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: email@example.com.
nih.gov (KM); firstname.lastname@example.org (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
PLoS ONE | www.plosone.org2September 2007 | Issue 9 | e955
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
PLoS ONE | www.plosone.org3 September 2007 | Issue 9 | e955
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
PLoS ONE | www.plosone.org4September 2007 | Issue 9 | e955
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.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org5 September 2007 | Issue 9 | e955
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
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org6 September 2007 | Issue 9 | e955
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.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org7 September 2007 | Issue 9 | e955
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.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org8 September 2007 | Issue 9 | e955
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.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org9 September 2007 | Issue 9 | e955
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
PLoS ONE | www.plosone.org 10 September 2007 | Issue 9 | e955
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.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org 11September 2007 | Issue 9 | e955
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.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org12 September 2007 | Issue 9 | e955
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 .
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org 13 September 2007 | Issue 9 | e955
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
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org 14September 2007 | Issue 9 | e955
scavenge a subset of IR-induced ROS that target proteins. Because
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 .
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org 15 September 2007 | Issue 9 | e955
modifications of a set of universal repair genes. With respect to the
impact of the whole-genome sequence of D. geothermalis on prevailing
models of extreme IR resistance, the results of the comparative
analysis weaken the arguments for a role of higher-order
chromosome alignment structures (Hypothesis I); more clearly
define and substantially revise downward the number of unchar-
acterized genes that might participate in DNA repair and contribute
to resistance (Hypothesis II); and are consistent with the notion of
a predominant role in resistance of systems involved in cellular
protection and detoxification (cell-cleaning) (Hypothesis III).
In the hierarchy of DNA lesions caused in vivo by radiation, DSBs
are the least frequent ones, but the most lethal . Since the
number of genomic DSBs induced by a given dose of IR in resistant
and sensitive bacteria is about the same [5,19], a legitimate
question is whether resistant and sensitive bacteria are also equally
susceptible to DNA base damage. Setlow and Duggan showed that
D. radiodurans and E. coli are similarly susceptible to DNA thymine-
dimers caused by UV . For IR and UV, the differences
reported in resistance of DNA to radiation damage are not nearly
sufficient to account for the relative resistance of D. radiodurans.
Thus, it seems surprising that the recombination and excision
repair systems of D. geothermalis and D. radiodurans did not proliferate
compared to sensitive cells . The DNA repair and damage
signaling systems of these radiation reisistant bacteria appear
quantitatively and qualitatively even less complex and diverse than
those reported for some sensitive bacteria [5,144]. Instead, the
stress-resistance phenotypes of the Deinococcus lineage appear to
have evolved progressively by accumulation of cell-cleaning
systems which eliminate organic and inorganic cell components
that become toxic under radiation or desiccation [12,23,46,92]. In
D. geothermalis and D. radiodurans, this form of cell-cleaning appears
to manifest itself as protein protection during exposure to IR 
or desiccation [JFK, EKG, MJD, unpublished], where proteins in
Deinococci are substantially more resistant to oxidative damage than
proteins in sensitive bacteria . Our finding that many genes in
the predicted Deinococcus damage response regulon are the same as
those found in SOS regulons of sensitive bacteria, but are regulated
differently, is easily reconciled with the idea that enzymes and
biochemical pathways in resistant bacteria survive and function
more efficiently because they are less prone to interference from the
toxic byproducts of IR and desiccation [12,17,23].
More generally, our findings place constraints on the degree to
which functional inferences can be made from whole-genome
transcriptome analyses based on a single organism. For example,
two independent analyses of gene induction in D. radiodurans
recovering from different IR doses revealed numerous genes that
are upregulated during the post-irradiation recovery, many of
which were viewed as plausible candidates for a significant role in
resistance [46,92]. The hierarchy of induced genes in both
transcriptome analyses was very similar, however, most of the
highly induced D. radiodurans genes have no orthologs in D.
geothermalis, and knockout of many of the uncharacterized unique
D. radiodurans genes that were strongly induced by IR had little
effect on IR resistance. A similar paradigm is emerging from the
analysis of other systems, where the cellular transcriptional
response to stress was largely stochastic, frequently involving
genes known to be unrelated to the mechanisms under in-
vestigation [145-147]. Thus, it stands to reason that any
comprehensive bioinformatics effort aimed at deciphering a com-
plex, multi-gene phenotype using whole-genome, transcriptome
and proteome approaches should aim to study at least two closely-
related species. In the present context of understanding the
genomic basis of extreme resistance phenotypes and the nature of
the common ancestor of the Deinococcus-Thermus group, we consider
Truepera radiovictrix an appropriate next candidate for whole-
genome sequencing. T. radiovictrix is a recently discovered, deeply
branching representative of the Deinococcus branch that is both
thermophilic and extremely IR-resistant .
