Permanent draft genome of Thermithiobaclillus tepidarius DSM 3134T, a moderately thermophilic, obligately chemolithoautotrophic member of the Acidithiobacillia

Thermithiobacillus tepidarius DSM 3134T was originally isolated (1983) from the waters of a sulfidic spring entering the Roman Baths (Temple of Sulis-Minerva) at Bath, United Kingdom and is an obligate chemolithoautotroph growing at the expense of reduced sulfur species. This strain has a genome size of 2,958,498 bp. Here we report the genome sequence, annotation and characteristics. The genome comprises 2,902 protein coding and 66 RNA coding genes. Genes responsible for the transaldolase variant of the Calvin-Benson-Bassham cycle were identified along with a biosynthetic horseshoe in lieu of Krebs’ cycle sensu stricto. Terminal oxidases were identified, viz. cytochrome c oxidase (cbb3, EC 1.9.3.1) and ubiquinol oxidase (bd, EC 1.10.3.10). Metalloresistance genes involved in pathways of arsenic and cadmium resistance were found. Evidence of horizontal gene transfer accounting for 5.9 % of the protein-coding genes was found, including transfer from Thiobacillus spp. and Methylococcus capsulatus Bath, isolated from the same spring. A sox gene cluster was found, similar in structure to those from other Acidithiobacillia – by comparison with Thiobacillus thioparus and Paracoccus denitrificans, an additional gene between soxA and soxB was found, annotated as a DUF302-family protein of unknown function. As the Kelly-Friedrich pathway of thiosulfate oxidation (encoded by sox) is not used in Thermithiobacillus spp., the role of the operon (if any) in this species remains unknown. We speculate that DUF302 and sox genes may have a role in periplasmic trithionate oxidation.


Introduction
Thermithiobacillus tepidarius DSM 3134 T [1,2] is a moderately thermophilic sulfur-oxidising obligately chemolithoautotrophic member of the Acidithiobacillia originally published as 'Thiobacillus tepidarius' and assigned to the Betaproteobacteriathis has since been resolved by proteogenomic studies and the species moved firstly to a new genus -Thermithiobacillus - [3] in the Gammaproteobacteria and later to a separate Class, along the the genus Acidithiobacillus [4]. To date it is the only species of the genus with a validly published name and one of only two strains in cultivation [5]. The obligately aerobic chemolithoautotroph was demonstrated [1,2] to use sulfur oxyanions as sole energy sources. It has a temperature optimum of 44°C, reflecting its environment of isolation. Chemostat-based studies have demonstrated unusually high specific growth yields compared to other chemolithoautotrophs and biochemical studies have demonstrated the presence of a range of sulfur oxidation enzymes including rhodanese (EC 2.8.1.1), trithionate hydrolase (EC 3.12.1.1), thiosulfate dehydrogenase (EC 1.8.2.2), a tetrathionate-proton symport system [6] and at least 3 of each cytochromes c and b [7]. Proton translocations per mole of energy source were significantly higher than in other sulfur-oxidising autotrophs, potentially explaining the high yields. T. tepidarius DSM 3134 T was selected for genome sequencing as part of the Department of the Environment DOE-CSP 2012 initiativeas a type species of a genus.

