Deinococcus geothermalis: The Pool of Extreme
Radiation Resistance Genes Shrinks
Kira S. Makarova1*, Marina V. Omelchenko1, Elena K. Gaidamakova2, Vera Y. Matrosova2, Alexander Vasilenko2, Min Zhai2, Alla Lapidus3,
Alex Copeland3, Edwin Kim3, Miriam Land3, Konstantinos Mavromatis3, Samuel Pitluck3, Paul M. Richardson3, Chris Detter4, Thomas Brettin4,
Elizabeth Saunders4, Barry Lai5, Bruce Ravel5, Kenneth M. Kemner5, Yuri I. Wolf1, Alexander Sorokin1, Anna V. Gerasimova6, Mikhail S.
Gelfand7,8, James K. Fredrickson9, Eugene V. Koonin1, Michael J. Daly2*
1National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of
America, 2Department of Pathology, Uniformed Services University of the Health Sciences (USUHS), Bethesda, Maryland, United States of America,
3US Department of Energy, Joint Genome Institute, Walnut Creek, California, United States of America, 4US Department of Energy, Joint Genome
Institute, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America, 5Environmental Research Division and Advanced
Photon Source, Argonne National Laboratory, Argonne, Illinois, United States of America, 6Research Institute of Genetics and Selection of Industrial
Microorganisms, Moscow, Russia, 7Institute for Information Transmission Problems of RAS, Moscow, Russia, 8Faculty of Bioengineering and
Bioinformatics, M. V. Lomonosov Moscow State University, Moscow, Russia, 9Biological Sciences Division, Pacific Northwest National Laboratory,
Richland, Washington, United States of America
Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The
mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of
the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes
underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the
thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D.
radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in
resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast,
most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D.
geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was
identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We
present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-
resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different
sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-
Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus.
Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in
resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate
in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and
Citation: Makarova KS, Omelchenko MV, Gaidamakova EK, Matrosova VY, Vasilenko A, et al (2007) Deinococcus geothermalis: The Pool of Extreme
Radiation Resistance Genes Shrinks. PLoS ONE 2(9): e955. doi:10.1371/journal.pone.0000955
Deinococcus geothermalis belongs to the Deinococcus-Thermus group,
which is deeply branched in bacterial phylogenetic trees and has
putative relationships with cyanobacteria [1,2]. The extremely
radiation-resistant family Deinococcaceae is comprised of greater than
twenty distinct species  that can survive acute exposures to
ionizing radiation (IR) (10 kGy), ultraviolet light (UV) (1 kJ/m2),
and desiccation (years) [4,5]; and can grow under chronic IR
(60 Gy/hour) . D. geothermalis was originally isolated from a hot
pool at the Termi di Agnano, Naples, Italy , and subsequently
identified at other locations poor in organic nutrients including
industrial paper machine water , deep ocean subsurface
environments , and subterranean hot springs in Iceland .
D. geothermalis is distinct from most members of the genus
Deinococcus in that it is a moderate thermophile, with an optimal
growth temperature (Topt) of 50uC , is not dependent on an
exogenous source of amino acids or nicotinamide for growth
[11,12], is capable of forming biofilms , and possesses
membranes with very low levels of unsaturated fatty acids
compared to the other species . Based on the ability of wild-
type and engineered D. geothermalis and D. radiodurans to reduce
a variety of metals including U(VI), Cr(VI), Hg(II), Tc(VII), Fe(III)
and Mn(III,IV) [11,13], these two species have been proposed for
Funding: The work of KSM, MVO, YIW, AS, and EVK was supported by the
Intramural Research Program of the National Institutes of Health, National Library
of Medicine. The work at USUHS was supported by grant DE-FG02-04ER63918 to
MJD from the U. S. Department of Energy (DOE), Office of Science, Office of
Biological and Environmental Research (BER), Environmental Remediation Sciences
Program (ERSP); and by grant FA9550-07-1-0218 to MJD from the Air Force Office
of Scientific Research. The work at the DOE-Joint Genome Institute was supported
by the DOE Office of Science. Work at the Advanced Photon Source was supported
by the DOE Office of Science, Office of Basic Energy Sciences, under Contract No.
DE-AC02-06CH11357. The work of MSG and AVG was supported by grants from
the Howard Hughes Medical Institute (55005610), INTAS (05-8028), and the
Molecular and Cellular Virology program of the Russian Academy of Sciences. D.
geothermalis was selected for genome sequencing by BER (http://www.science.
doe.gov/ober/RFS-2.pdf) with MJD as the Principal Investigator.
Competing Interests: The authors have declared that no competing interests
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
nih.gov (KM); email@example.com (MD)
Academic Editor: Michael Lichten, National Cancer Institute, United States of
Received July 24, 2007; Accepted September 4, 2007; Published September 26,
This is an open-access article distributed under the terms of the Creative
Commons Public Domain declaration which stipulates that, once placed in the
public domain, this work may be freely reproduced, distributed, transmitted,
modified, built upon, or otherwise used by anyone for any lawful purpose.
PLoS ONE | www.plosone.org1 September 2007 | Issue 9 | e955
bioremediation of radioactive waste sites maintained by the US
Department of Energy (DOE) [11,14,15]. These characteristics
were the impetus for whole-genome sequencing of D. geothermalis at
DOE’s Joint Genome Institute, and comparison with the
mesophilic D. radiodurans (Topt, 32uC), to date the only other
extremely IR-resistant bacterium for which a whole-genome
sequence has been acquired .
Chromosomal and plasmid DNAs in extremely resistant
bacteria are as susceptible to IR-induced DNA double strand
breaks (DSBs) as in sensitive bacteria [5,17–19] and broad-based
experimental and bioinformatic studies have converged on the
conclusion that D. radiodurans uses a conventional set of DNA
repair and protection functions, but with a far greater efficiency
than IR-sensitive bacteria [17,20,21]. This apparent contradiction
is exemplified by work which showed that the repair protein DNA
polymerase I (PolA) of D. radiodurans supports exceptionally
efficient DNA replication at the earliest stages of recovery from
IR, and could account for the high fidelity of RecA-mediated
DNA fragment assembly . Paradoxically, however, IR-, UV-,
and mitomycin-C (MMC)-sensitive D. radiodurans polA mutants are
fully complemented by expression of the polA gene from the IR-
sensitive Escherichia coli .
The reason why repair proteins, either native or cloned, in D.
radiodurans function so much better after irradiation than in
sensitive bacteria is unknown. The prevailing hypotheses of
extreme IR resistance in D. radiodurans fall into three categories:
(i) chromosome alignment, morphology and/or repeated se-
quences facilitate genome reassembly [5,21,23,24]; (ii) a subset
of uncharacterized genes encode functions that enhance the
efficiency of DNA repair ; and (iii) non-enzymic Mn(II)
complexes present in resistant bacteria protect proteins, but not
DNA, from oxidation during irradiation, with the result that
conventional enzyme systems involved in recovery survive and
function with far greater efficiency than in sensitive bacteria
[17,23]. The extraordinary survival of Deinococcus bacteria
following irradiation has also given rise to some rather whimsical
descriptions of their derivation, including that they evolved on
Mars . On the basis of whole-genome comparisons between
two Deinococcus genomes and two Thermus genomes, we present
a reconstruction of evolutionary events that are inferred to have
occurred both before and after the divergence of the D. radiodurans
and D. geothermalis lineages. We revise down substantially the
number of potential genetic determinants of radiation resistance,
predict a Deinococcus radiation response regulon, and consider the
implications of these comparative-genomic findings for different
models of recovery.
