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

An Erratum to this article was published on 11 October 2016


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 (cbb 3, EC and ubiquinol oxidase (bd, EC 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.


Thermithiobacillus tepidarius DSM 3134T [1, 2] is a moderately thermophilic sulfur-oxidising obligately chemolithoautotrophic member of the Acidithiobacillia originally published as ‘ Thiobacillus tepidarius ’ and assigned to the Betaproteobacteria – this 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, trithionate hydrolase (EC, thiosulfate dehydrogenase (EC, 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 3134T was selected for genome sequencing as part of the Department of the Environment DOE-CSP 2012 initiative – as a type species of a genus.

Organism information

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 7.5–8.0 – a hallmark of the genus – before 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 type – and only – strain 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.

Table 1 Classification and general features of Thermithiobacillus tepidarius DSM 3134T according to MIGS recommendations [8]
Fig. 1

Maximum-likelyhood phylogenetic tree based on CLUSTALW alignment of 16S rRNA gene sequences of the Acidithiobacillia. Type strains of each species of Acidithiobacillus are used, along with that of Thermithiobacillus (emboldened). Thermithiobacillus sp. NCIMB 8349 (the only other Thermithiobacillus sp. in culture) is given for the sake of completeness. Sequences pertaining to organisms for which a publically available genome sequence exists are underlined. Accession numbers for the GenBank database are in parentheses. Alignment and tree were constructed in MEGA 6 [30] using 1,509 positions and pairwise deletion. Tree was drawn using the Tamura-Nei model for maximum-likelyhood trees [31]. Values at nodes are based on 5,000 bootstrap replicates. Scale-bar indicates 2 substitutions per 100. Thiobacillus thioparus DSM 505T is used as the outgroup

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 3134T 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), C1 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 tetrathionate-accumulation in the early phases of growth make this strain a very interesting model organism for elucidation of sulfur oxidation pathways and their evolution.

Fig. 2

Transmission electron micrograph of T. tepidarius from a thiosulfate-limited chemostat (20 mM, 0.15 h-1). Cells were obtained from a chemostat-culture at steady-state by centrifugation and were washed and suspended in sterile 150 mM sodium chloride solution and applied to Formvar® and carbon coated copper grid before washing with further saline and staining in 50 mM uranyl acetate for 5 mins and washing again. Stained grids were visualized in a JEOL JEM-1400Plus transmission electron microscope, operating at 120 kV

Genome sequencing information

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.

Table 2 Project information

Growth conditions and genomic DNA preparation

T. tepidarius DSM 3134T 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 3134T 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 no –cov cutoff 10); wgsim (−e 0 –1 100 –2 100 –r 0 –R 0 –X 0); Allpaths–LG (PrepareAllpathsInputs: PHRED_64 = 1 PLOIDY = 1 FRAG_COVERAGE = 125 JUMP_COVERAGE = 25 LONG_JUMP_COV = 50, RunAllpathsLG: THREADS = 8 RUN = std_shredpairs TARGETS = standard VAPI_WARN_ONLY = True OVERWRITE = True). The final draft assembly contained 44 contigs in 43 scaffolds. The total size of the genome is 2.96 Mbp and the final assembly is based on 3,44.8 Mbp of Illumina data, which provides an average 116.4× coverage of the genome.

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 GenBank® 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 3134T is 2,958,498 bp-long 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 pseudogenes – the 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).

Table 3 Genome statistics
Table 4 Number of genes associated with general COG functional categories
Table 5 Genome properties of obligately chemolithoautotrophic members of the Proteobacteria

Insights from the genome sequence

As an obligate autotroph, it would be anticipated that genes encoding a complete Calvin-Benson-Bassham cycle and, in lieu of Krebs’ cycle, a biosynthetic horseshoe [24] would be present. A complete CBB cycle is present, and owing to the presence of a transaldolase (EC and absence of a sedoheptulose-1,7-bisphosphatase (EC gene, we can conclude that it is a transaldolase-variant CBB cycle [25]. Of Krebs’ cycle genes, citrate synthase (EC, aconitase (EC, isocitrate dehydrogenase (NADP+, EC, succinyl coenzyme A synthase (ADP-forming, EC and malate dehydrogenase (oxaloacetate decarboxylating, NADP+, EC were present. No fumarase or succinate dehydrogenase genes could be identified. The E1 subunit of α-ketoglutarate dehydrogenase was missing and the closest BLASTp match to the E2 subunit is annotated as a pyruvate dehydrogenase. These lesions are consistent with other obligate autotrophs and confirm the presence of a biosynthetic horseshoe in T. tepidarius [24].

