Skip to main content

Permanent draft genome of Thiobacillus thioparus DSM 505T, an obligately chemolithoautotrophic member of the Betaproteobacteria


Thiobacillus thioparus DSM 505T is one of first two isolated strains of inorganic sulfur-oxidising Bacteria. The original strain of T. thioparus was lost almost 100 years ago and the working type strain is Culture CT (=DSM 505T = ATCC 8158T) isolated by Starkey in 1934 from agricultural soil at Rutgers University, New Jersey, USA. It is an obligate chemolithoautotroph that conserves energy from the oxidation of reduced inorganic sulfur compounds using the Kelly-Trudinger pathway and uses it to fix carbon dioxide It is not capable of heterotrophic or mixotrophic growth. The strain has a genome size of 3,201,518 bp. Here we report the genome sequence, annotation and characteristics. The genome contains 3,135 protein coding and 62 RNA coding genes. Genes encoding the transaldolase variant of the Calvin-Benson-Bassham cycle were also identified and an operon encoding carboxysomes, along with Smith’s biosynthetic horseshoe in lieu of Krebs’ cycle sensu stricto. Terminal oxidases were identified, viz. cytochrome c oxidase (cbb3, EC and ubiquinol oxidase (bd, EC There is a partial sox operon of the Kelly-Friedrich pathway of inorganic sulfur-oxidation that contains soxXYZAB genes but lacking soxCDEF, there is also a lack of the DUF302 gene previously noted in the sox operon of other members of the ‘Proteobacteria’ that can use trithionate as an energy source. In spite of apparently not growing anaerobically with denitrification, the nar, nir, nor and nos operons encoding enzymes of denitrification are found in the T. thioparus genome, in the same arrangements as in the true denitrifier T. denitrificans.


In 1902, Thiobacillus thioparus was one of the first two obligately chemolithoautrophic sulfur-oxidizing Bacteria to be isolated, (along with what is now Halothiobacillus neapolitanus ), and was named in 1904 [1, 2]. The original isolates were lost, but Thiobacillus thioparus is now the type species of the genus (a member of the Betaproteobacteria ) and the type strain is an isolate from Starkey (1934) (= Culture CT = DSM 505 T = ATCC 8158 T = CIP 104484 T = JCM 3859 T = NBRC 103402 T) [3, 4]. Originally, the characteristic of utilizing inorganic sulfur compounds as an energy source was thought to be a taxonomic trait unique to Thiobacillus and at its height the genus contained in at least 32 different ‘species’ [4]. With phylogenetic methods however, many of these strains have since been reassigned to different, often new genera with T. thioparus DSM 505 T being one of four species with validly published names left in the genus. Compared to other inorganic sulfur-oxidisers, surprisingly little research has been conducted on T. thioparus DSM 505 T in terms of physiology and biochemistry or genetics, possibly due to the low growth yields of this species when compared to T. denitrificans and T. aquaesulis [5, 6] making it more challenging to study. It may, however, give extended and contrasted insights into autotrophic sulfur-oxidation in the Bacteria. It was selected for genome sequencing as part of the Department of Energy DOE-CSP 2012 initiative – as type species of a genus.

Organism information

Classification and features

Thiobacillus thioparus DSM 505 T was isolated from sandy loam soil from the New Jersey Agricultural Experimental Station planted with unspecified vegetable crops using 20mM thiosulfate as sole energy source in basal medium at pH 8.5 by Starkey (1934) [3] and is currently one of four species with validly published names within the genus. It forms small white colonies of 1–3 mm diameter after 2–3 days that turn pink or brown with age and which become coated with yellow-ish elementary sulfur. When grown in liquid media, finely divided (white) elementary sulfur is formed during early stages of growth, particularly when thiosulfate is used as the energy source. This disappears when growth approaches stationary phase. Thiosulfate is oxidized stoichiometrically to tetrathionate after 24 h accompanied by a rise in pH, characteristic of the Kelly-Trudinger pathway. Tetrathionate is subsequently oxidized to sulfate with culture pH falling to pH 4.8 by stationary phase. During continuous culture, no intermediates are detected in the medium once steady-state has been established. If the dilution rate of a thiosulfate limited chemostat is increased, a large production of elementary sulfur is observed, which disappears as a new steady-state is established at the faster dilution rate. General features of T. thioparus DSM 505 T are summarized in Table 1. A phylogenetic tree based on the 16S rRNA gene sequence showing this organisms position within the Betaproteobacteria and rooted with Thermithiobacillus tepidarius is given in Fig. 1.