RESULTS AND DISCUSSION
Resistance to IR and UV
One approach to delineating a minimal set of genes involved in
extreme resistance is to compare the whole-genome sequences of
two phylogenetically related but distinct species that are equally
resistant, whereby genes that are unique to both organisms are
ruled out, whereas shared genes are pooled as candidates for
involvement in resistance. We show that D. geothermalis (DSM
11300) and D. radiodurans (ATCC BAA-816) are equally resistant
to IR (60Co) (Figure 1A) and UV (254 nm) (Figure 1B) when
pre-grown and recovered at their optimal growth temperatures,
50uC and 32uC, respectively. When recovered at 50uC, the
survival of D. geothermalis exposed to 12 kGy was 1,000 times
greater than at 32uC (Figure 1A) . The extreme resistance to
desiccation of D. geothermalis recovered at 50uC was demon-
strated previously . Thus, D. geothermalis and D. radiodurans
Figure 1. Radiation resistance and genome structure of D.
geothermalis and D. radiodurans. A, IR (60Co, 5.5 kGy/h). B, UV
(254 nm) (3 J/m2s21). Open circle, D. radiodurans (32uC); open triangle,
D. geothermalis (50uC); and open square, D. geothermalis (32uC). Values
are from three independent trials with standard deviations shown. At
near-optimal growth temperatures, the 10% survival values (D10)
following IR for D. radiodurans (32uC) and D. geothermalis (50uC) are
15 kGy; for E. coli, 0.7 kGy (37uC) ; and for T. thermophilus (HB27) 0.8
kGy (65uC) . C, PFGE of genomic DNA prepared from irradiated
(0.2 kGy) D. radiodurans (DR+IR) and D. geothermalis (DG+IR); and
genomic DNA from non-irradiated D. geothermalis digested with SpeI
(DG+SpeI). (M) PFGE DNA size markers. PFGE was as described
Deinococcus Genome Analysis
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are well-suited to defining a conserved set of genes responsible for
Genome Sequence and Structure: General Features
The random shotgun method  was used to acquire the
complete sequence of the D. geothermalis (DSM 11300) genome,
that is comprised of a main chromosome (2,467,205 base pairs
(bp)), and two megaplasmids (574,127 bp and 205,686 bp). The
general structure of the predicted D. geothermalis genome was tested
by pulsed field gel electrophoresis (PFGE) of genomic DNA
linearized in vivo by exposure to IR (0.2 kGy), and by restriction
endonuclease (SpeI) cleavage (Figure 1C). The IR-treatment
revealed the existence of a ,570 kb megaplasmid in D. geothermalis,
and the SpeI-treatment yielded the expected number of chromo-
somal bands:3 singlets (632 kb,376 kband282 kb) and onedoublet
(574/579 kb); the plasmids do not contain a SpeI site. In comparison,
IR-treated D. radiodurans (ATCC BAA-816) subjected to PFGE
displayed the presence of the DR412 (412 kb) and DR177 (177 kb)
megaplasmids, previously observed . The approximately 206 kb
D. geothermalis megaplasmid was not visualized by PFGE although its
size lies between the two D. radiodurans megaplasmids, which were
readily observed (Figure 1C). Consistently, the abundance of DNA
clones for the 206 kb megaplasmid was significantly lower than the
574 kb megaplasmid during construction of the D. geothermalis
genome-library used for sequencing (data not shown). Thus, the
574 kb megaplasmid of D. geothermalis exists at higher copy-number
than the 206 kb megaplasmid.
Genome Comparison: General Features
Comparison of the general genome features of D. geothermalis and
D. radiodurans revealed major differences in genome partitioning,
and in the number of noncoding repeats (SNRs) (Table 1).
We previously demonstrated homo-
logous relationships between the DR412 megaplasmid of D.
radiodurans and the sole 233 kb megaplasmid (pTT27) of T.
thermophilus . Based on the gene contents of DR412 and
pTT27, we concluded that these megaplasmids evolved from
a common ancestor (Figure S1), are essential to the survival of both
species, and appear to serve as a sink for horizontally transferred
genes . In contrast, the 574 kb megaplasmid (DG574) of D.
geothermalis is distinct from pTT27, and appears to have been
derived from a fusion of DR412 and DR177 (Table S1), followed
by numerous rearrangements. Levels of gene order conservation
for the D. geothermalis and D. radiodurans chromosomes and
megaplasmids were determined by genomic dot plots 
(Figure S2). The dot plots of the chromosomes showed a clear
pattern characteristic of chromosomes of relatively closely related
bacteria that retain significant colinearity of the gene order. The
X-shape patternis thought
a chromosomal segment around the origin of replication .
By contrast, DR412 and DR177 did not display any discernable
colinearity (Figure S2B), indicating substantially greater levels of
rearrangement in the megaplasmids.
Repeats and Prophages
Dozens of small noncoding repeats
(SNRs) of an unusual, mosaic structure have been identified in the
D. radiodurans genome, suggesting a possible role in resistance .
In stark contrast, no mosaic-type SNRs were found in the D.
geothermalis genome (Table 1), suggesting that SNRs are not
involved in recovery from radiation or desiccation [26,29,30].
Further, there are about 20 DNA repeats in D. radiodurans that
contain oligoG stretches (Figure S3). Such DNA sequences might
adopt an ordered helical structure (G-quadruplex), predicted to
form parallel four-stranded complexes capable of promoting
chromosomal alignment . However, the absence of such
oligoG stretches in the G-rich sequence of D. geothermalis (G+C
content, 66%) indicates that G-quartets are not essential for
resistance. In contrast, the D. geothermalis genome contains
CRISPR repeats , whereas D. radiodurans does not (Table 1).
CRISPR repeats are part of a predicted RNA-interference-based
system implicated in immunity to phages and integrative plasmids
[33,34]. Since no homologous prophages were identified in the
two deinococci, and no CRISPR repeats are present in D. radiodurans,
these sequences apparently have no role in determining levels of
The 206 kb D. geothermalis megaplasmid (DG206), predicted by
genome sequencing, is in lower copy-number than DG574
(Figure 1C). The presence of DG206 in genomic DNA prepara-
tions was confirmed in D. geothermalis (DSM 11300) DNA samples
used for sequencing and from independent preparations by
polymerase chain reaction (PCR) using DG206-specific primers
that yielded DNA products of the predicted sizes (Figure S4).
DG206 contains 205 predicted open reading frames (ORFs), of
which 103 have significant similarity to genes in current databases;
approximately 40 are identical to genes in either the D. geothermalis
chromosome or DG574; and 28 have homologs in D. radiodurans,
including 3 ORFs encoding highly diverged single-stranded DNA-
binding proteins. Among other sequences of interest in DG206 are
22 transposon-related ORFs; 11 ORFs related to phage proteins;
and 5 ORFs related to conjugal plasmid replication systems. In
summary, DG206 is enriched for phage-, integrative plasmid- or
transposon-related ORFs, but encodes no known metabolic
enzymes and very few replication or repair proteins. Thus,
DR206 seems to mimic the trend seen for ORFs in the smallest
plasmid (46 kb) of D. radiodurans [16,21], with no predicted genes
implicated in resistance.
to arisefrom inversions of
The Deinococcus-Thermus Group: Gene-Gain and
Our previous analysis of the major events in the evolution of the
Deinococcus-Thermus group was based on D. radiodurans (ATCC
BAA-816) and T. thermophilus strain HB27 . The current study
includes additional comparisons with D. geothermalis (DSM 11300)
and a second strain of T. thermophilus (HB8). Based on the standard
approach of COGs (clusters of orthologous groups of proteins)
[35,36], COGs for Deinococcus and Thermus (tdCOGs) were
constructed (Table S2). The tdCOGs were used as a framework
for the whole-genome comparisons and evolutionary reconstruc-
tions (Figure 2). Using a weighted parsimony method and distantly
related bacteria as outgroups, the evolutionary reconstructions
Table 1. General Characteristics
D. geothermalis 3.27 Mbp D. radiodurans 3.28 Mbp
Main Chromosome2.46 Mbp (2,335 ORFs)2.65 Mbp (2,629 ORFs)
0.574 Mbp (522 ORFs)0.412 Mbp (368 ORFs)
0.206 Mbp (205 ORFs) 0.177 Mbp (145 ORFs)
PlasmidNot present 0.046 Mbp (39 ORFs)
Prophages1 region (,70 ORFs)2 regions (,75 ORFs)
,84 (,80 kb)52 (,62 kb)
CRISPRs6 regions (2 types)Not present
SNRsNot present295 (at least 9 types)
Deinococcus Genome Analysis
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revealed significant and independent expansion of the repertoire of
genes in the Deinococcus and Thermus lineages following their
divergence from a common ancestor. The expansion appears to
have occurred through both lineage-specific duplications and gene
acquisition via horizontal gene transfer (HGT). The high level of
protein family expansion (paralogy), and the larger complement of
species-specific genes acquired principally by HGT, could account
for the existence of 600–900 more genes in Deinococcus than
The Common Ancestor of the Deinococcus Lineage:
Trends of Gene-Gain and Gene-Loss
Our previous comparative analysis of T. thermophilus and D.
radiodurans identified several evolutionary trends that correlate with
the distinct phenotypes of these bacterial lineages . These
trends were further refined through the analysis of the D.
geothermalis sequence, and the unique features of the Deinococcus
lineage were used to better define the pathways implicated in
extreme radiation resistance (Table S2). One such trend in
Deinococcus, in comparison to the inferred common ancestor of the
Deinococcus-Thermus group, is the acquisition of a set of genes
involved in transcriptional regulation and signal transduction.
Examples of acquired transcriptional regulators include two
proteins of the AsnC family, two proteins of the GntR family,
and one protein of the IclR family. These families likely are
involved in amino acid degradation and metabolism [37–39].