In terms of respiration, 2 cytochrome c oxidases (cbb 3 EC and 2 ubiquinol oxidases (bd, EC could be identified, which is consistent with previous physiological studies [7]. Three cytochromes b 561 and three cytochromes c 553 were identified, along with other cytochromes c, again constant with previous studies [7].

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 closeby – this 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, 46] do not use the Kelly-Friedrich or “Sox” pathway of thiosulfate oxidation, and instead oxidise thiosulfate to tetrathionate via a poorly understood dehydrogenase – more 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, gene unknown) of Acidiphilium acidophilum was 35 kDa [27].

Fig. 3

Structure of the sox cluster in T. tepidarius and other chemolithoautotrophic Proteobacteria. A reference sox operon encoding the Kelly-Friedrich pathway of thiosulfate oxidation from Paracoccus denitrificans ATCC 17741T (Alphaproteobacteria) is given, showing soxXYZABCDEF genes and intergenic spacers, against gene clusters from T. tepidarius DSM 3134T, A. thiooxidans ATCC 19377T and A. caldus ATCC 51756T (the only sulfur-oxidising Acidithiobacillus spp.) of the Acidithiobacillia; Thiohalorhabdus denitrificans DSM 15699T (Gammaproteobacteria) and Thiobacillus thioparus DSM 505T (Betaproteobacteria). The DUF302-family hypothetical protein gene is indicated where present. 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

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 4255T (= ATCC 43788T , 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.


The genome of T. tepidarius DSM 3134T 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 present – and also found across obligate chemolithoautotrophs in the Proteobacteria that use the Kelly-Trudinger (aka S4I 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 evolutionary relationship with the Gammaproteobacteria . Thus far, the type species of each genus of the Acidithiobacillia is now sequenced, along with several other Acidithiobacillus spp. and other obligate chemolithoautotrophic Bacteria such as Thiobacillus spp. and Halothiobacillus spp. (Table 5), of these, T. tepidarius DSM 3134T 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.



1,000 microbial genomes


Tetrathionate intermediate pathway (aka Kelly-Trudinger pathway)


Sulfur oxidation pathway (aka Kelly-Friedrich pathway)


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We acknowledge Dr Hans-Peter Klenk at the DSMZ for the provision of genomic DNA for sequencing and thank Dr Ann P Wood (formerly of King’s College London, UK) for access to unpublished data pertaining to T. tepidarius DSM 3134T and its isolation.


The sequencing and annotation was performed under the auspices of the United States Department of Energy Joint Genome Institute (JGI), a DOE Office of Science User Facility and is supported by the Office of Science of the United States Department of Energy under Contract Number DE-AC02-05CH11231. The authors wish to acknowledge the School of Biological Sciences, University of Plymouth, United Kingdom for Ph.D studentship funding to LH and the Royal Society for a research grant (RG120444) to RB that supported the analysis of the 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 annotation of the genome sequence. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Correspondence to Rich Boden.

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An erratum to this article can be found at

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Boden, R., Hutt, L.P., Huntemann, M. et al. Permanent draft genome of Thermithiobacillus tepidarius DSM 3134T, a moderately thermophilic, obligately chemolithoautotrophic member of the Acidithiobacillia . Stand in Genomic Sci 11, 74 (2016).

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  • Thermithiobacillus tepidarius
  • Acidithiobacillia
  • Sulfur oxidation
  • Chemolithoautotroph
  • Thiosulfate
  • DUF302