Table 1 Classification and general features of Thiobacillus thioparus DSM 505T according to MIGS recommendations [43]
Fig. 1

Maximum-likelyhood phylogenetic tree based on MUSCLE alignment of 16S rRNA gene sequences of the genus Thiobacillus and the closely related members of the Betaproteobacteria. Type strains of each species are used and only species with validly published names are shown. 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 [53]. Tree was drawn using the Tamura-Nei model for maximum-likelyhood trees [54]. Values at nodes are based on 5,000 bootstrap replicates, with values <70% omitted. Scale-bar indicates 2 substitutions per 100. Thermithiobacillus tepidarius DSM 3134T from the Acidithiobacillia is used as the outgroup

Cells are 1.5–2.0 by 0.6 to 0.8 μm and stain Gram negative. They are motile by means of a single polar flagellum between 6 and 10 μm in length, as shown in Fig. 2. The dominant respiratory quinone is ubiquione-8 [710] and fix carbon dioxide using the Calvin-Benson-Bassham cycle at the expense of inorganic sulfur oxidation. Cells accumulate polyphosphate (‘volutin’) granules in batch culture and to a lesser extent in an energy-limited chemostat [8]. Carboxysomes are present in cells regardless of growth conditions employed (Fig. 1), but are apparently not found in T. denitrificans cells, at least not under growth conditions employed in previous studies [9]. T. thioparus can only use oxygen as a terminal electron acceptor – nitrate, nitrite, thiosulfate, sulfate, elementary sulfur and ferric iron do not support growth as terminal electron acceptors. The genomic DNA G + C content has been estimated using the thermal denaturation [11] at 61–62 mol% [7, 10]. T. thioparus DSM 505 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), one-carbon 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 or horse blood agar, chocolate agar). Energy sources that support autotrophic growth of DSM 505 T include thiosulfate, trithionate, tetrathionate, pentathionate, hexathionate, thiocyanate and dithionate. Some bone fide strains of T. thioparus (Tk-m and E6) grow autotrophically on carbon disulfide, dimethylsulfide, dimethyldisulfide and Admidate (O,O-dimethylphosphoramidothioate) [4], but it is not known if the type strain DSM 505 T is capable of this. Autotrophic growth is not supported by Fe(II), Mn(II), Cu(I), U(IV), sulfite, dimethylsulfoxide, dimethylsulfone, pyrite or formate. During batch growth on thiosulfate the intermediate production of tetrathionate is observed during early stages of growth, indicative of the Kelly-Trudinger pathway [12]. Compared to the other two members of the genus [5, 6], T. thioparus DSM 505 T has relatively low growth yields on thiosulfate (Hutt and Boden, manuscript in preparation) which may give insight into the physiological variances of Kelly-Trudinger pathway organisms even within one genus.

Fig. 2

Transmission electron micrographs of T. thioparus DSM 505T cells obtained from a thiosulfate-limited chemostat (20 mM, D = 0.07 h−1) visualized in a JEOL JEM-1400Plus transmission electron microscope, operating at 120 kV. a Negatively stained cells. Cells were applied to Formvar® and carbon coated copper grid before washing with saline and staining in 50 mM uranyl acetate for 5 mins and washing again. b Sectioned cells showing the presence of an electron dense polyphosphate (‘volutin’) granule and numerous polyhedral carboxysomes that are paler in comparison

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 GEBA-KMG project at the U.S. Department of Energy JGI. The genome project is deposited in the Genomes OnLine Database [13] and a high-quality permanent draft genome sequence in IMG (the annotated genome is publically available in IMG under Genome ID 2515154076) [14]. Sequencing, finishing and annotation were performed by the JGI using state of the art sequencing technology [15]. A summary of the project information is given in Table 2.

