Genome analysis and genome-wide proteomics of Thermococcus gammatolerans, the most radioresistant organism known amongst the Archaea.

Abstract

Thermococcus gammatolerans was isolated from samples collected from hydrothermal chimneys. It is one of the most radioresistant organisms known amongst the Archaea. We report the determination and annotation of its complete genome sequence, its comparison with other Thermococcales genomes, and a proteomic analysis.
T. gammatolerans has a circular chromosome of 2.045 Mbp without any extra-chromosomal elements, coding for 2,157 proteins. A thorough comparative genomics analysis revealed important but unsuspected genome plasticity differences between sequenced Thermococcus and Pyrococcus species that could not be attributed to the presence of specific mobile elements. Two virus-related regions, tgv1 and tgv2, are the only mobile elements identified in this genome. A proteogenome analysis was performed by a shotgun liquid chromatography-tandem mass spectrometry approach, allowing the identification of 10,931 unique peptides corresponding to 951 proteins. This information concurrently validates the accuracy of the genome annotation. Semi-quantification of proteins by spectral count was done on exponential- and stationary-phase cells. Insights into general catabolism, hydrogenase complexes, detoxification systems, and the DNA repair toolbox of this archaeon are revealed through this genome and proteome analysis.
This work is the first archaeal proteome investigation done at the stage of primary genome annotation. This archaeon is shown to use a large variety of metabolic pathways even under a rich medium growth condition. This proteogenomic study also indicates that the high radiotolerance of T. gammatolerans is probably due to proteins that remain to be characterized rather than a larger arsenal of known DNA repair enzymes.

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Genome Biology 2009, 10:R70
Open Access
2009Zivanovic et al.Volume 10, Issue 6, Article R70
Research
Genome analysis and genome-wide proteomics of Thermococcus
gammatolerans, the most radioresistant organism known amongst
the Archaea
Yvan Zivanovic¤*, Jean Armengaud¤†, Arnaud Lagorce*, Christophe Leplat*,
Philippe Guérin†, Murielle Dutertre*, Véronique Anthouard‡,
Patrick Forterre§, Patrick Wincker‡ and Fabrice Confalonieri*
Addresses: *Laboratoire de Génomique des Archae, Université Paris-Sud 11, CNRS, UMR8621, Bât400 F-91405 Orsay, France. †CEA, DSV,
IBEB Laboratoire de Biochimie des Systèmes Perturbés, Bagnols-sur-Cèze, F-30207, France. ‡CEA, DSV, Institut de Génomique, Genoscope,
rue Gaston Crémieux CP5706, F-91057 Evry Cedex, France. §Laboratoire de Biologie moléculaire du gène chez les extrêmophiles, Université
Paris-Sud 11, CNRS, UMR8621, Bât 409, F-91405 Orsay, France.
¤ These authors contributed equally to this work.
Correspondence: Fabrice Confalonieri. Email: fabrice.confalonieri@u-psud.fr
© 2009 Zivanovic et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Thermococcus gammatolerans proteogenomics<p>The genome sequence of Thermococcus gammatolerans, a radioresistant archaeon, is described; a proteomic analysis reveals that radi-oresistance may be due to unknown DNA repair enzymes.</p>
Abstract
Background: Thermococcus gammatolerans was isolated from samples collected from hydrothermal chimneys. It
is one of the most radioresistant organisms known amongst the Archaea. We report the determination and
annotation of its complete genome sequence, its comparison with other Thermococcales genomes, and a
proteomic analysis.
Results: T. gammatolerans has a circular chromosome of 2.045 Mbp without any extra-chromosomal elements,
coding for 2,157 proteins. A thorough comparative genomics analysis revealed important but unsuspected
genome plasticity differences between sequenced Thermococcus and Pyrococcus species that could not be
attributed to the presence of specific mobile elements. Two virus-related regions, tgv1 and tgv2, are the only
mobile elements identified in this genome. A proteogenome analysis was performed by a shotgun liquid
chromatography-tandem mass spectrometry approach, allowing the identification of 10,931 unique peptides
corresponding to 951 proteins. This information concurrently validates the accuracy of the genome annotation.