Classification and features
This strain was isolated from sulfidic groundwater flowing into a Roman bathhouse (Temple of Sulis-Minerva, now The Roman Baths, Bath, UK)the only other strain of this genus held in a culture collection (Thermithiobacillus sp. NCIMB 8349) came from decomposing concrete in the Melbourne sewers in the 1940s [5]. The authors have detected at least 6 OTUs representing probably other Thermithiobacillus spp. in 16S rRNA gene libraries from the Roman Baths and have isolated a number of strains to date, indicating that Thermithiobacillus spp. are no more difficult to isolate than other sulfur-oxidising autotrophs and may thus simply be rare or confined to rare ecosystems. It forms white colonies of 2-5 mm diameter in 48 h that smell faintly of elementary sulfur if grown on thiosulfate-containing basal salts agar. In batch cultures, thiosulfate is oxidized stoichiometrically to tetrathionate early in the exponential phase, resulting in an increase in culture pH from pH 6.8 to pH 7.5-8.0a hallmark of the genusbefore being fully oxidized to sulfate, with concomitant fall in culture pH, usually ending at pH 5.2. In continuous cultures, no intermediates accumulate in the medium. In the authors' hands, trithionate has also been observed very early in the growth phase in batch culture, prior to tetrathionate production. Substrate-level phosphorylation appears not to participate in the energy conservation of this strain and all ATP is thus formed through oxidative phosphorylation [2]. The typeand onlystrain was isolated from an enrichment culture comprising water obtained from the inflow of the Great Bath (Roman Baths, Bath, UK) in 1983 (Ann P. Wood, personal communication) added to a basal salts medium supplemented with thiosulfate and monomethylamine hydrochloride, before plating onto basalt salts agar containing 5 mM thiosulfate as sole energy source and incubated under air enriched with 5 % (v/v) carbon dioxide as sole carbon source. Key features of this organism are summarized in Table 1. A phylogenetic tree based on the 16S rRNA gene sequence, showing the position of the organism with regard to the Acidithiobacillia, rooted with Thiobacillus thioparus, is given in Fig. 1.
Cells are 0.6 -1.0 by 0.2 to 0.4 μm and stain Gram negative. They are rapidly motile by means of a single polar flagellum up to 4 μm in length, as shown in Fig. 2. Ubiquinone-8 is the dominant respiratory quinone and cells fix carbon dioxide via the Calvin-Benson-Bassham cycle at the expense of inorganic sulfur oxidation. Cells accumulate polyphosphate ('volutin') granules when grown in batch culture but are typically free from storage granules when grown in energy-source-limited chemostats. Anaerobic growth is not observed with tetrathionate as the electron donor and nitrate, nitrite, nitrous oxide, elementary sulfur, sulfate, tetrathionate or pyruvate as terminal electron acceptors, but cultures can reduce nitrate to nitrite. Experimental estimations of G + C content of genomic DNA are 66.6 ± 0.5 mol% by buoyant density [1] or 65.9 ± 0.8 mol% by acid denaturation [9] in our hands. Dry biomass is 47 % (w/w) C regardless of the energy source used. T. tepidarius DSM 3134 T does not grow on any organic carbon compound tested, including sugars (glucose, ribose, fructose, sucrose), intermediates of Krebs cycle (citrate, succinate, fumarate, malate, oxaloacetate), carboxylates (glycolate, formate, acetate, propionate, pyruvate), C 1 compounds (monomethylamine, dimethylamine, trimethylamine, methanol, methane), structural amino acids (all 20), substituted thiophenes (thiophene-2-carboxylate, thiophene-3-carboxylate) or complex media (yeast extract, nutrient broth, brain-heart infusion, Columbia sheep blood agar, chocolate agar). Energy sources that support autotrophic growth are elementary sulfur, sulfide, trithionate, tetrathionate, hexathionate, heptathionate and thiosulfate. Fe(II), Mn(II), Cu(I), U(IV), pentathionate, dithionate, thiocyanate, sulfite, carbon disulfide, carbonyl sulfide, dimethylsulfide, dimethylsulfoxide, dimethylsulfone and formate do not support autotrophic growth as energy sources. The high growth yields and tetrathionateaccumulation in the early phases of growth make this strain a very interesting model organism for elucidation of sulfur oxidation pathways and their evolution.

Genome project history
This organism was selected for sequencing on the basis of its role in sulfur cycling, physiological, biochemical, evolutionary and biogeochemical importance, and is part of the Genomic Encyclopedia of Bacteria and Archaea, 1,000 Microbial Genomes project at the U.S. Department of Energy, Joint Genome Institute (JGI). The genome project is deposited in the Genomes OnLine Database [10] and a high-quality permanent draft genome sequence in IMG [11]. Sequencing, finishing and annotation were performed by the JGI using state of the art sequencing technology [12]. A summary of the project information is shown in Table 2.