Further, the Deinococcus lineage acquired at least six TetR and
MerR family regulators dedicated to diverse stress response
pathways [40,41]. Among the acquired signal transduction genes,
the most notable examples are two-component regulators of the
NarL family (four distinct tdCOGs) involved in the regulation of
a variety of oxygen and nitrate-dependent pathways of Escherichia
coli ; and the presence of several diguanylate cyclase (GGDEF)
domain-containing proteins supports an increased role of cyclic
diGMP in Deinococci. A second evolutionary trend in Deinococcus is
the acquisition of genes encoding proteins involved in nucleotide
metabolism, in particular, degradation and salvage [43–45]. For
example, this group includes genes for xanthine dehydrogenase,
urate oxidase, deoxynucleoside kinases, thymidine kinase, FlaR-
like kinase, and two UshA family 59-nucleotidases.
Other gene-gains in Deinococcus relative to Thermus include genes
for enzymes of amino acid catabolism and the tricarboxylic acid
(TCA) cycle (Table S2). Beyond the differences reported pre-
viously [11,12], the new reconstructions indicate that several
catabolic genes of Deinococcus were already present in the
Deinococcus-Thermus common ancestor. Following their divergence,
however, the Thermus lineage appears to have lost many of these
systems, including all enzymes involved in histidine degradation.
By contrast, the Deinococcus lineage not only retained a majority of
the predicted ancestral catabolic functions, but acquired new
pathways including ones involved in the degradation of tryptophan
and lysine, and several peptidases (Table S2). A hallmark of the
Deinococcus lineage is the presence of two predicted genes for malate
synthase, an enzyme of the glyoxylate bypass which converts
isocitrate into succinate and glyoxylate, allowing carbon that
enters the TCA cycle to bypass the formation of a-ketoglutarate
and succinyl-CoA . It has been proposed that the strong
upregulation of the glyoxylate bypass observed in D. radiodurans
following irradiation facilitates recovery by limiting the production
of metabolism-induced reactive oxygen species (ROS) .
Dgeo_2616/DRA0277 is the malate synthase ortholog present
in the Thermus lineage, but the second predicted deinococcal
malate synthetase (Dgeo_2611/DR1155) is unique and only
distantly related to homologs in other bacteria. Although the
two predicted deinococcal malate synthetases could have similar
functions, the genomic context of Dgeo_2611/DR1155 indicates
otherwise; Dgeo_2611/DR1155 are both located in a predicted
operon with two cyclic amidases of unknown biochemical
In a broader context, the present reconstruction indicates that
many expanded families of paralogous genes in D. radiodurans
proliferated before the emergence of the common ancestor of the
Deinococci, but the expansions were not present in the ancestor of
the Deinococcus-Thermus group (Table 2). Such Deinococcus-specific
expanded families include the Yfit/DinB family of proteins,
acetyltransferases of the GNAT family, Nudix hydrolases, a/
b superfamily hydrolases, calcineurin family phosphoesterases, and
others. Many of these expansions are for predicted hydrolases,
phosphatases in particular, but their substrate specificities are
either unknown or the affinity of known substrates is extremely low
. It has been proposed, therefore, that the majority of these
predicted enzymes perform cell-cleaning functions including
degradation of damaged nucleic acids, proteins and lipids, and/
or other stress-induced cytotoxins . The global proliferation of
these enzymes in the Deinococcus lineage (Table S3) supports the
acquisition of chemical stress-resistance determinants early in its
evolution; and the independent proliferation of determinants
within these deinococcal species (e.g., calcinurin phosphatses,
Figure S5) might represent secondary adaptations to specific stress
environments. In summary, these findings indicate that the
Deinococcus stress-resistance phenotypes evolved continuously, both
by lineage-specific gene duplications and by HGT from various
sources (Table S3, S4 and S5) .
Individual Deinococcus Species: Gene-Gain and
The comparison of gene-gain and gene-loss events in the D.
radiodurans and D. geothermalis lineages reveals numerous differences,
many of which correlate with their distinct metabolic phenotypes
The most notable, distinctive feature of D.
geothermalis is a greater abundance of genes for sugar metabolism
enzymes, which could have been acquired after the divergence of
the two Deinococci. The largest group within this set of genes is
predicted to be involved in xylose utilization, needed for growth on
Figure 2. Whole genome evolutionary reconstructions for D. radio-
durans, D. geothermalis, T. thermophilus (HB8) and T. thermophilus
(HB27). For each internal node of tree (open boxes), the inferred
number of tdCOGs is shown. For each tree branch the inferred number
of tdCOGs lost (minus sign) and gained (plus sign) is shown. For the
deep ancestor of the Cyanobacteria, Actinobacteria and Deinococcus-
Thermus group (shaded box), the inferred number of COGs is shown.
For the extant species, the number of tdCOGs, the number of proteins
in tdCOGs (in parentheses), and the number of ‘‘free’’ (not assigned to
tdCOGs) proteins (plus sign) are shown.
Deinococcus Genome Analysis
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plant material. D-xylose, which forms xylan polymers, is a major
structural component of plant cell walls , and the presence of
genes for aldopentose (xylose)-degradation explains why D.
geothermalis is a persistent contaminant in paper mills .
Specifically, D. geothermalis contains genes encoding xylanases
(Dgeo_2723; Dgeo_2722), an ABC-type xylose transport system
(Dgeo_2691). Several of the genes that encode enzymes of xylose
metabolism form paralogous families (Table S4), most of which
form a cluster on the megaplasmid DG574 (Dgeo_2703-
Dgeo_2687), which also contains two gene clusters predicted to
be involved in carbohydrate utilization (Dgeo_2669-Dgeo_2693,
Dgeo_2832-Dgeo_2812). By comparison, there are no large
clusters of functionally related genes on the D. geothermalis
chromosome; approximately 80 and 120 encoding proteins
involved in sugar-metabolism were identified on DG574 and the
chromosome, respectively. The putative xylose metabolism
functions of D. geothermalis appear to represent an expansion of
a pre-existing, broad and diverse set of functions underlying the
saccharolytic phenotypes of all Deinococci [7,11,49,50]. In contrast,
D. radiodurans has a proteolytic lifestyle, where a loss of various
accompanied by a gain of several predicted peptidases (DR0964,
DR1070, DR2310, DR2503) and a urease system (DRA0311-
DRA0319) . Thus, the evolutionary processes underlying the
emergence of extreme resistance in Deinococci appear not to be
dependent on a particular set of genes for sugar- or nitrogen-
metabolism. In summary, these findings support that DG574 is
essential to the natural growth modes of D. geothermalis, which is
a proficient saccharolytic organism [7,49,50] and strengthen the
case that the megaplasmids in the Deinococcus-Thermus group are
major receptacles of horizontally acquired genes, as proposed
(Figure3)  was
Table 2. Ancestral expansions: paralogous gene families expanded in the Deinococcus lineage (DD) versus the Thermus lineage (TT)
Description COG numbers
Number of tdCOGs: in DD only/in
TT only/in TT and DD combined
Number of proteins
MutT-like phosphohydrolases (Nudix) COG0494 COG10513/2/6 12/18/8
Calcineurin-like phosphoesteraseCOG0639 COG1408 COG1768 COG1692 7/0/412/11/4
Lipase-like alpha/beta hydrolaseCOG0596 COG10736/0/613/16/5.5
Subtilisin-like proteaseCOG1404 2/0/4 7/10/3
Acetyltrasferases GNAT familyCOG0454 COG1670 12/0/722/33/7
DinB family (DNA damage and stress
COG2318 no COG7/0/29/13/2
Figure 3. Gene-gain and gene-loss for different functional groups for D. radiodurans and D. geothermalis. Designations of functional groups
(from the COG database): J–Translation, ribosomal structure and biogenesis; K–Transcription; L–DNA replication, recombination and repair; D–Cell
division and chromosome partitioning; O–Posttranslational modification, protein turnover, chaperones; M–Cell envelope and outer membrane
biogenesis; N–Cell motility and secretion; P–Inorganic ion transport and metabolism; T–Signal transduction mechanisms; C–Energy production and
conversion; G–Carbohydrate transport and metabolism; E–Amino acid transport and metabolism; F–Nucleotide transport and metabolism; H–
Coenzyme metabolism; I–Lipid metabolism; Q–Secondary metabolites biosynthesis, transport and catabolism; V–genes involved in stress response
and microbial defense.
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Further supporting the notion that a distinct set of metabolic
genes is not a prerequisite for high levels of radioresistance, there
are patent differences between sulfate and energy metabolism in D.
geothermalis and D. radiodurans. In agreement with previously
published results [7,11,51], the prototrophic D. geothermalis has
orthologs of the nadABCD genes that are required for nicotinamide
adenine dinucleotide (NAD) biosynthesis, whereas the auxotrophic
D. radiodurans lacks these genes and is dependent on an exogenous
source of this coenzyme [21,51]. Another example illustrating the
relationship in D. radiodurans between gene-loss and its growth
requirements is that of cobalamine (vitamin B12). Whereas D.
geothermalis and T. thermophilus are not dependent on B12 in
minimal medium, D. radiodurans can utilize inorganic sulfate as the
sole source of sulfur only when vitamin B12 is present .