MATERIALS AND METHODS
The strains used were as follows: Deinococcus radiodurans (ATCC
BAA-816), Deinococcus geothermalis (DSM 11300), and Escherichia coli
Cell Growth, Irradiation, Mutant Construction, and
D. radiodurans strain ATCC BAA-816 was grown at 32uC in
undefined liquid nutrient-rich medium TGY (1% tryptone/0.1%
glucose/0.5% yeast extract) or on TGY solid medium . In liquid
culture, cell density was determined at 600 nm by a Beckman
Coulter spectrophotometer. For acute IR (60Co Gammacell
irradiation unit, J. L. Shepard and Associates, Model 109) or UV
(254 nm) exposures, late logarithmic-phase D. radiodurans cultures
[OD600=0.9, 16108colony-forming units (cfu)/ml] were irradiated
to the indicated doses (Figure 1). Cell viability and cell numbers were
determined by plate assay as described previously . Three
independent cellcultures andirradiation treatments of the samekind
were performed and served as biological replicates for determining
irradiation resistance profiles. To test the predicted involvement of
the indicated genes, a mutant (Figure S6) was generated using
previously developed D. radiodurans disruption protocols . PCR
was carried out as described previously .
Whole-Genome Sequencing, Assembly and
The complete genome of D. geothermalis (DSM 11300) was
sequenced at the Joint Genome Institute (JGI) using a combination
of 3 kb-, 8 kb- and fosmid- (40 kb) libraries. Library construction,
sequencing, finishing, and automated annotation steps were
carried out as follows.
DNA shearing and sub-cloning
isolated DNA was randomly sheared to 3 kb fragments in a 100 ml
volume using a HydroShearTM(Genomic Solutions, Ann Arbor,
MI). The sheared DNA was immediately blunt end-repaired at
room temperature for 40 min using 6 U of T4 DNA Polymerase
(Roche Diagnostics, Indianapolis, IN), 30 U of DNA Polymerase I
Klenow Fragment (NEB, Beverly, MA), 10 ml of 10 mM dNTP
mix (GE Healthcare, Piscataway, NJ), and 13 ml of 106Klenow
Buffer in a 130 ml total volume. After incubation, the reaction was
heat-inactivated for 15 min at 70uC, cooled to 4uC for 10 min,
and then frozen at 220uC for storage. The end-repaired DNA was
run on a 1% Tris/Borate/EDTA (TBE) agarose gel for ,60 min
at 120 volts. Using ethidium bromide stain and UV illumination,
3 kb sheared fragments were extracted from the agarose gel and
purified using QIAquickTM
Gel Extraction Kit (QIAGEN,
Valencia, CA). Approximately 300 ng of purified fragment was
blunt-end-ligated overnight at 16uC into the Sma I site of 100 ng of
pUC18 cloning vector (Roche) using 12 U T4 DNA Ligase, 3.2 ml
106buffer (Roche), and 4.8 ml 30% PEG in a 32 ml total reaction
volume. A very similar process was carried out to create an 8 kb
library in pMCL200 with 10 mg of isolated genomic DNA.