Growth conditions and genomic DNA preparation
T. tepidarius DSM 3134 T DNA was obtained from Dr Hans-Peter Klenk at the Deutsche Sammlung von Mikroorganismen und Zellkulturen Gmbh (DSMZ) having been grown on basal salts medium pH 6.9, supplemented with 10 mM tetrathionate as the sole energy source (DSM Medium 333). DNA was extracted using the JETFLEX Genomic DNA Purification Kit from Genomed (Löhne, Germany) into TE Buffer.

Genome sequencing and assembly
The draft genome of Thermithiobacillus tepidarius DSM 3134 T was generated at the DOE Joint Genome Institute (JGI) using the Illumina technology [13]. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 13,370,056 reads totaling 2,005.5 Mbp. Library construction and sequencing were performed at the JGI -details are on their website [14]. All raw Illumina sequence data was passed through JGI's DUK filtering program, which removes known Illumina sequencing and library preparation artifacts (Mingkun L, Copeland A, Han J, Unpublished). Filtered Illumina reads were assembled using Velvet (version 1.1.04) [15]; 1-3 Kbp simulated paired end reads were created from Velvet contigs using wgsim [16] and Illumina reads were assembled with simulated read pairs using Allpaths-LG (version r42328) [17]. Parameters for assembly steps were: Velvet (velveth: 63 -shortPaired and velvetg: −very clean yes -exportFiltered yes -min contig lgth 500 -scaffolding nocov cutoff 10); wgsim (−e 0 -1 100 -2 100 -r 0 -R 0 -X 0); Evidence codes -IDA Inferred from direct assay, 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 the Gene Ontology project [28,29] Allpaths-LG (

Genome annotation
Genes were identified using Prodigal [18] as part of the DOE-JGI genome annotation pipeline [19]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information non-redundant database, UniProt, TIGR-Fam, Pfam, KEGG, COG, and InterPro database. These data sources were combined to assert a product description for each predicted protein. tRNAScanSE was used to find tRNA genes and rRNA genes were found using searches against models of the ribosomal RNA genes built from SIVLA [20,21]. Additional gene prediction analysis and functional annotation was performed within the IMG-ER platform [22,23]. For each gene discussed in this publication, the annotation was manually checked against the Gen-Bank® databased manual searches using the BLASTn and BLASTp algorithms -both of the gene from T. tepidarius and using the equivalent gene from members of the Acidithiobacillia or Escherichia coli.

Genome properties
The genome of T. tepidarius DSM 3134 T is 2,958,498 bplong with a 66.8 mol% G + C content (Table 3). Of the 2,968 predicted genes, 2,902 were protein-coding genes and 66 were RNA genes, including 2 rRNA operons. A total of 2,348 genes (79.1 %) were assigned a putative function. A total of 3.4 % were identified as pseudogenesthe remainder annotated as hypothetical proteins. The properties and the statistics of the genome are given in Table 3. The distribution of genes into COGs functional categories is presented in Table 4. The genome is one of the smaller genomes of those sequenced thus far from chemolithoautotrophic Proteobacteria (Table 5).   The total is based on the total number of protein coding genes in the genome