Semi-quantification of proteins by spectral count was done on exponential- and stationary-phase cells. Insights
into general catabolism, hydrogenase complexes, detoxification systems, and the DNA repair toolbox of this
archaeon are revealed through this genome and proteome analysis.
Conclusions: This work is the first archaeal proteome investigation done at the stage of primary genome
annotation. This archaeon is shown to use a large variety of metabolic pathways even under a rich medium growth
condition. This proteogenomic study also indicates that the high radiotolerance of T. gammatolerans is probably
due to proteins that remain to be characterized rather than a larger arsenal of known DNA repair enzymes.
Published: 26 June 2009
Genome Biology 2009, 10:R70 (doi:10.1186/gb-2009-10-6-r70)
Received: 24 March 2009
Revised: 29 May 2009
Accepted: 26 June 2009
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2009/10/6/R70

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Genome Biology 2009, Volume 10, Issue 6, Article R70 Zivanovic et al. R70.2
Genome Biology 2009, 10:R70
Background
Thermococcales are strictly anaerobic and hyperthermophilic
archaea belonging to the Euryarchaeota phylum. In this
order, three genera are distinguished: Pyrococcus [1], Ther-
mococcus [2] and Palaeococcus [3]. With about 180 different
species listed to date, the Thermococcus genus is the largest
archaeal group characterized so far. They have been isolated
from terrestrial hot springs, deep oil reservoirs, and are
widely distributed in deep-sea environments [4,5]; they are
considered as key players in marine hot-water ecosystems.
Thermococcus species are able to grow anaerobically on vari-
ous complex substrates, such as yeast extract, peptone, and
amino acids in the presence of elemental sulfur (S°), and yield
hydrogen sulfide. Several species are also capable of ferment-
ing peptides, amino acids or carbohydrates without sulfur
producing acids, CO2 and H2 as end products [6,7]. Recently,
some species such as Thermococcus strain AM4 and Thermo-
coccus onnurineus NA1 were shown to be capable of litho-
trophic growth on carbon monoxide [8,9]. In this case, the CO
molecule, probably oxidized into CO2, is used as energy and/
or carbon source.
Five Thermococcales genomes have been sequenced and
annotated so far: Pyrococcus horikoshii [10], Pyrococcus
furiosus [11], Pyrococcus abyssi [12], Thermococcus kodaka-
raensis KOD1 [13] and T. onnurineus NA1 [8]. Although their
respective gene contents are highly conserved, synteny analy-
ses have shown an extensive frequency of genomic DNA rear-
rangements in Thermococcales [14,15]. The relatively low
fraction of insertion sequence elements or repeats in Thermo-
coccus genomes contrasts with the fact that genome rear-
rangements are faster than normal protein sequence
evolution [13].
Some hydrothermal chimneys in which many thermophilic
prokaryotes were isolated were shown to be especially rich in
heavy metals [16,17] and exposed to natural radioactivity
doses hundreds of times higher than those found on the
Earth's surface [18]. Although such extreme conditions were
likely to have been much more common during the first
stages of life on Earth, they are deleterious and few data are
currently available regarding the strategies that thermophiles
use to live in such environments. The hyperthermophilic
archaeon Thermococcus gammatolerans was recently iso-
lated from samples collected from hydrothermal chimneys
located in the mid-Atlantic Ridge and at the Guyamas basin
[19,20]. T. gammatolerans EJ3 was obtained by culture
enrichment after irradiation with gamma rays at massive
doses (30 kGy). It was described as an obligatory anaerobic
heterotroph organism that grows optimally at 88°C in the
presence of sulfur or cystine on yeast extract, tryptone and
peptone, producing H2S. This organism withstands 5 kGy of
radiation without any detectable lethality [21]. Exposure to
higher doses slightly reduces its viability whereas cell survival
of other thermophilic radioresistant archaea drastically
decreases when cells are exposed to such radiation doses
[20]. Based on these data, T. gammatolerans is one of the
most radioresistant archaeon isolated and characterized thus
far. As Archaea and Eukarya share many proteins whose
functions are related to DNA processing [22], the radioresist-
ant T. gammatolerans EJ3 species is a unique model organ-
ism along the Archaea/Eukarya branch of the phylogenetic
tree of life. In contrast to the well-characterized Deinococcus
radiodurans, the radioresistant model amongst Bacteria
[23,24], the lack of knowledge on T. gammatolerans EJ3
urges us to further characterize this archaeon using the most
recent OMICs-based methodologies.
