Standards in Genomic Sciences
Permanent draft genome of Thiobacillus thioparus DSM 505T, an obligately
chemolithoautotrophic member of the Betaproteobacteria
--Manuscript Draft-Manuscript Number:
SIGS-D-16-00109R3
Full Title:
Permanent draft genome of Thiobacillus thioparus DSM 505T, an obligately
chemolithoautotrophic member of the Betaproteobacteria
Article Type:
Short genome report
Funding Information:
U.S. Department of Energy
(DE-AC02-05CH11231)
Royal Society
(RG120444)
Dr Nikos C Kyrpides
Dr Rich Boden
Abstract:
Thiobacillus thioparus DSM 505T is one of first two isolated strains of inorganic sulfuroxidising 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 1.9.3.1) and ubiquinol oxidase (bd, EC 1.10.3.10).
There is a partial sox operon of the Kelly-Friedrich pathway of inorganic sulfuroxidation 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.
Corresponding Author:
Rich Boden, Ph.D B.Sc
University of Plymouth
Plymouth, Devon UNITED KINGDOM
Corresponding Author Secondary
Information:
Corresponding Author's Institution:
University of Plymouth
Corresponding Author's Secondary
Institution:
First Author:
Lee P Hutt
First Author Secondary Information:
Order of Authors:
Lee P Hutt
Marcel Huntemann
Alicia Clum
Manoj Pillay
Krishnaveni Palaniappan
Neha Varghese
Natalia Mikhailova
Dimitrios Stamatis
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Tatiparthi Reddy
Chris Daum
Nicole Shapiro
Natalia Ivanova
Nikos C Kyrpides
Tanja Woyke
Rich Boden, Ph.D B.Sc
Order of Authors Secondary Information:
Response to Reviewers:
In response to request to add 2 percentages to one of the tables (which could have
been added in proof tbh), they are done. Hopefully this is now final.
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Manuscript
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12 1 Permanent draft genome of Thiobacillus thioparus
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15 2 DSM 505 , an obligately chemolithoautotrophic
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33 9 [1] School of Biological and Marine Sciences, University of Plymouth, Drake Circus, Plymouth, PL4 8AA,
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[2] Sustainable Earth Institute, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, UK.
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* Corresponding author ([email protected])
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Abstract
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 CalvinBenson-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 1.9.3.1) and ubiquinol oxidase (bd, EC 1.10.3.10). 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.
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Keywords:
Thiobacillus thioparus, Betaproteobacteria, sulfur oxidation,
chemolithoautotroph, carboxysome, denitrification
Abbreviations
ATCC – American Type Culture Collection
BLAST – Basic Linear Alignment Search Tool
CIP – Collection de L’institut Pasteur
COG – Clusters of Orthologous Groups
DSMZ - Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
GEBA – Genomic Encyclopedia of Bacteria and Archaea.
IMG – Integrated Microbial Genomes database.
JCM – Japan Collection of Microorganisms
JGI – Joint Genome Institute
KMG – 1,000 Microbial Genomes project
NBRC – NITE Biological Resource Center
NCBI – National Center for Biotechnology Information
RuBisCO - ribulose-1,5-bisphosphate carboxylase/oxygenase
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Introduction
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 505T = ATCC 8158T = CIP 104484T = JCM 3859T = NBRC 103402T) [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 505T 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 505T 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 sulfuroxidation 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 505T 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,
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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 505T 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 Figure 1.
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 - 10 μm in length, as shown in Figure 2. The dominant respiratory quinone is
ubiquione-8 [7-10] 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 505T 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
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sheep or horse blood agar, chocolate agar). Energy sources that support autotrophic growth of DSM 505T
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 505T 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 505T 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.
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.
Growth conditions and genomic DNA preparation
T. thioparus DSM 505T 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
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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 505T 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 http://www.jgi.doe.gov. All raw
Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes
known Illumina sequencing and library preparation artifacts [17]. Following steps were then performed
for assembly: (1) filtered Illumina reads were assembled using Velvet (version 1.1.04) [18], (2) 1–3 Kbp
simulated paired end reads were created from Velvet contigs using wgsim [19], (3) Illumina reads were
assembled with simulated read pairs using Allpaths–LG (version r41043) [20]. 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 [21], followed by a round of manual curation using GenePRIMP
[22] for finished genomes and Draft genomes in fewer than 10 scaffolds. The predicted CDSs were
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translated and used to search the NCBI nonredundant database, UniProt, TIGRFam, Pfam, KEGG, COG,
and InterPro databases. The tRNAScanSE tool [23] was used to find tRNA genes, whereas ribosomal
RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [24].
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 [25].
Additional gene prediction analysis and manual functional annotation was performed within the IMG
platform [26] developed by the JGI, Walnut Creek, CA, USA [27, 28].
Genome Properties
The genome of T. thioparus DSM 505T 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 12475T [12].