Extended insights
Two pairs of genes encoding ribulose-1,6-bisphosphate carboxylase (RuBisCO) could be identified, each comprising a large and small subunit gene. One pair is found close to cbbO and cbbQ genes, with no other cbb genes closebythis is consistent with Acidithiobacillus spp. and other obligate chemolithoautotrophs and indicates a Form IAq RuBisCO. The other pair is found close to cbb genes and in that sense is perhaps more similar to Form II RuBisCO [26]. Metalloresistance genes including those for arsenite efflux and arsenate reductase (arsB and arsC, respectively) were identified along with those implicated in tellurite, cadmium, cobalt, zinc, copper and silver resistance. Sulfur-oxidation genes are obviously of paramount interest in an obligate chemolithoautotroph, however, a number of proposed enzymes of sulfur metabolism have no genes identified thus far. It is known that the Acidithiobacillia [1, 2, 4-6] do not use the Kelly-Friedrich or "Sox" pathway of thiosulfate oxidation, and instead oxidise thiosulfate to tetrathionate via a poorly understood dehydrogenasemore than one form of which may exist. Some Kelly-Friedrich pathway genes are present in the genome and these are given in Fig. 3, showing comparison with those from other organisms that do not use the Kelly-Friedrich pathway versus one (Paracoccus denitrificans) that does. It can be seen from Fig. 3 that the non-Kelly-Friedrich organisms lack the soxC and soxD genes that are involved in a 6-electron capture during thiosulfate oxidation and all contain a gene encoding DUF302-family protein of unknown function 191 amino acids in length (G579DRAFT_01426 in T. tepidarius). Assuming these proteins are found in the periplasm of T. tepidarius as they are in Paracoccus spp., they could play a role in trithionate or higher polythionate oxidation (tetrathionate being oxidized solely in the cytoplasm [6]. The DUF302 protein of T. tepidarius would have a mass of 20.6 kDa based on the amino acyl sequence but contains a potential dimerization domain, so could be 41.2 kDa. It is worth noting that the periplasmic trithionate hydrolase (EC 3.12.1.1, gene unknown) of Acidiphilium acidophilum was 35 kDa [27].
One hundred seventy eight genes (5.9 % of genome) were flagged as potentially horizontally transferred from the species Thiobacillus thioparus, Thiobacillus denitrificans and Sulfuricella denitrificans in the Hydrogenophilaceae. This is particularly interesting since Thiobacillus aquaesulis DSM 4255 T (= ATCC 43788 T , no genome available) is closely related to these 3 species and was isolated originally from the Roman Baths and thus inhabits the exact same location [28]. A further 55 genes (1.9 %) were potentially transferred from Methylococcus capsulate, a strain of which (Bath = NCIMB 11132) was also isolated from the Roman Baths [25]. There is no clear pattern in the proteins encoded by the genes marked as potentially transferred.

Conclusions
The genome of T. tepidarius DSM 3134 T is the first for this genus and one of very few available for the Class Acidithiobacillia. The genome gives evidence and insight into the carbon dioxide fixation pathway, biosynthesis and sulfur oxidation as well as metal resistance and potential gene transfer from other species also isolated from the Roman Baths from which this organism was obtained. These data confirm that a transaldolase variant of the Calvin-Benson-Bassham cycle is used for carbon dioxide fixation. Sulfur oxidation genes of the sox operon are present but soxC and soxD are missing, though a DUF302-family protein was presentand also found across obligate chemolithoautotrophs in the Proteobacteria that use the Kelly-Trudinger (aka S 4 I pathway) of sulfur oxidation, rather than the Kelly-Friedrich (aka Sox) pathway. This genome sequence has already been utilized to propose the Class Acidithiobacillia [4] for Thermithiobacillus and Acidithiobacillus and to determine their  (Table 5), of these, T. tepidarius DSM 3134 T has one of the smaller genomes, presumably because it lacks the salt-tolerance systems of Halothiobacillus spp. or the iron-oxidation or acid-tolerance of Acidithiobacillus spp. This genome sequence will enable further evolutionary studies into the nature of the Acidithiobacillia and chemolithoautotrophs in general, along with ecological studies including organism-organism interactions in the environment owing to the evidence for horizontal gene transfer evident in this genome. Authors' contributions RB and LH analysed and mined the genome data in public databases for genes of interested and performed BLASTn/BLASTp searches to verify and validate the annotation etc and made comparisons of the sulfur oxidation operons with those in other organisms. RB constructed the phylogenetic tree. LH grew the organism and performed analyses thereof, and performed electron microscopy at the Electron Microscopy Centre, University of Plymouth. All other authors contributed to the sequencing, assembly and It is worth noting that P. denitrificans and T. thioparus do not grow on trithionate and that thiosulfate oxidation in Thermithiobacillus and Thiobacillus has been unequivocally shown not to proceed via the periplasmic Kelly-Friedrich oxidation pathway and instead occurs via tetrathionate as an intermediate, which is then oxidized to sulfate in the cytoplasm (the Kelly-Trudinger pathway [32,33]). The highly conserved soxXYZAB cluster occurs in all of the genomes examined and the DUF302 gene appears highly conserved in the Acidithiobacillia. The function is as-yet unknown, as is that of the sox genes in these Kelly-Trudinger pathway organisms. Analysis of conserved domains indicates that DUF302 may form a homodimer