Although more than 50 archaeal genomes have been
sequenced so far, only a few archaea have been analyzed in
depth at both the genome and proteome levels. Halobacte-
rium sp. NRC-1 was the first archaeon to be analyzed for its
proteome on a genome-wide scale. A partial proteome shot-
gun revealed 57 previously unannotated proteins [25]. A set
of 412 soluble proteins from Methanosarcina acetivorans
was identified with a two-dimensional gel approach [26]. In
Aeropyrum pernix K1, 19 proteins that were not previously
described in the genomic annotation were discovered [27].
Halobacterium salinarum and Natronomonas pharaonis
proteomes were scrutinized with a special focus on amino-
terminal peptides or low molecular weight proteins [28-30].
Although labor-intensive, proteogenomic re-annotation of
sequenced genomes is currently proving to be very useful
[31]. Moreover, genome-scale proteomics reveals whole pro-
teome dynamics upon changes in physiological conditions.
Here we present a genome analysis of T. gammatolerans EJ3
and a detailed comparison with other Thermococcales
genomes. To gain real insights into the physiology of T. gam-
matolerans, we analyzed the proteome content of exponen-
tial- and stationary-phase cells by a liquid chromatography
(LC)-tandem mass spectrometry (MS/MS) shotgun approach
and semi-quantification by spectral counting. T. gammatol-
erans is the first archaeon whose genome and proteome have
been analyzed jointly at the stage of primary annotation. With
these results in hand and its remarkable radiotolerance, T.
gammatolerans is now a model of choice amongst the
Archaea/Eukarya lineage.
Results and discussion
Genome sequence
The complete genome sequence of T. gammatolerans has
been determined with good accuracy, with final error rate lev-
els of less than 2.4 × 10-05 before manual editing of 48 remain-
ing errors. It is composed of a circular chromosome of
2,045,438 bp without extra-chromosomal elements, and a
total of 2,157 coding sequences (CDSs) were identified (Table
S1 in Additional data file 1). Their average size is 891 nucle-
otides, comprising CDSs ranging from 32 (tg2073, encoding a
conserved hypothetical protein) to 4,620 amino acids
(tg1747, encoding an orphan protein).

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Genome Biology 2009, 10:R70
Genome annotation accuracy as evaluated by
proteomics
We analyzed the proteome content of T. gammatolerans
grown in optimal conditions (rich medium supplemented
with S°) at two stages, exponential and stationary. Total pro-
teins were resolved by one-dimensional SDS-PAGE and iden-
tified by nanoLC-MS/MS shotgun analysis. From the large
corpus of MS/MS spectra (463,840) that were acquired,
170,790 spectra could be assigned to 11,056 unique peptides
(Table S6 in Additional data file 2). A total of 951 proteins
were identified with very stringent search parameters (at
least two peptides with P < 0.001; Table S7 in Additional data
file 2). Our experimental results clearly show that all MS/MS
identified peptides map to an entry in both the TGAM_ORF0
and TGAM_CDS1 databases (see Materials and methods),
corresponding to 44% of the theoretical proteome and to a
polypeptide coverage of 33% on average. Accordingly, all con-
fident MS/MS spectra protein assignments confirmed the
predicted genes, but we cannot exclude that a few new genes
encoding small and non-abundant proteins may be present as
such polypeptides typically resulted in a limited number of
trypsic peptides that can be difficult to detect. While 45% of
the theoretical proteome, composed of proteins ranging
between 10 and 40 kDa, is detected by mass spectrometry,
only 23% of proteins below 10 kDa are detected. This strong
bias indicates that there may be some doubt regarding the
real existence of some short annotated genes. Alternatively,
most of them may correspond to non-abundant proteins.