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, 29, 30, 33]. Smith’s horseshoe representing a very
near-complete Krebs’ cycle was identified, in which citrate synthase (EC 2.3.3.16), aconitase (EC 4.2.1.3),
isocitrate dehydrogenase (NADP+, EC 1.1.1.42), succinyl coenzyme A synthase (ADP-forming, EC
2.6.1.5), succinate dehydrogenase (EC 1.3.5.1) and malate dehydrogenase (oxaloacetate decarboxylating,
NADP+, EC 1.1.1.40) genes are present, but fumarase (EC 4.2.1.2) and the E3 subunit of α-ketoglutarate
dehydrogenase (NAD+, EC 1.2.4.2) genes are absent – E1 and E2 are present. This is a comparatively
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large version of Smith’s horseshoe [33], 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 3134T [12, 31,
32] of the Acidithiobacillia. Activities of Krebs’ cycle enzymes assayed by Smith et al. [33] 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 505T, 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 25259T [34], though enzyme activity was also absent in this strain [33,
35]. 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 505T - clearly this is needed in order to identify if genomic data reflect
true activities in vivo [29].
A complete Calvin-Benson-Bassham cycle is present, with a single copy of the large (cbbL) and small
(cbbS) form I RuBisCO (EC 4.1.1.39) subunits and, owing to the presence of a transaldolase (EC 2.2.1.2)
and absence of a sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) gene, we can conclude that it uses the
transaldolase-variant Calvin-Benson-Bassham cycle [36]. Adjacent to these genes are cbbO and cbbQ,
consistent with form IAq RuBisCO [37], 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) [34]. 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
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if it is an operon or not) in T. thioparus DSM 505T does not start with cbbLS, (as would be found in
Halothiobacillus, Acidithiobacillus and Thermithiobacillus spp. [38]) - the lack of these genes has also
been observed in T. denitrificans ATCC 25259T [34] and may be a diagnostic property of this genus.
Whilst T. thioparus DSM 505T 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 [56] was found
With regard to respiration, five cytochromes c553, one cytochrome c556 and three cytochrome b were
present. The two high-affinity terminal oxidases were both found – with multiple gene copies of
cytochrome c oxidase (cbb3, EC 1.9.3.1) and a single copy of cytochrome bd-type quinol oxidase (EC
1.10.3.10). It is worth noting that T. denitrificans also possess the aa3 variant of cytochrome c oxidase
(EC 1.9.3.1), 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 cbb3 or bd-type high-affinity terminal oxidases [56] 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 1.8.2.2), one gene (tsdA) for which was identified in Allochromatium
1
2
3
4
5
6
7
8
9
10
11218
12
13219
14220
15
16221
17
18222
19
223
20
21224
22
23225
24
226
25
26227
27
28228
29
30229
31230
32
33231
34
35232
36233
37
38234
39
40235
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236
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43237
44
45238
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239
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48240
49
50241
51
52242
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65
vinosum, a member of the Gammaproteobacteria [39]. However, tsdA is not present in T. thioparus DSM
505T 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 [4042]. 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 c551
and c552.5 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 505T 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 3134T, Acidithiobacillus
thiooxidans ATCC 19377T and Acidithiobacillus caldus ATCC 51756T, and Thiohalorhabdus
denitrificans DSM 15699T, 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 505T, 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 1.8.2.3) 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
1
2
3
4
5
6
7
8
9
10
11243
12
13244
14
15245
16
17246
18
19247
20248
21
22249
23
24250
25
251
26
27252
28
29253
30
254
31
32255
33
34
35
36256
37
38257
39258
40
41259
42
43260
44
261
45
46262
47
48263
49
264
50
51265
52
53266
54
55
56
57
58
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65
reductase (aprAB, EC 1.8.99.2) genes, the presence of which has also been confirmed in T. aquaesulis
[57].
It was previously noted that 5.9% of the genome (178 genes) of Thermithiobacillus tepidarius DSM
3134T 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 505T with only 6 genes from Ttb. tepidarius DSM 3134T, 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.
Conclusions
The genome of Thiobacillus thioparus DSM 505T 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 [34] 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
[55]. 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
1
2
3
4
5
6
7
8
9
10
11267
12
13268
14269
15
16270
17
18271
19
272
20
21
22
23273
24
25274
26
27
28275
29
30
31276
32
33277
34
278
35
36279
37
38280
39
40281
41
42282
43
44283
45
46284
47
285
48
49286
50
51287
52
53288
54
55
56
57
58
59
60
61
62
63
64
65
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
[29,33].
Competing interests
The authors have no competing interests.
Funding
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.
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24295
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34300
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38302
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Acknowledgements
We acknowledge Dr Hans-Peter Klenk at the DSMZ for the provision of genomic DNA.
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13421
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17423
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19424
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21425
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426
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25427
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13429
14430
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45432
433
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Table 1
Table 1. Classification and general features of Thiobacillus thioparus DSM 505T according to
MIGS recommendations [43]
Property
Term
Evidence
codea
Classification
Domain Bacteria
TAS [48]
Phylum ‘Proteobacteria’
TAS [49]
Class Betaproteobacteria
TAS [50]
Order Hydrogenophilales
TAS [51]
Family Hydrogenophilaceae
TAS [52]
Genus Thiobacillus
TAS [2, 3]
Species Thiobacillus thioparus
TAS [2, 3]
(Type) strain: DSM 505T
TAS [53]
Gram stain
Negative
TAS [3]
Cell shape
Rod
TAS [2, 3]
Motility
Motile
TAS [2, 3]
Sporulation
None
TAS [3, 55]
Temperature range
N.D.