Conversely, D. geothermalis has lost several genes for enzymes of
protoheme biosynthesis (HemEZY) , which in D. geothermalis
likely yields siroheme under the microaerophilic conditions which
predominate at the Toptof D. geothermalis; the solubility of dioxygen
in water at 50uC is significantly lower than at 32uC, the Toptof
There are also important differences between the systems for
enzymes implicated in energy transformation in D. geothermalis and
D. radiodurans. The D. geothermalis chromosome encodes two heme-
copper cytochrome oxidases of types ba3 and caa3 ; and
Dgeo_2704), known to be expressed under oxygen-limiting
conditions , is encoded by DG574. In contrast, D. radiodurans
encodes only the caa3 oxidase system (DR2616-DR2620), which
apparently was present in the Deinococcus-Thermus common
ancestor. Furthermore, D. geothermalis encodes genes for proteins
that comprise an assimilatory nitrite NAD(P)H reductase and
a molybdopterin-cofactor-dependent nitrate reductase system
(Dgeo2392-Dgeo_2389), which also is known to be expressed
under anaerobic conditions [56,57]; and D. geothermalis encodes
several predicted multi-copper oxidases (Dgeo_2590, Dgeo_2559,
Dgeo_2558) that are not present in D. radiodurans and are most
similar to homologs from nitrogen-fixing bacteria. Since nitrogen
fixation in D. geothermalis has not yet been studied, the possibility
remains that these enzymes are involved in dissimilatory anaerobic
reduction of nitrate or nitrite [58,59]. D. geothermalis, but not D.
radiodurans, also encodes a formate dehydrogenase, which is related
to nitrate reductase and has a possible role in energy transfer
under anaerobic conditions .
In general, the evolutionary trends in D.
radiodurans lineage appear to mimic closely those of the Deinococcus
lineage, which is evident from the analysis of expanded families of
paralogous genes (Table S5). In particular, proliferation of genes
for the Yfit/DinB family, Nudix enzymes, acetyltransferases of the
GNAT superfamily, and the a/b hydrolase superfamily was
observed (Table 2). Plausible resistance-related functions readily
can be proposed for these and other expanded families of
deinococci. For example, hydrolases might degrade oxidized
lipids; Yfit/DinB proteins might be involved in cell damage-
related pathways ; subtilisin-like proteases might degrade
proteins oxidized during irradiation [17,61]; and the Nudix-
related hydrolase, diadenosine polyphosphatase (ApnA), yields
adenosine, a molecule that has been implicated in cytoprotection
from oxidative stress and radiation [62,63].
Several families expanded in D. radiodurans are predicted to
possess functions potentially relevant to stress response, but are not
conserved in D. geothermalis; most likely, non-conserved families can
be disqualified as major contributors to the extreme IR and
desiccation resistance phenotypes. Families that are specifically
expanded in D. radiodurans include the TerZ family of proteins,
which are predicted to confer resistance to various DNA damaging
agents [64,65]; secreted proteins of the PR1 family, whose
homologs are involved in the response to pathogens in plants,
and resistance to hydrophilic organic solvents in yeast [66,67];
PadR-like regulators, which are implicated in the regulation of
amino acid catabolism and cellular response to chemical stress
agents and drugs [68–70]; TetR/AcrR transcriptional regulators,
which are involved in antibiotic resistance regulation ; and
KatE-like catalases, which would decompose hydrogen peroxide
[71–73]. In contrast, there are family expansions which are shared
by D. radiodurans and D. geothermalis, but have no obvious role in
radiation or desiccation resistance. These include SAM-dependent
metyltransferases (COG0500) and an uncharacterized family of
predicted P-loop kinases (COG0645). In some bacteria, homologs
of these kinases are fused to phosphotransferases that mediate
resistance to aminoglycosides .
Since the IR-, UV- and desiccation-resistance profiles of D.
radiodurans and D. geothermalis are identical (Figure 1) , the subset
of stress response genes in D. radiodurans that are not unique, but
exist in excess compared to D. geothermalis are unlikely to be
required for extreme resistance either (Figure 3). This subset
includes two Cu-Zn superoxide dismutases (SOD), a peroxidase,
two HslJ-like heat shock proteins, and many genes implicated in
antibiotic resistance (Table S5). Consistently, SodA and KatA of
D. radiodurans can be disrupted with almost no loss in radiation
resistance , and antibiotics have little effect on survival
following irradiation provided corresponding antibiotic resistance
genes are present [18,76–79].
The Deinococcus lineage
Considerable independent gene-
gain was detected in both D. geothermalis and D. radiodurans lineages
in several other functional categories including transcriptional
regulation, signal transduction, membrane biogenesis, inorganic
ions metabolism, and to a lesser extent DNA replication and repair
(Figure 3). In general, regulatory functions mirror the metabolic
and stress-response-related differentiation of these two species
outlined above. For instance, among the 12 genes for predicted
transcriptional regulators that apparently were acquired in the D.
geothermalis lineage, five are similar to ones known to be involved in
the regulation of sugar metabolism in other bacteria, two of the
RpiR family and three of the AraC family [80,81]. By contrast, D.
radiodurans has at least 25 unique genes for transcriptional
regulators: three of the ArcR family; 16 of the Xre family; one
of the CopG/Arc/MetJ family; and five of a species-specific
expanded family reported previously  that likely is responsible
for stress-response control [82-85]. Other potentially independent
gains involve genes predicted to be involved in signal transduction
systems. D. radiodurans, for example, encodes photochromic
histidine kinase, a protein that has been extensively studied in D.
radiodurans and plays a role in the regulation of pigment
biosynthesis [86,87],but is
Alternatively, D. geothermalis encodes a putative negative regulator
of sigma E, a periplasmic protein of the RseE/MucE family
(Dgeo_2271). So far, RseE/MucE-members have been detected
only in proteobacteria, where it regulates the synthesis of alginate,
an extracellular polysaccharide which plays a key role in the
formation of biofilms . D. geothermalis, however, likely does not
produce alginate itself since it has no orthologs of the genes of the
alignate pathway . On the other hand, D. geothermalis has
clusters of genes implicated in exopolysaccharide biosynthesis,
with the most notable cluster located on DG574 (Dgeo_2671-
Dgeo_2646). It seems likely that this cluster is involved in the
biosynthesis of exopolysaccharides, which might facilitate biofilm
formation in D. geothermalis, and the Dgeo_2271 protein could be
a regulator of this process. Overall, D. radiodurans encodes
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approximately 470 unique, uncharacterized proteins, for which no
function could be predicted, compared to approximately 286 such
proteins in D. geothermalis. Thus, an additional 756 unique,
uncharacterized genes of the Deinococcus lineage can be excluded
from the pool of putative determinants of the extreme IR, UV and
desiccation resistance phenotype.
Reassessment of the Genetic Determinants of
Previously Implicated in the Radiation Resistance of D.
Over the last two decades, extensive experimental
and comparative-genomic analyses have been dedicated to the
identification and evolutionary origin of the genetic determinants
of radiation resistance in D. radiodurans. Early on, it became evident
that the survival mechanisms underlying extreme radiation
resistance in D. radiodurans probably were not unique. In 1994,
for example, IR-sensitive D. radiodurans polA mutants were fully
complemented by expression of the polA gene from the IR-sensitive
E. coli ; and in 1996, UV-sensitve D. radiodurans uvrA mutants
were complemented by uvrA from E. coli , suggesting that these
recombination and excision repair genes are necessary but not
sufficient to produce extreme DNA damage resistance. Following
the whole-genome sequencing of D. radiodurans in 1999 ,
comparative-genomic analysis revealed many distinctive genomic
features that subsequently became the focus of high throughput
experiments, including the analysis of transcriptome and proteome
dynamics of D. radiodurans recovering from IR [46,91,92].
Surprisingly, the cellular transcriptional response to IR in D.
radiodurans appeared largely stochastic, and mutant analyses
confirmed that many of the highly induced uncharacterized
genes were unrelated to survival. So far, those correlative studies
have failed to produce a coherent, comprehensive picture of the
complex interactions between different genes and systems that
have been thought to be important for the resistance phenotype.