Translation start codon verification by mass
spectrometry and amino-terminal modifications
After checking for trypsin and semi-trypsin specificities, we
found 290 different amino-terminal peptidic signatures
(Table S9 in Additional data file 3). They correspond to 173
different proteins. The start codon of 20 genes was incorrectly
predicted and was corrected. Out of the 173 proteins, 70
exhibit a methionine at their amino terminus, 98 start with
another amino acid, and 5 are found in both forms (Table S10
in Additional data file 3). The pattern for initial methionine
cleavage is standard and depends on the steric hindrance of
the second amino acid residue. As a result, polypeptides start
with Ala (29 cases), Gly (18 cases), Pro (14 cases), Ser (12
cases), Thr (12 cases) and Val (18 cases).
A restricted set (13%) of these proteins (23 of 173) were found
acetylated at their amino-terminal residue (Table S10 in
Additional data file 3). This post-translational modification
occurs for both cytosolic and membrane proteins. In contrast
to halophilic organisms [32], we found in T. gammatolerans
that the presence of an acidic amino acid (mainly Glu) in the
second (when Met is not removed) or the third position of the
polypeptide (when Met is removed) enhances the acetylation
process (8 cases out of 11, and 10 cases out of 12, respectively).
However, such a pattern does not imply acetylation as 25 pro-
teins were found exclusively unacetylated. Remarkably, both
acetylated and unacetylated amino termini were detected in
11 cases. In eukaryotes, three amino-terminal acetyltrans-
ferases, NatA, NatB, and NatC, have been described with
preferential substrates [33]. We did not find any homologues
of these acetyltransferase complexes in the T. gammatoler-
ans genome but did find three putative N-acetyltransferases
encoded by tg0455, tg1315, and tg1588. From the amino-ter-
minal peptidic signatures that were recorded in our shotgun
analysis, we deduced that T. gammatolerans encodes at least
a functional analogue of NatA, because acetylation occurs on
Ala, Gly, and Ser residues when the amino-terminal Met is
removed (12 cases out of 12 different acetylated proteins), and
a functional analogue of NatB that acetylates the Met residue
when a Met-Glu, Met-Asp, or Met-Met dipeptide is located at
the amino terminus of the protein. Such dipeptides are found
for 9 out of 11 acetylated proteins; the remaining 2 acetylated
proteins start with Met-Gln.
Genome features
Table 1 summarizes the general features of T. gammatoler-
ans compared with those of other sequenced Thermococca-
les. No significant differences in gene composition statistics
were seen for these genomes. Amongst Thermococcales, a
specific trait of Thermococcus genomes was noted when com-
paring the GC percentages of coding and inter-gene regions:
this difference rises to 10% for Thermococcus compared to
about 5% for Pyrococcus. As expected, average CDS identity
values reflect the phylogenetic distance relationships within
Thermococcales.
T. gammatolerans shares 1,660 genes with T. kodakaraensis
KOD1 whereas only 1,489 genes were found to be common
with T. onnurineus NA1, a number similar to that obtained
when T. gammatolerans is compared to Pyrococcus species.
This result is due to the lower size of the T. onnurineus NA1
genome, which is about 200 kb shorter than the other
sequenced Thermococcus genomes. Consequently, the three
Thermococcus genomes share only 1,416 common genes
(Table S2 in Additional data file 1). Remarkably, two-thirds of
the 74 genes conserved in T. gammatolerans and T. onn-
urineus NA1 but missing in T. kodakaraensis KOD1 encode
putative hydrogenase complexes that are present in several
copies in T. gammatolerans and T. onnurineus NA1
genomes, or encode conserved proteins of unknown function.
Among the six Thermococcales genomes, 1,156 genes are con-
served (Table S3 in Additional data file 1). They were obvi-
ously present in the common ancestor before the divergence
of Thermococcus and Pyrococcus. After searching for
sequence similarities and specific motifs and domains in pub-
lic databases, as defined in the Materials and methods, we are
able to propose a function for 1,435 T. gammatolerans CDSs.
Among the 722 remaining genes encoding hypothetical pro-
teins, 214 are conserved in all the six sequenced Thermococ-
cales. The products of one-sixth (120) of these genes were
experimentally detected by our proteomic detection
approach. T. gammatolerans possess a set of 326 genes
absent in other sequenced Thermococcales (Table S4 in Addi-

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tional data file 1). Among them, 98 are distributed in diverse
functional categories as predicted by sequence similarity, the
most important features being discussed below.