NAS
Optimum temperature
28 °C
TAS [3, 54]
pH range; Optimum
N.D.; 7.0
NAS
Carbon source
Carbon dioxide
TAS [3, 54]
MIGS-6
Habitat
Agricultural soil (sandy loam)
TAS [3]
MIGS-6.3
Salinity
N.D.
NAS
MIGS-22
Oxygen requirement
Aerobic
TAS [3, 54]
MIGS-15
Biotic relationship
Free-living
TAS [3, 54]
MIGS-14
Pathogenicity
Non-pathogen
NAS
MIGS-4
Geographic location
New Jersey, United States of America
TAS [3]
MIGS-5
Sample collection
1934
TAS [3]
MIGS-4.1
Latitude
40° 28' 57'' N
TAS [3]
MIGS-4.2
Longitude
74° 26' 14'' E
TAS [3]
MIGS-4.4
Altitude
28 m
TAS [3]
MIGS ID
a
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 [44,45].
1
2
3
4
5
6
7
8
9
10
11438
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
1
2
3
4
5
6
7
8
9
10
11439
12
13440
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29441
30442
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Table 2
Table 2. Project information.
MIGS ID
Property
Term
MIGS 31
Finishing quality
Improved High-Quality Draft
MIGS-28
Libraries used
Illumina Standard PE
MIGS 29
Sequencing platforms
Illumina
MIGS 31.2
Fold coverage
122.7
MIGS 30
Assemblers
Allpaths/Velvet
MIGS 32
Gene calling method
NCBI Prokaryotic Genome Annotation Pipeline
Locus Tag
B058
Genbank ID
ARDU00000000
GenBank Date of Release
April 16th, 2013
GOLD ID
Ga0025551
BIOPROJECT
PRJNA169730
Source Material Identifier
DSM 505T
Project relevance
GEBA-KMG
MIGS 13
1
2
3
4
5
6
7
8
9
10
11443
12
13444
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32455
33
34456
35
36457
37
38458
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Table 3
Table 3. Genome statistics.
Attribute
Genome size (bp)
DNA coding (bp)
DNA G+C (bp)
DNA scaffolds
Total genes
Protein coding genes
RNA genes
Pseudo genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
CRISPR repeats
Value
3,201,518
2,937,381
1,994,510
18
3,197
3,135
62
538
267
2,597
2,258
2,700
376
767
2
% of Total 445
100.00
446
91.75
62.30
447
100.00
448
100.00
98.06
449
1.94
16.83
450
8.35
81.23
451
70.63
84.45
452
11.76
453
23.99
0.04
454
Commented [BSBCL2]: Please include the percentage of
the total number of bp that this genome attribute accounts
for
Commented [BSBCL3]: Please add the percentage of
the total number of bp that this genome attribute accounts
for to the adjacent column
1
2
3
4
5
6
7
8
9
10
11459
12
13460
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40461
462
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Table 4
Table 4. Number of genes associated with general COG functional categories.
Code
J
Value
%age
Description
205
8.2
Translation, ribosomal structure and biogenesis
A
1
0.0
RNA processing and modification
K
120
4.8
Transcription
L
83
3.3
Replication, recombination and repair
B
1
0.0
Chromatin structure and dynamics
D
38
1.5
Cell cycle control, Cell division, chromosome partitioning
V
61
2.4
Defense mechanisms
T
156
6.2
Signal transduction mechanisms
M
225
9.0
Cell wall/membrane biogenesis
N
101
4.0
Cell motility
U
56
2.2
Intracellular trafficking and secretion
O
144
5.8
Posttranslational modification, protein turnover, chaperones
C
207
8.3
Energy production and conversion
G
86
3.4
Carbohydrate transport and metabolism
E
153
6.1
Amino acid transport and metabolism
F
63
2.5
Nucleotide transport and metabolism
H
144
5.8
Coenzyme transport and metabolism
I
76
3.0
Lipid transport and metabolism
P
206
8.2
Inorganic ion transport and metabolism
Q
37
1.5
Secondary metabolites biosynthesis, transport and catabolism
R
165
6.6
General function prediction only
S
143
5.7
Function unknown
-
939
29.4
Not in COGs
The total is based on the total number of protein coding genes in the genome.
1
2
3
4
5
6
7
8
9
10
11463
12464
13465
14
15466
16
17467
18
19468
20
21469
22
23470
24471
25
26472
27
28473
29
30474
31
32475
33
34476
35
36
477
37
38478
39
40
41479
42
480
43
44
45481
46
47482
48
49483
50484
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Figure legends
Figure 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 [46]. Tree was drawn using the Tamura-Nei model for maximum-likelyhood
trees [47]. 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.
Figure 2. Transmission electron micrographs of T. thioparus DSM 505T cells obtained from a thiosulfatelimited chemostat (20mM, 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.
Figure 1
Click here to download Figure R1 new tree.tif
Figure 2
Click here to download Figure higher qual Figure EM images.tif