The complete sets of orthologous genes in D. radiodurans and D.
geothermalis are listed in Table S2. Within the subgroup of genes in
D. radiodurans previously implicated in resistance by transcriptional
induction following exposure to IR  (3 hours after irradiation
and displaying more than a 2-fold induction), 45% have no
othologs in D. geothermalis. This raises the possibility that many
genes induced in irradiated D. radiodurans do not functionally
participate in recovery, or that D. geothermalis carries a distinct set of
ofthe Genomic Features
resistance determinants. From the subgroup of putative resistance
genes lacking counterparts in D. geothermalis, we constructed D.
radiodurans knockouts of four representative genes: i) a ligase
predicted to be involved in DNA repair (DRB0100) ; ii)
a LEA76 desiccation resistance protein homolog (DR0105) ;
iii) a predicted protein implicated in stress response (DR2221)
; and iv) a protein of unknown function (DR0140) .
Homozygous disruptions of each of these genes in D. radiodurans
(Figure S6) had no significant effect on IR resistance (Figure 4).
By contrast, most of the genes whose mutants display radiation-
sensitive phenotypes in D. radiodurans [4,20,46,92,93] are con-
served in D. geothermalis. To date, 15 single-gene mutants of D.
radiodurans have been reported to be moderately to highly
radiation-sensitive; of these, 13 genes have orthologs in D.
geothermalis (Table 3). The exceptions are DR0171 and DR1289,
which encode the DNA helicase RecQ and a transcriptional
regulator, respectively (Table 3). Remarkably, 10 of the 15 genes
are conserved in other bacteria and are well-characterized
components of DNA repair pathways. However, 5 of the 15
genes (DR0003, DR0070, DR0326, DR0423, DRA0346) are
unique to the Deinococcus lineage, supporting the existence of at
least a few novel resistance mechanisms.
Given that the two Deinococcus species are equally resistant to IR
(Figure 1A), genes dedicated specifically to the extreme radiation/
desiccation response are expected to belong to the set of tdCOGs.
D. radiodurans and D. geothermalis share 231 tdCOGs that are
relatively uncommon in other prokaryotes, and 63 of these are
unique to the Deinococcus lineage. Using the most sensitive methods
available to predict function, we reanalyzed these tdCOGs by
using a remote sequence similarity search, and genomic context
analysis [94–96]. Interpretation of such analyses, however, is
constrained by the complexity and ambiguities inherent in the
approach, and by the knowledge base. In contrast, many cytosolic
proteins (e.g., RecA, PolA, SodA and KatA) are known to be
intimately involved in resistance, so we present functional
predictions for 50 genes (Table S6). Among the predictions for
cytosolic proteins, several are new and potentially relevant to
resistance. For example, DR0644 (Figure 5A) is predicted to be
a distinct Cu/Zn superoxide dismutase that could defend against
metabolism-induced oxidative stress during recovery (Table S7);
and DR0449 (Figure 5B) is a divergent member of the RNAse H
family that is fused to a novel domain, a combination that is
currently unique to Deinococcus. Other functional insights were for
Figure 4. IR resistance of wild-type (ATCC BAA-816) and D. radiodurans mutants lacking orthologs in D. geothermalis (DSM 11300). Survival
values following 9 kGy (60Co) are from three independent trials with standard deviations shown. The structure of the homozygous mutants DRB0100,
DR2221, DR105 and DR0140 are presented in Figure S6.
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Table 3. D. radiodurans genes implicated in radiation resistance
Description and Comments
 ; [46,92]
Holliday junction resolvasome, helicase subunit, RuvB.
RecQ family of DNA helicase. The mutant is sensitive to IR, UV, H2O2and MMC. In
D. geothermalis there is a protein Dgeo_1226, which contains one Helicase
superfamily C-terminal domain and one HDRC domain, which are similar to the
corresponding domains of DR1289, but not the complete DR1289 ortholog.
Excinuclease ATPase subunit, UvrA.
Helicase subunit of the DNA excision repair complex, UvrB.
DNA gyrase (topoisomerase II) A subunit.
DNA gyrase (topoisomerase II) B subunit.
Tellurium resistance protein TerB.
Tellurium resistance protein TerZ/TerD.
; This work
Tellurium resistance protein TerZ/TerD.
CinA ortholog, MoeA family.
29R59 RNA ligase, LigT.
Ro-like RNA binding protein.
Molecular chaperone (small heat shock protein).
NRAMP family membrane transporter.
Uncharacterized conserved protein, two low-complexity regions.
Uncharacterized conserved protein.
Regulatory Zn-dependent protease fused to HTH transcriptional regulator domain.
Zn-dependent protease, HTPX superfamily.
Predicted protein, probably secreted.
Predicted low-complexity protein.
Predicted DNA single-strand annealing protein, containing HHH motif, Rad42/
Uncharacterized conserved protein, probably secreted.
Uncharacterized DsbA-like thioredoxin fold protein.
HTH transcription factor, CAP family.
Uncharacterized protein conserved in bacteria.
Cytochrome C-related, CXXC motif.
HTH transcription factor, phage type.
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Description and Comments
PprA protein, involved in DNA damage resistance mechanisms.
Bacteria Archaea Eukarya
[46,92] ; This work;
Homolog of eukaryotic DNA ligase III.
HicB family protein.
HTH transcriptonal regulator, specific for DR.
Yellow protein (Drosophila) or royal jelly protein (honey bee).
Acyl-CoA-binding protein, ACBP.
Archaea Bacteria Eukarya
LEA14-like desiccation-induced protein.
Archaea Bacteria Eukarya
Desiccation-induced protein. The mutant is resistant to radiation but sensitive to
LEA76/LEA26-like desiccation-induced protein. The mutant is resistant to radiation
but sensitive to desiccation.
; This work
LEA76/LEA26-like desiccation-induced protein.
; This work
Protein kinase of RIO1 family.
Homolog of a tymocyte protein cThy28kD.
Uncharacterized protein, uma2.
DNA polymerase of the X family with C terminal PHP hydrolase domain.
RecR, the mutant is sensitive to DNA interstrand cross-linking agents but resistant
to UV and IR.
DNA Polymerase A, PolA.
Diverged LexA homolog. Has a distinct DNA binding domain. Its mutant is slightly
more resistant to IR.
PLP-binding enzyme fused to HRD domain.
AAbbreviations: DR, D. radiodurans; DG, D. geothermalis.
BInduction in DR whole-genome microarrays reported by Tanaka et al  versus DR microarray results by Liu et al ; +, induced; 2, not induced; NA, microarray result is not available.
CMutant phenotype: +, IR sensitive; 2, IR resistant; n/a, not applicable. Corresponding mutant in D. radiodurans reported as referenced.
DReferences include original papers where the gene was inferred to be involved in radiation resistance or the corresponding mutant of the gene has been studied.
Table 3. cont.
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DR0041/Dgeo_0188, that is a paralog of DR0432 (DdrA)
(Figure 5C); and a member of the RAD22/Rad52 family
(Figure 5C) of single-stranded annealing proteins , that yields
a moderately sensitive phenotype in D. radiodurans upon disruption
. Interestingly, the radiation-sensitive T. thermophilus encodes
a homolog of DdrA (TTC1923), indicating that this protein had
an ancestral role that was not directly related to radiation
resistance. Notably, we continue to find proteins in Deinococcus
species which are only remotely similar to well-characterized
enzymes in other organisms, and it is difficult to predict their role
in the cell or radiation resistance. For example, we have identified
a protein that is conserved in both D. geothermalis and D. radiodurans
and is distantly related to enzymes of the QueF/FolE family,
which are involved in queuosine/folate biosynthesis (Figure 5D),
Figure 5. Multiple alignments of selected families conserved in two Deinococcus species. The multiple alignments were constructed for
selected representative sets of sequences by the MUSCLE program . Where necessary, alignments were modified manually on the basis of PSI-
BLAST outputs . The positions of the first and the last residue of the aligned region in the corresponding protein are indicated for each sequence.
The numbers within the alignment refer to the length of inserts that are poorly conserved between all the families. Secondary structure elements are
denoted as follows: E-b-strand; and H-a-helix. The coloring scheme is as follows: predominantly hydrophobic residues are high-lighted in yellow;
positions with small residues are in green; positions with turn-promoting residues are in cyan; positions with polar residues are in red; hydroxyl-group
containing residues are in blue; aromatic residues are in magenta; and invariant, highly conserved groups are in boldface. A, DR0644-Dgeo_0284
conserved pair of orthologs belong to the copper/Zinc superoxide dismutase family; shaded letters refer to amino acids that play an important role in
the Cu2+/Zn2+coordination environment and in the active site region. The bottom line shows the correspondence between the most conserved
regions corresponding to the b-stand structural core and conserved in most family members as denoted in Bordo et al . B, Dgeo_0137-DR0449
are highly specific for the Deinococcus lineage proteins that have an RNAse H-related domain. Catalytic residues conserved in the RNAse H family are
shaded. Secondary structure elements are shown for E. coli RNase HI (PDB:2rn2). C, DR0041-Dgeo_0188 is another conserved pair (DdrA-related) of
proteins belonging to the Rad52 family of DNA single-strand annealing proteins . Secondary structure elements are shown for human RAD52
(PDB:1KN0) . sak is a phage gene described previously ; D, DR0381-Dgeo_0373 are diverged homologs of NADPH-dependent nitrile
reductase (GTP cyclohydrolase I family) that might be involved in nucleotide metabolism. The conserved Cys and Glu found in the substrate binding
pocket of both protein families and specific zinc-binding and catalytic residues in the FolE family are shaded. The QueF family motif is boxed. Other
catalytic residues in FolE not found in QueF are in yellow. Genbank Identifier (gi) numbers are listed on the right.