Table 2 Project information

Growth conditions and genomic DNA preparation

T. thioparus DSM 505 T DNA was obtained from Dr Hans-Peter Klenk at the DSMZ, having been grown on basal salts medium pH 6.6, supplemented with 40 mM thiosulfate as the sole energy source (DSM Medium 36), under air at 26 °C for 72 h. DNA was extracted using the JETFLEX Genomic DNA Purification Kit from Genomed (Löhne, Germany) into TE Buffer. Quality was checked by agarose gel electrophoresis.

Genome sequencing and assembly

The draft genome of Thiobacillus thioparus DSM 505 T was generated at the DOE JGI using the Illumina technology [16]. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 11,161,382 reads totalling 1,674.2 Mbp. All general aspects of library construction and sequencing performed at the JGI can be found at All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts (Mingkun L, Copeland A, Han J, Unpublished). Following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet (version 1.1.04) (Mingkun, L., Unpublished), (2) 1–3 Kbp simulated paired end reads were created from Velvet contigs using wgsim [17], (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG (version r41043) [18]. Parameters for assembly steps were: 1) Velvet (velveth: 63 –shortPaired and velvetg: −very clean yes –export-Filtered yes –min contig lgth 500 –scaffolding no –cov cutoff 10) 2) wgsim (−e 0–1 100–2 100 –r 0 –R 0 –X 0) 3) 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 = OVERWRITE = True). The final draft assembly contained 20 contigs in 20 scaffolds. The total size of the genome is 3.2 Mbp and the final assembly is based on 392.6 Mbp of Illumina data, which provides an average 122.7× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [19], followed by a round of manual curation using GenePRIMP [20] for finished genomes and Draft genomes in fewer than 10 scaffolds. The predicted CDSs were translated and used to search the NCBI nonredundant database, UniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. The tRNAScanSE tool [21] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [22]. Other non–coding RNAs such as the RNA components of the protein secretion complex and the RNase P were identified by searching the genome for the corresponding Rfam profiles using INFERNAL [23]. Additional gene prediction analysis and manual functional annotation was performed within the IMG platform [24] developed by the JGI, Walnut Creek, CA, USA [25, 26].

Genome properties

The genome of T. thioparus DSM 505 T is 3,201,518 bp-long with a 62.3 mol% G + C content (Table 3). Of the 3,197 predicted genes, 3,135 were protein-coding genes and 62 were RNA genes. A total of 2,597 genes (81.23%) have predicted function. A total of 538 (16.83%) 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 COG functional categories is given in Table 4. The genome is the second largest genome of obligate chemolithoautotrophs sequenced to date and is 89% (bp/bp) or 90% (protein coding genes/protein coding genes) of the size of that of T. denitrificans DSM 12475 T [12].

Table 3 Genome statistics
Table 4 Number of genes associated with general COG functional categories

Insights from the genome sequence

As an obligate autotroph, it would be expected for a complete Calvin-Benson-Bassham cycle and, in lieu of Krebs’ cycle, Smith’s biosynthetic horseshoe [12, 2729]. Smith’s horseshoe representing a very near-complete Krebs’ cycle was identified, in which citrate synthase (EC, aconitase (EC, isocitrate dehydrogenase (NADP+, EC, succinyl coenzyme A synthase (ADP-forming, EC, succinate dehydrogenase (EC and malate dehydrogenase (oxaloacetate decarboxylating, NADP+, EC genes are present, but fumarase (EC and the E3 subunit of α-ketoglutarate dehydrogenase (NAD+, EC genes are absent – E1 and E2 are present. This is a comparatively large version of Smith’s horseshoe [29], which varies between Classes of the ‘ Proteobacteria ’, for example, genes for fumarase, succinate dehydrogenase and the E1 subunit of α-ketoglutarate dehydrogenase were recently found to be missing from Thermithiobacillus tepidarius DSM 3134 T [12, 30, 31] of the Acidithiobacillia . Activities of Krebs’ cycle enzymes assayed by Smith et al. [29] in cell-free extracts of T. thioparus found activities of all enzymes except for α-ketoglutarate dehydrogenase, indicating the E1 and E2 subunits alone were not sufficient for activity. Interestingly, Smith detected fumarase activity; however, this strain of T. thioparus was isolated by the authors themselves and was not DSM 505 T, so there may be further variation of Smith’s horseshoe at strain level. With many species having been erroneously classified as strains of T. thioparus in the past that have subsequently been proven to belong to other genera [4] it is also plausible that Smith’s strain was from another specie, genus or even Class. The E1, E2 and E3 subunit genes for α-ketoglutarate dehydrogenase were identified in the genome of T. denitrificans ATCC 25259 T [32], though enzyme activity was also absent in this strain [29, 33]. It is important to stress that the full suite of enzymes of Krebs’ cycle or Smith’s horseshoe have never been assayed in T. thioparus DSM 505 T - clearly this is needed in order to identify if genomic data reflect true activities in vivo [27].