Following standard protocols, 1 ml of each ligation product
(3 kb or 8 kb) was electroporated into DH10B ElectromaxTMcells
(Invitrogen, Carlsbad, CA) using the GENE PULSERH II
electroporator (Bio-Rad, Hercules, CA). Transformed cells were
Approximately 3–5 mg of
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org16 September 2007 | Issue 9 | e955
transferred into 1 mL of SOC medium and incubated at 37uC in
a rotating wheel for 1 h. Cells (usually 20–50 ml) were spread on
22622 cm LB agar plates containing 100 mg/mL of ampicillin
(pUC19) or 20 mg/mL of chloramphenicol (pMCL200), 120 mg/
mL of IPTG, and 50 mg/mL of X-GAL. Colonies were grown for
16 h at 37uC. Individual white recombinant colonies were selected
and picked into 384-well microtiter plates containing LB/glycerol
(7.5% v/v) media containing 50 mg/mL of ampicillin or 20 mg/
mL of chloramphenicol using the Q-BotTMmultitasking robot
(Genetix, Dorset, U.K.). To test the quality of the library, 48
colonies were directly PCR-amplified with pUC m13–28 and –40
primers using standard protocols. Libraries passed PCR quality
control if they had .90% 3 kb inserts or 8 kb inserts, respectively.
For more details, see research protocols at www.jgi.doe.gov.
One ml-aliquots of saturated E. coli
cultures (DH10B) containing (pUC19 vector with random 3 kb
DNA inserts or pMCL200 vector with random 8 kb DNA inserts)
were added to 5 ml of a 10 mM Tris-HCl pH 8.2 and 0.1 mM
EDTA denaturation buffer. The mixtures were heat-lysed at 95uC
for 5 min then placed at 4uC for 5 min. To these denatured
products, 4 ml of a rolling circle amplification (RCA) reaction
mixture (TempliphiTMDNA Sequencing Template Amplification
Kit, GE Healthcare) were added. The amplification reactions were
carried out at 30uC for 12–18 h. The amplified products were
heat-inactivated at 65uC for 10 min then placed at 4uC until used
as template for sequencing .
Aliquots of the 10 ml amplified plasmid
RCA products were sequenced with standard pUC m13–28 or –40
primers. The reactions typically contained 1 ml of the RCA product,
sequenced with 4 pmoles (1 ml) of standard M13–28 or –40 primers,
0.5 ml 56buffer, 1.75 ml H2O, and 0.75 ml BigDye sequencing kit
30 sec,50uCfor20 sec,60uC for4 min,and finallyheldat 4uC.The
reactions were then purified by a magnetic bead protocol (see
research protocols, www.jgi.doe.gov) and run on an ABI PRISM
3730xl (Applied Biosystems) capillary DNA sequencer.
Fosmid Library Construction
isolated DNA was randomly sheared to 40 kb fragments (25 cycles at
speed code 17 using the large assembly, part # JHSH204007) in
a 60 mLvolumeusingaHydroShearTM(GeneMachines,San Carlos,
CA). The sheared DNA was immediatelyblunt end-repaired at room
temperature for 45 min using the End-It end-repair kit (Epicentre,
Madison, WI). The end-repair reaction contained 60 mL sheared
DNA,8 mLof106End-Itbuffer,8 mLof2.5 mMEnd-ItdNTPmix,
8 mL of 10 mM End-It ATP, and 4 mL of End-It Enzyme mix in
a 80 mL total volume. After 45 min of incubation, the reaction was
heat-inactivated for 10 min at 70uC, cooled to 4uC for 10 min and
then frozen at 220uC for storage. The end-repaired DNA was run
on a 1% TBE low melting point agarose gel for 13 hours using the
following conditions (Temperature: 14uC, Voltage: 4.5 V/cm, Pulse
initial: 1.0–final: 7.0 sec, Angle: 120u) on a BioRad Chef-DR IIITM
System PFGE system.Using standard procedures,the gelwasstained
with ethidium bromide, destained, and visualized under UV for less
than 10 seconds while the 40 kb band was excised. DNA was
extracted from the agarose gel and blunt-end ligated into pCC1FOS
following the Copy Control Fosmid Kit (Epicentre) protocol. With
minimal modifications to the Copy Control Fosmid Kit (Epicentre)
protocol, the ligated DNA was packaged, infected and plated for
picking and end-sequencing. For detailed JGI protocols used, please
see research protocols at www.jgi.doe.gov.