Paradoxical genome plasticity in Thermococcales
The six closely related and fully sequenced Thermococcales
species (three Thermococcus, T. gammatolerans, T. kodaka-
raensis, and T. onnurineus, and three Pyrococcus, P. abyssi,
P. horikoshii, and P. furiosus) enable insights into ongoing
genome evolution at a global scale since limited sequence
divergence enables the fate of most genes in each considered
lineage to be specifically tracked (Table 1 and Figure 1a). Most
rearrangement mechanisms identified so far are non-random
(for example, symmetry for replication-linked recombina-
tions [34,35], site specificity for mobile elements [15,36,37],
and recombination hotspots). For example, uneven fragmen-
tation rates were described in archaea from pairwise compar-
isons at replication termini regions of Pyrococcus species
[38], a situation already noted for bacterial genomes [39],
although this does not preclude that random recombination
prevails on a global genome scale. Determination of the chro-
nology of genome recombination events among the three
Pyrococcus species showed that, as a consequence, nucleo-
tidic sequences can evolve at increased rates [15]. Here, we
take advantage of the very high fraction of conserved genes
between six Thermococcales (approximately 58 to 73%; Fig-
ure 1b) to deduce the global number of reciprocal recombina-
tion events and their distribution patterns.
Pairwise genome scatter plots were determined to analyze
recombination patterns between genomes. They show two
different types of pattern (Figure 2, upper right), one in which
chromosomes co-linearity is recognizable (see Pyrococcus
pairs pab/ph/, pab/pf and ph/pf plots), and another where all
genes seem randomly scattered, except for a few islands of
syntenic blocks (see Pyrococci/Thermococci pairs plots: tg,
tk, ton versus pab, ph, pf, and Thermococcus pairs: tg/tk, tg/
ton and tk/ton). This is unexpected for Thermococcus pairs,
since the overall number of similar genes is very close in Ther-
mococcus and Pyrococcus species (approximately 71 to 73%
and approximately 67 to 73%, respectively; Figure 1b), and
their sequence similarity is very high (intra-Thermoccocus
identity range 69 to 77%; intra-Pyrococcus identity range 81
to 85%; Figure 1a).
In order to determine the global recombination trends, we
modeled a chromosome as a finite length segment disrupted
by N hits (recombination events), each hit generating N + 1
intervals (fragments or synteny blocks) whose size frequency
distribution obeys a power law when hits are at random (in
which Frequency = a × Fragment_sizeb, a and b being con-
stants). We determined synteny block length distribution for
every genome pair (Figure 2, bottom left), and, in all cases,
real distributions can be fitted to a power law model with
good statistical support (R2 range 0.88 to 0.95; coefficients of
determination given by least square regression analysis; Fig-
ure 1c). We conclude that all six Thermococcales genomes
exhibit random recombination distribution over the entire
genome. Although unlikely, it could result from the summa-
tion of several local and mutually compensating recombina-
tion hot spots/regions, but there is no evidenced for this at
this resolution. If we equate fragments of the model with real
synteny blocks, the random hits hypothesis allows us to deter-
mine the number of recombination events yielding the
observed distributions by summing the number of synteny
blocks (minus 1). The absolute number of recombination
events (Figure 1d) spans a rather narrow range (612 to 982
overall hits), which increases slightly when comparing intra-
Table 1
General features of the six sequenced Thermococcales species*
T. gammatoleransT. onnurineusT. kodakaraensisP. abyssiP. horikoshii P. furiosus
Genome size (nt)
Percentage coding
regions
GC%
Intergene GC%
Number of CDSs
Gene overlaps†
Mean CDS length
(nt)
Average CDS
identity with T.