A complete Calvin-Benson-Bassham cycle is present, with a single copy of the large (cbbL) and small (cbbS) form I RuBisCO (EC subunits and, owing to the presence of a transaldolase (EC and absence of a sedoheptulose-1,7-bisphosphatase (EC gene, we can conclude that it uses the transaldolase-variant Calvin-Benson-Bassham cycle [34]. Adjacent to these genes are cbbO and cbbQ, consistent with form IAq RuBisCO [35], which is canonically cytoplasmic, rather than carboxysomal. A cluster of 11 genes that encode carboxysome shell proteins and a carboxysome carbonic anhydrase were also identified. This cluster is located on the forward strand while on the reverse strand the RuBisCO cluster is located c.9 kb upstream, between which is a divergently transcribed transcriptional regulator (cbbR) [32]. Further evidence of carboxysome expression can be seen in transmission electron micrographs (Fig. 1). The carboxysome gene cluster (experimental data would be required to demonstrate if it is an operon or not) in T. thioparus DSM 505 T does not start with cbbLS, (as would be found in Halothiobacillus , Acidithiobacillus and Thermithiobacillus spp. [36]) - the lack of these genes has also been observed in T. denitrificans ATCC 25259 T [32] and may be a diagnostic property of this genus.

Whilst T. thioparus DSM 505 T cannot grow anaerobically with denitrification but T. denitrificans does, putative operons encoding the nitrate reductase (nar), nitrite reductase (nir), nitric oxide reductase (nor) and nitrous oxide reductase (nos) proteins of canonical denitrification are found in both T. denitrificans and T. thioparus – this may indicate a potential for the latter to grow with denitrification albeit not under conditions previously employed – for example, it may occur only under micro-oxic conditions rather than fully anoxic conditions, alternatively, some unknown factor may prevent detection of nitrate and/or the expression of these genes. No evidence for any alternative denitrification pathways [37] was found

With regard to respiration, five cytochromes c 553, one cytochrome c 556 and three cytochrome b were present. The two high-affinity terminal oxidases were both found – with multiple gene copies of cytochrome c oxidase (cbb 3, EC and a single copy of cytochrome bd-type quinol oxidase (EC It is worth noting that T. denitrificans also possess the aa 3 variant of cytochrome c oxidase (EC, which may permit it greater metabolic diversity, for example, growth under more variable oxygen partial pressures - this could potentially explain why T. thioparus cannot denitrify under anoxic conditions, even though it possesses the operons encoding enzymes of denitrification. It is known that microaerophilic organisms usually employ cbb 3 or bd-type high-affinity terminal oxidases [37] and this may further evidence that T. thioparus and T. denitrificans may grow under such conditions, potentially with denitrification, though we cannot find any studies that demonstrate this in vivo thus far.