Deinococcus Genome Analysis
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but their role in the Deinococci remains undefined. Collectively,
these results support the conclusion that many genes that are
significantly induced in irradiated D. radiodurans are not involved in
recovery (Table 3). Thus, the genome of D. geothermalis is a resource
of major importance in delineating a reliable minimal set of
resistance determinants, by corroborating those that are conserved
and ruling out those which are unique.
Delineation of the Deinococcus Radiation Response
A potential radiation-desiccation response regulon
and the corresponding regulator common to D. radiodurans and
D. geothermalis were identified using the approach developed by
Mironov et al [99,100]. In the search for such a regulator, we used
a training-set comprised of sequences flanking D. radiodurans genes
that were strongly upregulated by IR, and for which the
corresponding mutants were radiosensitive (Table 3) . The
upstream regions of several genes from the training set (DR0326,
ddrD; DR0423, ddrA; DRA0346, pprA; DR0070, ddrB) revealed
a strong palindromic motif, designated the radiation/desiccation
response motif (RDRM). Using a positional weight matrix, the
RDRM was used to generate the initial profile and to scan the
entire D. radiodurans genome. This genome survey picked up
a similar motif in the upstream regions of other genes upregulated
after irradiation . The upstream regions with the highest
scores (DR0219, DR0906, DR1913 and DR0659) were then used
to better define the RDRM, and the complete genomes of D.
radiodurans and D. geothermalis were scanned with the updated motif.
Using moderately relaxed parameters (Materials and Methods),
approximately 120 genes in each of the Deinococcus genomes were
selected by the screen. The final, most conservative prediction of
the radiation/desiccation response (RDR) regulon consisted of two
groups: (i) a set of orthologous genes present in both Deinococcus
species that contain the RDRM; and (ii) a set of unique genes of D.
radiodurans that contain the RDRM and are upregulated during the
recovery from irradiation [46,92]. Since microarray data for D.
geothermalis are not available, it was not possible to predict a set of
unique RDRM-dependent genes for this species. Table 4 lists the
set of genes predicted to comprise the regulon together with the
corresponding RDRM sites (Figure 6). Collectively, the RDR
regulon is predicted to consist of a minimum of 29 genes in D.
radiodurans and 25 genes in D. geothermalis, contained within 20
operons in each species.
The RDR regulon is dominated by DNA repair genes,
including the recombinational repair proteins RecA and RecQ
[101,102]; the mismatch repair proteins MutS and MutL, that are
located in one operon in D. geothermalis; and the UvrB and UvrC
proteins, which are involved in nucleotide excision repair (Table 4).
In addition, the predicted RDR regulon includes the transketolase
gene. In bacteria, transketolase is a key enzyme of the pentose-
phosphate pathway for carbohydrate metabolism and is known to
be induced by a variety of stress conditions including cold shock,
and mutagens that trigger the SOS response . Moreover, the
pentose-phosphate pathway in D. radiodurans is reported to
facilitate DNA excision repair induced by UV irradiation and
hydrogen peroxide (H2O2) . The RDRM also precedes
a conserved histidine catabolism operon . Several bacterial
biodegradative and related operons are known to be differentially
induced in response to a decline in biosynthetic and energy-
generating activities under oxidative stress . For example, the
TCA cycle in D. radiodurans is strongly down-regulated following
irradiation , whereas the glyoxylate bypass of the TCA cycle,
and the His operon are induced . Several studies have
provided direct evidence that survival of D. radiodurans following
exposure to IR depends on a coordinated metabolic response and
a high level of respiratory control [46,107].
The regulation of gene expression in D. radiodurans during
recovery from IR has been the subject of considerable in-
vestigation. Recently, it has been shown that the induction of recA
in irradiated D. radiodurans is regulated by the IrrE/PprI protein
[108,109], which consists of two domains, a Xre-like HTH
domain and a Zn-dependent protease. In both D. radiodurans and
D. geothermalis, the irrE gene is located upstream of the folate
biosynthesis operon, but appears to be regulated independently
. Since recA in D. radiodurans is strongly induced following
irradiation [46,111], it was surprising that the irrE gene of D.
radiodurans was constitutively expressed, showing no post-irradia-
tion induction [46,92,110]. Furthermore, the IrrE/PprI protein
has an unusual domain structure and does not appear to bind the
promoter region of recA or other induced genes .
Compared to radiosensitive bacteria, the regulatory mech-
anisms underlying the response to radiation in D. radiodurans are
still poorly characterized. For example, the LexA-regulated SOS-
dependent radiation response regulon of E. coli is well-defined
[103,112–115], but an equivalent system in D. radiodurans has not
been identified. D. geothermalis has one lexA gene (DG1366) and D.
radiodurans has two lexA paralogs (DRA0344, DRA0074). However,
the lexA genes in D. radiodurans are not induced after irradiation,
are not involved in RecA induction , and are not preceded by
RDRM sites [46,92]. Therefore, LexA is not a candidate for the
role of the regulator of the Deinococcus RDR regulon. In the
microarray experiments of Liu et al, several putative regulators
were upregulated in D. radiodurans following exposure to 15,000 Gy
. In contrast, at lower doses (3,000 Gy), the D. radiodurans
microarray experiments of Tanaka et al detected only one
upregulated putative regulator (DdrO) (DR2574) . An
orthologous gene for DdrO is present in D. geothermalis
(Dgeo_0336). DdrO is a Xre family protein and is the only
Deinococcus gene for a predicted regulator that is preceded by
a RDRM site (Table 4). This arrangement is common to many
stress response regulators, e.g., the lexA genes of many species
. Thus, we propose that DdrO is the global regulator of the
RDR regulon in the Deinococcus lineage.
Impact of the comparative-genomic analysis of the
two Deinococcus genomes on Resistance Models
In 1971, Moseley and Mattingly reported the first mutant analyses
for D. radiodurans that showed that its recovery from radiation is
dependent on DNA repair . Subsequent research confirmed
that DNA repair enzymes, which are central to recovery of
irradiated bacteria in general, were key to D. radiodurans survival.
Remarkably, several highly radiation-sensitive D. radiodurans DNA
repair mutants were fully complemented by expression of
orthologous genes from radiosensitive bacteria [4,90,119–121].
Thus, the extreme resistance phenotype appeared to be de-
pendent, at least in part, on a conventional set of DNA repair
functions [5,17,21]. Generally, this view has been supported by the
analysis of the complete genome sequence of D. radiodurans ,
and subsequently, by whole-transcriptome and whole-proteome
analyses for D. radiodurans recovering from IR [46,91,92]. Central
to current models of extreme resistance are hypotheses that aim to
reconcile the seemingly paradoxical findings that DNA repair
proteins in D. radiodurans function extremely efficiently, yet appear
structurally unremarkable, and often can be complemented by
orthologs from radiosensitive bacteria. Within this conceptual
framework, we examined the impact of the inferences on gene-
gain and gene-loss derived from the comparative-genomic analysis
of the two Deinococcus species on prevailing models of extreme
radiation and desiccation resistance.
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Table 4. The predicted radiation and desiccation resistance regulon of Deinococci
Site in DR
Site in DG
Description and Comments
Uncharacterized conserved protein
Single-stranded DNA-binding protein
DNA gyrase (topoisomerase II) A subunit
Similar to DR1142, but with a frameshift
DNA gyrase (topoisomerase II) B subunit
Predicted DNA single-strand annealing protein, containing
a HHH motif, Rad42/Rad22/RecT/erf family
Predicted low complexity protein
PprA protein, involved in DNA damage resistance
DNA mismatch repair ATPase MutS
DNA mismatch repair enzyme, Hexb/MutL
UvrD Superfamily I helicase
Helicase subunit of the DNA excision repair complex, UvrB
Holliday junction resolvasome, helicase subunit, RuvB
CinA ortholog, MoeA family, first gene in operon containing
RNA ligase ligT and RecA
Excinuclease ATPase subunit, UvrA
HTH transcription factor, phage type
Urocanate hydratase (and three more genes in the same
operon for histidine degradation)
Uncharacterized DsbA-like thioredoxin fold protein
SbcD, DNA repair exonuclease
HTH transcriptional regulator
Ro-like RNA binding protein
AAbbreviations: DR, D. radiodurans; DG, D. geothermalis.
