T. marianensis 7p75a T share 98.3 to 98.6% sequence identity with the other type strains of the genus Thermaerobacter [2,8] and T. nagasakiensis being the closest relative. Outside the genus members of the recently proposed genus "Calditerricola" (88.6%) [9] and the genus Moorella (88.1%) [10] share the highest degree of sequence similarity. The genomic survey sequence database (gss) contains as best hits several 16S rRNA gene sequence from The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific [11] at a similarity level of only 84%. No phylotypes from environmental samples database (env_nt) could be linked to the species T. marianensis or even the genus Thermaerobacter, indicating a rather rare occurrence of members of this genus in the habitats screened thus far (as of November 2010). A representative genomic 16S rRNA sequence of T. marianensis 7p75a T was compared using NCBI BLAST under default values (e.g., considering only the best 250 hits) with the most recent release of the Greengenes database [12] and the relative frequencies of taxa and keywords, weighted by BLAST scores, were determined. The five most frequent genera were Thermaerobacter (39.7%), Moorella (35.3%), Geobacillus (6.4%), Thermoactinomyces (3.6%) and Bacillus (3.4%). Regarding hits to sequences from other members of the genus, the average identity within HSPs (high-scoring segment pairs) was 98.1%, whereas the average coverage by HSPs was 96.3%. The species yielding the highest score was Thermaerobacter subterraneus. The five most frequent keywords within the labels of environmental samples which yielded hits were 'compost(ing)' (6.1%), 'municipal' (2.9%), 'scale' (2.7%) and 'process/stages' (2.6%). 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. marianensis 7p75a T in a 16S rRNA based tree. The sequences of the two 16S rRNA gene copies differ from each other by two nucleotides, and differ by up to two nucleotides from the previously published 16S rRNA sequence (AB011495), which contains one ambiguous base call.
The cells of T. marianensis are generally rod-shaped (0.3-0.6 × 2-7 µm), straight to slightly curved with rounded ends (Figure 2). The cells can be arranged in pairs [2]. T. marianensis is a Gram-positive, spore-forming bacterium (Table 1). At stationary phase cells, may stain Gram-negative. Motility and flagella have not been observed [2], but genes for biosynthesis and assembly of flagella have been identified in the here reported genome sequence. The organism is a strictly aerobic chemoheterotroph. T. marianensis is a typical marine bacterium and requires sea salts (0.5-5%, optimum 2%) in media for good growth [2]. The temperature range for growth is between 50°C and 80°C, with an optimum at 75°C [2]. The pH range for growth is 5.4-9.5, with an optimum at pH 7.0-7.5 [2]. T. marianensis is able to grow on yeast extract, peptone and casein. It utilizes carbohydrates like starch, xylan, chitin, maltose, maltotriose, cellobiose, lactose, trehalose, sucrose, glucose, galactose, xylose, mannitol, inositol. The strain is also able to grow on amino acids like casamino acids, valine, isoleucine, cysteine, proline, serine, threonine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine. T. marianensis is able to grow well on various carboxylic acids like propionate, 2aminobutyric acid, malate, pyruvate, tartarate, succinate, lactate, acetate and glycerol [2].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [32], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [33]. The genome project is deposited in the Genome OnLine Database [17,34] 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. Phylogenetic tree highlighting the position of T. marianensis 7p75a T relative to the other type strains within the family. The tree was inferred from 1,489 aligned characters [13,14] of the 16S rRNA gene sequence under the maximum likelihood criterion [15] and rooted in accordance with the current taxonomy. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates [16] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [17] are shown in blue, published genomes in bold.   Phylum Firmicutes TAS [20][21][22] Class Clostridia TAS [20,23,24] Order Clostridiales TAS [20,25,26] Family Incertae sedis XVII TAS [20] Genus Thermaerobacter TAS [2,5,27] Species , 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 [29]. 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 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. 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 mis-assemblies were corrected with ga-pResolution [42], Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [37]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 132 additional reactions and 4 shatter libraries 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 [38]. 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 431.8 × coverage of the genome. Final assembly contained 689,185 pyrosequence and 26,930,845 Illumina reads.

Genome annotation
Genes were identified using Prodigal [39] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [40]. 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) platform [41].

Genome properties
The genome consists of a 2,844,696 bp long chromosome with a GC content of 72.5% (Table 3 and Figure 3). Of the 2,435 genes predicted, 2,375 were protein-coding genes, and 60 RNAs; 48 pseudogenes were also identified. The majority of the protein-coding genes (74.1%) 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.