Extended insights

Enzymes of the Kelly-Trudinger pathway remain poorly understood and many of the genes are yet to be identified [12]. The oxidation of thiosulfate to tetrathionate takes place via a cytochrome c-linked thiosulfate dehydrogenase (EC, one gene (tsdA) for which was identified in Allochromatium vinosum , a member of the Gammaproteobacteria [38]. However, tsdA is not present in T. thioparus DSM 505 T supporting the hypothesis that more than one thiosulfate dehydrogenase is present in the ‘ Proteobacteria ’ or could vary at Class level. Numerous Kelly-Friedrich pathway genes (soxXYZAB) were present but the remaining of the conserved soxTRS-VW-XYZABCDEFGH genes being absent [3941]. In Paracoccus spp. ( Alphaproteobacteria ) soxYZ encodes a protein complex that binds thiosulfate via a cysteine residue in the initial stage of the Kelly-Friedrich pathway while soxXA encode cytochromes c 551 and c 552.5 which capture two electrons from thiosulfate oxidation. Finally soxB encodes a hydrolase that removes the terminal sulfone group as sulfate. Missing the SoxCD or sulfur dehydrogenase protein from the multi-enzyme system would leave a sulfur atom attached to the SoxYZ residue, preventing the action of SoxB to liberate this residue to act further with another thiosulfate molecule. An unidentified protein may participate in the release of these sulfur atoms and potentially may explain the deposits of sulfur seen during initial stages of growth. This does not explain the production of tetrathionate which still would require the activity of thiosulfate dehydrogenase. If soxXYZAB genes are being expressed then they may be required as a functional part of the Kelly-Trudinger pathway. The action of Sox proteins (if any) in T. thioparus DSM 505 T in conjunction and potentially collaboration with additional Kelly-Trudinger pathway proteins would undoubtedly be essential in resolving its chemolithoautotrophic metabolism, which remains poorly understood. An unidentified gene encoding a putative DUF302-family protein is present between soxA and soxB genes of Thermithiobacillus tepidarius DSM 3134 T, Acidithiobacillus thiooxidans ATCC 19377 T and Acidithiobacillus caldus ATCC 51756 T, and Thiohalorhabdus denitrificans DSM 15699 T, the function of which may be important in the Kelly-Trudinger pathway [12]; however, DUF302 is not present on the sox operon of T. thioparus DSM 505 T, although six unidentified genes annotated as DUF302 family proteins are present elsewhere in the genome. The soxEF genes encode and a flavocytochrome c sulfide dehydrogenase (EC and are both found in the T. denitrificans genome, separate from the main sox gene cluster, but they are not found in T. thioparus , whereas dissimilatory sulfite reductase (dsr) genes are found in both genomes, as are adenylyl sulfate reductase (aprAB, EC genes, the presence of which has also been confirmed in T. aquaesulis [42].

It was previously noted that 5.9% of the genome (178 genes) of Thermithiobacillus tepidarius DSM 3134 T were potential horizontally transferred genes from Thiobacillus thioparus , Thiobacillus denitrificans and Sulfuricella denitrificans of the Betaproteobacteria [12]; 96 of these genes came from the two Thiobacillus spp. However, very little gene transfer has taken place from members of Acidithiobacillia to T. thioparus DSM 505 T with only 6 genes from Ttb. tepidarius DSM 3134 T, 4 from Acidithiobacillus spp. This is perhaps not surprising due to the thermophilic and acidophilic nature of these three Acidithiobacillia compared to the mesophilic requirements of T. thioparus and the unlikelihood of these species co-inhabiting the same environments. A far larger portion (143 genes; 4.82%) of genes were attributed to transfer from members of Gammaproteobacteria , many of which grow at more neutral pH and mesophilic temperatures. There was no distinct pattern of any particular metabolism pathways or resistances etc. being encoded by these potentially transferred genes.


The genome of Thiobacillus thioparus DSM 505 T gives insights into many aspects of its physiology, biochemistry and evolution. This organism uses the transaldolase variant of the Calvin-Benson-Bassham cycle and produces carboxysomes for carbon dioxide fixation, evident from both the genome and transmission electron microscopy. The expression of both carboxysomes and RuBisCO may be regulated by the same divergently transcribed transcriptional regulator. Smith’s biosynthetic horseshoe is present in lieu of Krebs’ cycle sensu stricto, but this is unusually large as only 2 genes are missing, though T. denitrificans [32] is only missing 1 – this may in part explain the heterotrophic growth of T. aquaesulis since just one additional gene would convert Smith’s horseshoe into a functional version of Krebs’ cycle [6]. Many inorganic sulfur-oxidation genes of the sox cluster were found but soxC, soxD, soxE and soxF are absent. The tsdA gene for a thiosulfate dehydrogenase identified in Allochromatium vinosum is absent and confirmation of the presence of a different thiosulfate dehydrogenase enzyme and gene will require further study. The genome sequence will enable evolutionary studies into the nature of Thiobacillus and chemolithoautotrophs in general, in particular reigniting the obligate versus facultative and the autotrophic versus mixotrophic debates that have been largely absent from the literature in recent years, but genome sequences becoming available will now answer many questions proposed over 10 years ago [27, 29].