BInduction in whole-genome microarrays reported by Tanaka et al .
CInduction in whole-genome microarrays reported by Liu et al .
DIn D. geothermalis, MutS and MutL are in the same operon, therefore RDRM information is shown only for Dgeo_1537 (the first gene in the operon).
*RDRM sites included in the final profile were used to scan the genomes of D. radiodurans and D. geothermalis.
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Hypothesis I: Chromosome Alignment, Morphology and
Repeated Sequences Facilitate Genome Reassembly
dependent homologous recombination occurs at hundreds of IR-
induced DSB sites in D. radiodurans during recovery from 17.5 kGy
IR [18,76–79]. In D. radiodurans, the alignment of its multiple
identical chromosomes is often tacitly assumed as the starting point
for a given repair model, yet little is known about how, or even if,
such chromosomal alignment occurs. The first model that
considered this possibility in the recovery of D. radiodurans was
published by Minton and Daly in 1995 . The model built on
the idea that alignment of identical chromosomes is a natural and
early consequence ofsemi-conservative
persistent chromosomal pairing was predicted to facilitate the
‘search for homology’ that precedes homologous recombination.
The model made two major predictions: first, transmission
electron microscopy (TEM) of chromosomal DNA from D.
chromosomes; and second,
separated genomic locations should show strong positional effects
upon irradiation. Both predictions have been tested and refuted:
no linking structures have been observed by TEM-based optical
mapping , and molecular studies have shown high levels of
recombination between homologous DSB fragments irrespective
of their genomic origin [76–79,122]. Thus, it has been concluded
that IR-induced DSB fragments in D. radiodurans are mobile and
that the structural form of its nucleoids does not play a key role in
radioresistance. These conclusions were subsequently strengthened
by cryoelectron microscopy of vitreous sections of D. radiodurans
[123,124], and by nucleoid morphology studies [5,12,24,125].
The genome of D. radiodurans contains numerous, unusual,
mosaic-type SNRs [16,21,29] which potentially could contribute to
genome assembly by holding together homologous DSB pairs .
showed that IR-induced DSB fragments in D. radiodurans were not
linked . Consistently, the present whole-genome comparison
detected none of these repeats in D. geothermalis, nor any other
expanded repeat families, including G-quadruplex sequences
(Table 1) (Figure S3). We did not identify any unusual features in
chromosome-binding proteins that are conserved in the two
Deinococcus genomes compared to the orthologous proteins from
other bacteria  (Table S7 and S8). Thus, our comparative
analysis does not seem to support Hypothesis I. More broadly, there
that structural alignment, aggregation or organization of the D.
radiodurans chromosomes has a significant effect on radiation/
desiccation resistance. However, we cannot rule out the possibility
that the genomes of sensitive bacteria have structural characteristics
that predispose them to inefficient genome reassembly.
Hypothesis II: A Subset of Uncharacterized Genes Encode
Functions that Enhance the Efficiency of DNA repair
general, bioinformatic and experimental studies suggest that genome
configuration and copy-number or the protection and repair
functions of sensitive bacteria do not have unique properties that
predispose them to DNA damage or inefficient DNA repair
[5,20,21]. More specifically, chromosomes in sensitive and
resistant bacteria are equally susceptible to IR-induced DSB
damage [5,19] and UV-induced base damage ; and DNA
repair and protection genes of T. thermophilus, a radio-sensitive
representative of the Deinococcus-Thermus group, and E. coli do not
show obvious differences from their counterparts in D. radiodurans or
D. geothermalis [5,21,27] (Table S8). Furthermore, several E. coli DNA
repair genes, including polA and uvrA, have been shown to restore the
corresponding radiation-sensitive D. radiodurans mutants to wild-type
levels of D. radiodurans resistance [4,90,120]; and the products of
irradiation are consistent with the canonical version of the DSB
repair model [76–79]. It has been proposed that D. radiodurans uses
a coreset of conventional DNA repairenzymes innovel ways, where
proteins. For example, Zahradka et al have recently proposed
a model called extended synthesis dependent strand annealing
(ESDSA) that utilizes PolA in an unprecedented way .
Under the ESDSA, DSB fragments formed in irradiated D.
radiodurans are first subject to a 59R39 exonuclease resection
mechanism that generates overhanging 39 tails. A 39 tail then
invades a homologous DSB fragment derived from a different
chromosomal copy, displacing the corresponding 59 strand as
a loop. Synthetic extension of the priming 39 terminus might then
proceed to the end of the invaded fragment, followed by annealing
of the newly synthesized long 39 extension with a complementary
strand of another fragment engaged in ESDSA (Figure S7). For
example, if the sequences of two priming fragments were ABCD
and GHIJ, then a bridging and templating fragment could be
DEFG, and the sequence of the assembled contig would be
ABCDEFGHIJ . The ESDSA model accounts for the
formation of large, interspersed blocks of old and new DNA
observed in repaired D. radiodurans chromosomes. Some aspects of
the ESDSA model, however, are difficult to reconcile with earlier
experimental findings for recA-independent single-stranded anneal-
ing (SSA) mechanisms in irradiated D. radiodurans  (Figure S7).
Zharadka et al conceded that the SSA model is a potential
alternative to ESDSA and could perhaps generate small blocks of
old and new DNA , but pointed out that the E. coli PolA
Klenow fragment, that lacks the 59R39 exonuclease, fully
complements D. radiodurans polA mutants for resistance to c-
radiation. The present analysis shows that, although D. radiodurans
and D. geothermalis do not encode recBCE, they both encode recJ,
in D. radiodurans following
Figure 6. Sequence signature of a predicted site of a radiation response regulator. Four different nucleotides are shown by four letters (A, G, C, T)
in different colors. The height of the letter is proportional to its contribution to the information content in the corresponding position of the multiple
alignment used for ‘‘sequence logo’’ construction. The figure was constructed by the ‘‘sequence logo’’ program described previously .
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a putative 59R39exonuclease that could potentially provide
nuclease activity missing in the Klenow fragment (Table S8).
The possibility that extreme resistance in D. radiodurans is
determined by novel genes that enhance conventional repair
functions has also been examined [20,46,98]. At least 12 genes of
D. radiodurans, which were implicated in resistance by transcrip-
tional profiling following IR, have been knocked out and the
resulting mutants were characterized for IR resistance (Table 3).
Remarkably, for most of the novel mutants, the IR resistances
remained high [20,46,98], indicating that few of the uncharacter-
ized genes, at least individually, makes a substantial contribution
to the recovery of irradiated D. radiodurans. For example, the
DR0423 protein has been reported to bind 39 ends of single-
stranded DNA molecules, perhaps, protecting 39 termini gener-
ated by SSA or ESDSA from nuclease degradation. A DR0423
knockout mutant, however, retained approximately half of the
wild-type level of IR resistance [92,98]. To date, only a few of the
uncharacterized genes selected for disruption analysis have
contained the RDRM (Table 3 and 4).
At least three Deinococcus proteins involved in repair show
features that stand out against the overall, ‘‘garden-variety’’ of
bacterial repair systems. First, D. radiodurans encodes a protein
(DR1289) of the RecQ helicase family, which contains three
Helicase and RNase D C-terminal (HRDC) domains, whereas
most of the other bacterial RecQ proteins have a single HRDC
domain. A D. radiodurans recQ knockout mutant is sensitive to IR,
UV, H2O2, and MMC, and it has been reported that all three
HRDC domains contribute to resistance . However, D.
geothermalis has no ortholog of the D. radiodurans RecQ, but does
encode the Dgeo_1226 protein that contains a helicase superfam-
ily II C-terminal domain and a second HDRC domain that has
high similarity to the corresponding domains of DR1289. Both
DR1289 and Dgeo_1226 belong to the predicted resistance
regulon (Table 4). A second exceptional case is RecA, the key
repair protein that is required for homologous DNA recombina-
tional repair following irradiation . The DNA strand-exchange
reactions promoted by the RecA proteins from all other bacteria
studied to date are ordered such that the single-stranded DNA is
bound first, followed by the double-stranded DNA. In contrast, the
D. radiodurans RecA binds the DNA duplex first and the
homologous single-stranded DNA substrate second . It seems
likely, however, that these unusual properties of RecA are
ancestral to the Deinococcus-Thermus group. Indeed, most of the
amino acid residues that are distinct in Deinococcus and could be
responsible for the structural and functional differences between
the RecA proteins of Deinococcus and other bacteria are also present
in the RecA sequence of Thermus (Figure S8). In this context, early
work by Carroll et al  reported that E. coli RecA did not
complement an IR-sensitive D. radiodurans recA point-mutant
(rec30) and that expression of D. radiodurans RecA in E. coli was
lethal. More recently, however, it has been reported that E. coli
recA can provide partial complementation to a D. radiodurans recA
null mutant , and that D. radiodurans recA fully complements E.
coli recA mutants . This suggests that the D. radiodurans RecA
protein is not as unusual as initially believed, but rather is more
analogous to polA and uvrA of D. radiodurans, which can be
functionally replaced by E. coli orthologs [4,90,93,120]. A third
example, the Deinococcus single-stranded binding protein (Ssb) has
a distinct structure, with two OB-fold domains in a monomer, but
this feature was apparently already present in the common
ancestor of Deinococcus/Thermus group and therefore cannot be
linked to radiation resistance directly .