gammatolerans%‡
tRNAs
rRNAs
2,045,438
94.0%
1,847,607
91.7%
2,088,737
93.2%
1,765,118
93.1%
1,738,505
95.0%
1,908,256
93.8%
53.6%
43.3%
2,157
237 (11%)
891
51.2%
42.4%
1,976
402 (20%)
857
52.0%
42.0%
2,306
557 (24%)
844
44.7%
39.6%
1,896
317 (17%)
918
41.9%
39.8%
1,955
712 (36%)
854
40,80%
35.8%
2,125
657 (31%)
842
100% 76.7%77.2%72.8% 71.2%71.5%
464646464646
2× 5S, 7S, 16S, 23S2× 5S, 7S, 16S, 23S2× 5S, 7S, 16S, 23S2× 5S, 7S, 16S, 23S 2× 5S, 7S, 16S, 23S 2× 5S, 7S, 16S, 23S
*Data for the five Thermococcales strains were from GenBank. †Total number of overlaping genes and fraction of genes with overlaps (percentage in
parentheses). ‡This refers to average identity percent values obtained by similarity matches with BLASTP. Nt, nucleotides.

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Thermococcales genome parameters defined in this study
Figure 1
Thermococcales genome parameters defined in this study. For each parameter, a chart for genome pairs (tg, T. gammatolerans; tk, T. kodakaraensis; ton, T.
onnurineus; pab, P. abyssi; pf, P. furiosus; ph, P. horikoshii) is shown in the upper part of the panel, and a table of data used to build the chart is shown in the
lower part of the panel. (a) Cross-genome average CDS identity. Values were determined by compiling identity percentage of each gene first hit in a
BLASTP full genome cross-match, using 80% alignment length and 0.3 of maximum bit score threshold values (see Materials and methods). Values were
then averaged by the total number of similar genes in each pair. (b) Percentage of similar (conserved) genes for each genome pair. Numbers of similar
genes were determined as in (a). The number of conserved genes in each genome pair was then averaged by half of the sum of the total number of genes
from both genomes. (c) Genome pair values of least squares line of best fit determination coefficients (R2) for synteny block length distribution (Figure 2,
left bottom). (d) Total number of recombination events for genome pairs. These numbers are actually the total number of synteny blocks + 1 within each
genome pair. (e) Average recombinations per gene (ARG) for genome pairs. The total number of recombination values (from (d)) was normalized by the
number of conserved genes in each pair.
tktonpabpfph
tg
tk
ton
pab
pf
50,0
55,0
60,0
65,0
70,0
75,0
80,0
85,0
90,0
tg
tk
ton
pab
pf
tg
tk
ton
pab
pf
(c)
76,777,2
68,8
72,8
75,9
58,0
71,2
75,1
59,0
81,6
71,5
75,3
56,8
85,4
82,0
tktonpabpfph
Cross-genome average CDS identity %
tg
tktonpabpf
tk
ton
pab
pf
ph
50,00
55,00
60,00
65,00
70,00
75,00
tk
ton
pab
pf
ph
tk
ton
pab
pf
ph
70,70
72,00
65,50
61,60
58,60
100,00
72,80
65,20
63,80
60,60
100,00
70,00
67,10
66,60
100,00
71,50
73,30
100,00
66,40
tg tkton pab pf

% Conserved genes (conserved genes / total genes)
tktonpabpfph
tg
tk
ton
pab
pf
0,8500
0,9000
0,9500
1,0000
tg
tk
ton
pab
pf
tg
tk
ton
pab
pf
0,92230,9074
0,9206
0,8817
0,9168
0,8803
0,9058
0,9461
0,8983
0,9192
0,9539
0,9492
0,8996
0,9403
0,9014
tktonpabpfph
Synteny block length frequency distribution coefficient of
determination
tktonpabpfph
tg
tk
ton
pab
pf
0
200
400
600
800
1000
Absolute number of recombination events
tg
tk
ton
pab
pf
tg
tk
ton
pab
pf
757
0
677
612
0
901
920
855
0
894
940
881
763
0
939
982
873
837
712
tktonpab pfph

tktonpabpfph
tg
tk
ton
pab
pf
0,00
0,10
0,20
0,30
0,40
0,50
0,60
0,70
0,80
Average recombinations per gene (ARG)
tg
tk
ton
pab
pf
tg
tk
ton
pab
pf
0,48
0,00
0,45
0,39
0,00
0,68
0,67
0,63
0,00
0,68
0,67
0,64
0,53
0,00
0,78
0,76
0,67
0,59
0,53
tk tonpab pfph
(a)
(b)
(d)
(e)