American type culture collection


Basic linear alignment search tool


Collection de L’institut Pasteur


Clusters of orthologous groups


Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH


Genomic encyclopedia of Bacteria and Archaea


Integrated microbial genomes database


Japan collection of microorganisms


Joint Genome Institute


1,000 microbial genomes project


NITE biological resource center


National center for biotechnology information


Ribulose-1,5-bisphosphate carboxylase/oxygenase


  1. 1.

    Nathanson A. Über eine neue Gruppe von Schwefelbakterien und ihren Stoffwechsel. Mitt zool Stn Neapel. 1902;15:655–80.

    Google Scholar 

  2. 2.

    Beijerinck MW. Ueber die Bakterien, welche sich im Dunkeln mit Kohlensäure als Kohlenstoffquelle ernähren können. Centralbl Bakteriol Parasitenkd Infektionskr Hyg Abt II. 1904;11:592–599.

  3. 3.

    Starkey RL. Isolation of some bacteria which oxidise thiosulfate. Soil Sci. 1934;39:197–219.

    Article  Google Scholar 

  4. 4.

    Boden R, Cleland D, Green PN, Katayama Y, Uchino Y, Murrell JC, Kelly DP. Phylogenetic assessment of culture collection strains of Thiobacillus thioparus, and definitive 16S rRNA gene sequences for T. thioparus, T. denitrificans, and Halothiobacillus neapolitanus. Arch Microbiol. 2011;194:187–95.

    Article  PubMed  Google Scholar 

  5. 5.

    Justin P, Kelly DP. Growth kinetics of Thiobacillus denitrificans in anaerobic and aerobic chemostat culture. J Gen Microbiol. 1978;107:123–30.

    CAS  Article  Google Scholar 

  6. 6.

    Wood AP, Kelly DP. Isolation and physiological characterisation of Thiobacillus aquaesulis sp. nov., a novel facultatively autotrophic moderate thermophile. Arch Microbiol. 1988;149:339–43.

    CAS  Article  Google Scholar 

  7. 7.

    Katayama-Fujimura Y, Tsuzaki N, Kuraishi H. Ubiquinone, fatty acid and DNA base composition determination as a guild to the taxonomy of the genus Thiobacillus. J Gen Microbiol. 1982;128:1599–611.

    CAS  Google Scholar 

  8. 8.

    Katayama-Fujimura Y, Enokizono Y, Kaneko T, Kuraishi H. Deoxyribonucleic acid homologies among species of the genus Thiobacillus. J Gen Appl Microbiol. 1983;29:287–95.

    CAS  Article  Google Scholar 

  9. 9.

    Katayama-Fujimura Y, Tsuzaki N, Hirata A, Kuraishi H. Polyhedral inclusion bodies (carboxysomes) in Thiobacillus species with reference to the taxonomy of the Genus Thiobacillus. J Gen Appl Microbiol. 1984;30:211–22.

    CAS  Article  Google Scholar 

  10. 10.

    Kelly DP, Wood AP. Confirmation of Thiobacillus denitrificans as a species of the genus Thiobacillus, in the β-subclass of the Proteobacteria, with strain NCIMB 9548 as the type strain. Int J Syst Evol Microbiol. 2000;50:547–50.

    Article  PubMed  Google Scholar 

  11. 11.

    Marmur J, Doty P. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol. 1962;5:109–18.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Boden R, Hutt LP, Huntemann M, Clum A, Pillay M, Palaniappan K, Varghese N, Mikhailova N, Stamatis D, Reddy T, Ngan CY, Daum C, Shapiro N, Markowitz V, Ivanova N, Woyke T, Kyrpides N. Permanent draft genome of Thermithiobaclillus tepidarius DSM 3134T, a moderately thermophilic, obligately chemolithoautotrophic member of the Acidithiobacillia. Standards in Genomic Sciences. 2016; 11. DOI: 10.1186/s40793-016-0188-0

  13. 13.

    Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, et al. The Genomes OnLine Database (GOLD) v. 4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2012;40:D571–9.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Markowitz VM, Chen I-MA, Palaniappan K, Chu K, Szeto E, Pillay M, et al. IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res. 2014;42:D560–7.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Mavromatis K, Land ML, Brettin TS, Quest DJ, Copeland A, Clum A, et al. The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation. PLoS One. 2012;7:e48837.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Bennett S. Solexa Ltd. Pharmacogenomics. 2004;5(4):433–8.

    Article  PubMed  Google Scholar 

  17. 17.

    Zerbino D, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Reads simulator wgsim. [].

  19. 19.

    Gnerre S, MacCallum I. High–quality draft assemblies of mammalian genomes from massively parallel sequence data. PNAS. 2011;108(4):1513–8.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Hyatt D, Chen GL, Lacascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11:119.

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010;7:455–7.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Lowe TM, Eddy SR. tRNAscan–SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25:955–64.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Pruesse E, Quast C, Fuchs BM, Knittel K, Ludwig W, Peplies J, Glckner FO. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nuc Acids Res. 2007;35:2188–7196.

    Article  Google Scholar 

  24. 24.

    INFERNAL. Inference of RNA alignments. [].

  25. 25.

    The Integrated Microbial Genomes (IMG) platform. [].

  26. 26.

    Markowitz VM, Mavromatis K, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009;25:2271–8.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Wood AP, Aurikko JP, Kelly DP. A challenge for 21st century molecular biology and biochemistry: what are the causes of obligate autotrophy and methanotrophy. FEMS Microbiol Rev. 2004;28:335–52.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Peeters TL, Liu MS, Aleem MIH. The tricarboxylic acid cycle in Thiobacillus denitrificans and Thiobacillus-A2. J Gen Microbiol. 1970;64:29–35.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Smith AJ, London J, Stanier RY. Biochemical basis of obligate autotrophy in blue- green algae and thiobacilli. J Bacteriol. 1967;94:972–83.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kelly DP, Wood AP. Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov. Int J Syst Evol Microbiol. 2000;50:511–6.

    Article  PubMed  Google Scholar 

  31. 31.

    Hudson C, Williams K, Kelly DP. Definitive assignment by multigenome analysis of the Gammaproteobacterial Genus Thermithiobacillus to the Class Acidithiobacillia. Pol J Microbiol. 2014;63:245–7.

    PubMed  Google Scholar 

  32. 32.

    Beller HR, Chain PSG, Letain TE, Chakicherla A, Larimer FW, Richardson PM, Coleman MA, Wood AP, Kelly DP. The genome sequence of the obligately chemolithoautotrophic, facultatively anaerobic bacterium Thiobacillus denitrificans. J Bacteriol. 2006;188:1473–88.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Taylor BF, Hoare DS. Thiobacillus denitrificans as an obligate chemolithoautotroph. Cell suspension and enzymic studies. Arch Mikrobiol. 1971;80:262–76.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Anthony C. The biochemistry of methylotrophs. 1st ed. London: Academic Press; 1982.

    Google Scholar 

  35. 35.

    Badger MR, Bek EJ. Multiple RuBisCO forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle. J Exp Bot. 2008;59:1525–41.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Sutter M, Roberts EW, Gonzalez RC, Bates C, Dawoud S, Landry K, Cannon GC, Heinhorst S, Kerfeld CA. Structural characterization of a newly identified component of α-carboxysomes: the AAA+ domain protein CsoCBBQ. Sci Rep. 2015;5:16243.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Chen J, Strous M. Denitrification and aerobic respiration, hybrid electron transport chains and co-evolution. Biochim Biophys Acta Bioenergetics. 2013;1827:136–44.

    CAS  Article  Google Scholar 

  38. 38.