It has been repeatedly proposed that nonhomologous end-
joining (NHEJ) occurs in D. radiodurans [20,131–136]. However,
experiments specifically designed to test for the occurrence of
NHEJ in D. radiodurans have shown that NHEJ of irradiation-
induced DSB fragments is extremely rare, if not absent . More
recent work also supports this conclusion . In the present and
a previous study, we did not identify any orthologs of genes from
other organisms that might encode NHEJ in D. geothermalis or D.
radiodurans . However, it cannot be ruled out that Deinococcus
encodes a unique NHEJ system. For example, DRB0100 encodes
an ATP-dependent ligase that contains domains that could
potentially contribute to NHEJ, namely, a predicted phosphatase
of the H2Macro superfamily and an HD family phosphatase and
polynucleotide kinase [46,92]. Furthermore, DRB0100 belongs to
a set of three genes comprising a putative operon (DRB0098-0100)
that is strongly induced by IR. A homozygous disruption of the
DRB0100 gene, however, is fully IR-resistant (Table 3) (Figure 4),
and genome comparison showed that D. geothermalis has no
orthologs of DRB0100 or any functionally related operons.
Despite the strong induction of DRB0100 following irradiation
and the apparent relevance of the predicted function of this
protein to D. radiodurans repair, DRB0100 appears not to
contribute to resistance (Figure 4), and when purified, does not
display DNA or RNA ligase activity in vitro . These findings,
therefore, reflect a broader paradox of Deinococcus: whereas
computational analyses have revealed an increasing number of
new proteins potentially involved in the extreme resistance
phenotype, very few of the corresponding D. radiodurans mutants
tested so far have had a significant effect on its IR resistance. The
present work leads to further shrinking of the set of genes
implicated as major contributors to the resistance phenotype by
showing that many of the original candidates are not conserved
between D. geothermalis and D. radiodurans. Thus, our comparative
analysis appears to be inconsistent with Hypothesis II, and
reinforces inferences from a growing body of experimental work
on Deinococcus species, which support that these organisms rely on
a relatively conventional set of DNA repair functions.
Hypothesis III: The level of Oxidative Protein Damage
during Irradiation Determines Survival
decade, several observations have challenged the DNA-centered
view of IR toxicity in eukaryotes and prokaryotes [5,17,23,138],
including (i) IR-induced bystander-effects in mammalian cells,
defined as cytotoxic effects elicited in non-irradiated cells by
irradiated cells, or following microbeam irradiation of cells where
the cytoplasm but not the nucleus is directly traversed by radiation
; (ii) the genomes of radiation-sensitive bacteria revealed
nothing obviously lacking in their repertoire of DNA repair and
protection systems compared to resistant bacteria [12,21]; and (iii)
for a group of phylogenetically diverse bacteria at the opposite
ends of IR resistance, the amount of protein damage, but not DNA
DSB damage, was quantifiably related to radioresistance [5,17].
Thus, while the etiological radicals underlying different oxidative
toxicities appear closely related , the pathway connecting the
formation of IR-induced ROS with endpoint biological damage is
still not definitively established . It has been proposed recently
that proteins in IR-sensitive cells are major initial targets, where
cytosolic proteins oxidized by IR might actively promote mutation
by transmitting damage to DNA , and IR-damaged DNA
repair enzymes might passively promote mutations by repair
malfunction . In comparison, Mn-dependent radioprotective
complexes in IR-resistant bacteria  appear to protect proteins
from oxidation during irradiation, with the result that enzymatic
systems involved in recovery survive and function with great
efficiency . The proposed mechanism of extreme IR resistance
requires a high intracellular Mn/Fe concentration ratio, where
redox-cycling of Mn(II) complexes in resistant bacteria [5,17]
Over the past
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scavenge a subset of IR-induced ROS that target proteins. Because Download full-text
the formation of ROS during irradiation is extremely rapid ,
an intracellular protection system that is ubiquitous, but not highly
dependent on the induction of enzymes, stage of growth, or
temperature over a range at which cells are metabolically active,
could provide a selective advantage to the host in diverse settings.
Since high intracellular Mn/Fe ratios have been implicated in
radiation and desiccation resistance [5,12,17,23], we examined the
intracellular concentrations and distributions of Mn, Fe and seven
other elements in D. geothermalis compared to D. radiodurans,
determined by x-ray fluorescence (XRF) microscopy (Figure 7)
. The XRF analyses showed that the intracellular levels of
Mn and Fe and their locations in D. geothermalis are essentially the
same as D. radiodurans , but very different from the
concentrations and distributions in IR-sensitive bacteria [5,142].
In this context, both D. radiodurans and D. geothermalis encode the
Mn(II) transporter Nramp (DR1709) and a putative Mn-de-
pendent transcriptional regulator TroR (DR2539) , but lack
many genes for Fe homeostasis common in other bacteria,
including for siderophore biosynthesis (COG3486, COG4264,
COG4771) and Fe transport (COG1629, COG0810) (Table S9)
. Consistently, D. radiodurans and D. geothermalis do not secrete
siderophores (Figure S9), the nramp gene of D. radiodurans is
essential and could not be disrupted, and the Fe uptake regulator
(Fur) in D. radiodurans was dispensable (Figure S10); a system for
gene disruption in D. geothermalis has not been developed. Other
recent work that has strengthened the argument for a critical role
of Mn(II) in the extreme resistance phenotypes of D. radiodurans
includes in vitro studies of Heinz and Marx . They have
shown that purified D. radiodurans PolA and E. coli PolA can bypass
certain forms of IR-induced DNA damage during replication in
the presence but not in the absence of 1 mM Mn(II), and
suggested that Mn(II) ions might serve as important modulators of
enzyme function . In summary, we conclude that our
genome comparison (Table S9), gene knockout (Figure S10) and
element analyses (Figure 7) appear to be consistent with
Hypothesis III, whereby survival is facilitated by systems which
regulate the concentration and distribution of intracellular Mn and
Fe. Based on recent work, it appears that the presence of globally-
distributed intracellular nonenzymic Mn(II) complexes in resistant
bacteria facilitates recovery by preventing a form of IR-induced
Fe-catalyzed protein oxidation known as carbonylation .
Based on their identical radiation resistance characteristics and
close phylogenetic relationship, D. geothermalis and D. radiodurans are
well-suited to defining a minimal set of conserved genes that could
be responsible for extreme resistance. The two major findings of
this analysis are (i) the characterization of the evolutionary trends
that led to the emergence of extreme stress resistance in the
Deinococcus lineage, in particular the finding that many families of
paralogous genes, previously shown to be expanded in D.
radiodurans, proliferated before the emergence of the common
ancestor of the Deinococci, but were not present in the ancestor of
the Deinococcus-Thermus group (Table 2); and (ii) delineation of a set
of genes that comprise the predicted Deinococcus radiation and
desiccation response regulon, which defines a new subgroup of
targets for investigation in the Deinococci (Table 4). These findings
have strengthened the view that Deinococci rely more heavily on the
high efficiency of their detoxifying systems, including enzymic and
nonenzymic ROS scavengers, than on the number and specificity
rule out the possibility that the exceptional efficiency of DNA repair
processes in both Deinococcus species is, at least in part, due to
Figure 7. X-ray fluorescence (XRF) microprobe element distribution
maps . A, D. geothermalis (diplococcus). B, D. radiodurans
(tetracocus). Cells were harvested from mid-logarithmic cultures in
undefined rich medium, imaged, and quantified as described previously
. The element distribution images are plotted to different scales
designated by a single color-box, where red represents the highest
concentration and black the lowest. ppm values in parentheses next to
the element symbol correspond to red. XRF microprobe analysis
measurements were made at beamline 2ID-D at the Advanced Photon
Source, Argonne National Laboratory as described recently .
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