    Denkmann K, Grein F, Zigann R, Siemen A, Bergmann J, van Helmont S, Nicolai A, Pereira IAC, Dahl C. Thiosulfate dehydrogenase: a widespread unusual acidophilic c-type cytochrome. Environ Microbiol. 2012;14:2673–88.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Wodara C, Kostka S, Egert M, Kelly DP, Friedrich CG. (1994) Identification and sequence analysis of the soxB gene essential for sulfur oxidation of Paracoccus denitrificans GB17. J Bacteriol. 1994;176:6188–91.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Wodara C, Bardischewsky F, Friedrich CG. Cloning and characterization of sulfite dehydrogenase, two c-type cytochromes, and a flavoprotein of Paracoccus denitrificans GB17: essential role of sulfite dehydrogenase in lithotrophic sulfur oxidation. J Bacteriol. 1997;179:5014–23.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Friedrich CG, Quentmeier A, Bardischewsky F, Rother D, Kraft R, Kostka S, Prinz H. Novel genes coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17. J Bacteriol. 2000;182:4677–87.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Mayer B, Kuever J. Molecular analysis of the distribution and phylogeny of dissimilatory adenosine 5′-phosphosulfate reductase-encoding genes (aprBA) among sulfur-oxidizing prokaryotes. Microbiology (UK). 2007;153:3478–98.

    Article  Google Scholar 

  43. 43.

    Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576 [PubMed].

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Garrity GM, Bell JA, Lilburn T, Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. Secondth ed. New York: Springer; 2005. p. 1. Part B.

    Google Scholar 

  46. 46.

    Garrity GM, Bell JA, Lilburn T. Class II. Betaproteobacteria class. nov. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM, editors. Bergeys Manual of Systematic Bacteriology, second edition, vol. 2 (The Proteobacteria) part C (The Alpha-, Beta-, Delta-, and Epsilonproteobacteria). New York: Springer; 2005. p. 575.

    Google Scholar 

  47. 47.

    Garrity GM, Bell JA, Lilburn T. Order II. Hydrogenophilales ord. nov. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM, editors. Bergeys Manual of Systematic Bacteriology, second edition, vol. 2 (The Proteobacteria), part C (The Alpha-, Beta-, Delta-, and Epsilonproteobacteria). New York: Springer; 2005. p. 763.

    Google Scholar 

  48. 48.

    Garrity GM, Bell JA, Lilburn T. “Family I. Hydrogenophilaceae fam. nov.”. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM, editors. Bergeys Manual of Systematic Bacteriology, second edition, vol. 2 (The Proteobacteria), part C (The Alpha-, Beta-, Delta-, and Epsilonproteobacteria). New York: Springer; 2005. p. 763.

    Google Scholar 

  49. 49.

    Skerman VBD, McGowan V, Sneath PHA. Approved list of bacterial names. Int J Syst Evol Microbiol. 1980;30:225–420.

    Article  Google Scholar 

  50. 50.

    Kelly DP, Wood AP, Stackebrandt E. Genus II. Thiobacillus Beijerinck 1904. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM, editors. The Proteobacteria, Part C, The Alpha-, Beta-, Delta-, and Epsilonproteobacteria, 2. 2nd ed. New York: Springer; 2005. p. 764–9.

    Google Scholar 

  51. 51.

    Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Guide to GO evidence codes. [].

  53. 53.

    Tamura K, Stecher G, Peterson D, Filipski A, Sumar S. MEGA6: Molecular Evolutionary Generics Analysis version 6.0. Mol Biol Evol. 2013;30:2725–19.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993;10:512–26.

    CAS  PubMed  Google Scholar 

Download references


We acknowledge Dr Hans-Peter Klenk at the DSMZ for the provision of genomic DNA.


The sequencing and annotation was performed under the auspices of the United States Department of Energy 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 and Marine Sciences, University of Plymouth for studentship funding to LH that supported the analysis of the genome and to the Royal Society Research Grant RG120444 awarded to RB to support the analysis of this genome.

Authors’ contributions

LPH and RB analysed and mined the genome data in public databases for genes of interest and performed BLASTn/BLASTp searches to verify and validate the annotation etc. and made comparisons of the sulfur oxidation, denitrification etc. operons with those in other organisms. RB constructed the phylogenetic tree. LPH grew the organism and performed electron microscopy at the Electron Microscopy Unit, 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.

Author information



Corresponding author

Correspondence to Rich Boden.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hutt, L.P., Huntemann, M., Clum, A. et al. Permanent draft genome of Thiobacillus thioparus DSM 505T, an obligately chemolithoautotrophic member of the Betaproteobacteria . Stand in Genomic Sci 12, 10 (2017).

Download citation


  • Thiobacillus thioparus
  • Betaproteobacteria
  • Sulfur oxidation
  • Chemolithoautotroph
  • Carboxysome
  • Denitrification