Assembly and Structural Analysis
based on 34,919 total reads. The Phred/Phrap/Consed software
package (http://www.phrap.com) was used for sequence assembly
and quality assessment [150,151]. After the whole-genome
Approximately 15–20 mg of
Draft assemblies were
shotgun stage, sequence reads were assembled with parallel
Phrap (High Performance Software, LLC). All mis-assemblies
were corrected by editing in Consed , and gaps between
contigs were closed by custom primer walk or PCR amplification
(Roche Applied Science, Indianapolis, IN). The completed
genome sequence of D. geothermalis (DSM 11300) contained
36,718 reads, achieving an average of 8-fold sequence coverage
per base with an error rate less than 1 in 100,000. The D.
geothermalis genome sequence can be accessed at GenBank, or at
the JGI Integrated Microbial Genomes website (http://img.jgi.
doe.gov). Predicted coding sequences were manually analyzed and
evaluated using an Integrated Microbial Genomes (IMG)
structure of the predicted D. geothermalis genome was examined
by PFGE as described previously for D. radiodurans [77,78]. For
structural analysis, D. geothermalis was exposed to 0.2 kGy, which
introduces approximately 0.013 DSB/Gy per genome, and the
cells were then embedded and lysed in agarose. For PFGE of
genomic DNA subjected to restriction endonuclease analysis, non-
irradiated D. geothermalis cells were used.
Orthologous Clusters and Evolutionary
Reconstructed clusters of orthologous genes for the Deinococcus and
Thermus genomes (tdCOGs) were constructed using a technique
based on the standard COG approach [35,36,153]. First, a coarse-
grained classification was obtained by assigning predicted genes to
the NCBI Clusters of Orthologous Groups of proteins (COGs)
using the COGNITOR method . Then, the genes were
organized into tight clusters, based on triangles of best hits .
Proteins belonging to the same cluster were aligned using the
MUSCLE program ; alignments were converted into PSI-
BLAST PSSMs . Subsequent PSI-BLAST searches using these
PSSMs against a database of Deinococcus and Thermus proteins were
used to merge homologous clusters and previously unclustered
proteins into tdCOGs. Cases when proteins assigned to different
COGs were automatically clustered into one tdCOG were
resolved by manual curation (either COG or tdCOG assignment
was changed to remove the contradiction).
Evolutionary events in the history of the Deinococcus-Thermus
group were reconstructed using an ad hoc parsimony approach
[27,155,156]. Presence/absence data from COG-based recon-
struction of the deep ancestor of Cyanobacteria, Actinobacteria
and Deinococcus-Thermus group  were added to the tdCOG
phyletic patterns. Simple parsimony rules were used to infer the
ancestral states and the evolutionary events in the history of the
Deinococcus and Thermus genomes (e.g. a gene present in both
Deinococci and in the deep ancestor but absent in both Thermus
species was considered to be present in the Deinococcus-Thermus
group ancestor and in the Deinococcus genus ancestor, but lost by
the Thermus genus ancestor). The only departure from the
straightforward parsimony inference was made for homologous
tdCOGs that form clade-specific expanded families, e.g. there are
several tdCOGs, all assigned into the same ancestral COG, with
genes present in both Deinococci but in neither of the Thermus
species. In this case, contrary to the formal parsimony assumption
of multiple losses in the Thermus ancestor, the scenario was
interpreted as multiple gains (due to duplications) in the Deinococcus
ancestor (Table S10).
XRF microscopy measurements were made at beamline 2ID-D at
the APS as described previously . Briefly, the 2ID-D is an
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org17September 2007 | Issue 9 | e955
undulator beamline with Fresnel zone plates focusing optics that
produced a focal spot with a FWHM (full width at half maximum)
spatial resolution of approximately 120 nm for these experiments.
For each pixel, the full XRF spectrum between approximately
2 keV and 10 keV was measured using a silicon drift detector.
Thus, the distribution of elements between phosphorus and zinc
on the periodic table of elements could be measured with 120-nm
resolution throughout a cell and its periphery (Figure 7). XRF
microprobe measurements were made on D. geothermalis cells
grown in TGY to OD6000.3 at 50uC; and D. radiodurans cells were
grown in TGY to OD6000.3 at 32uC. The cells were deposited on
grids as suspensions in TGY liquid medium, which served to help
maintain the structure and viability of the cells as they dried.
the Deinococcus-Thermus group.
Found at: doi:10.1371/journal.pone.0000955.s001 (0.08 MB
Proposed evolutionary history of genome partitions in
of D. radiodurans and D. geothermalis.
Found at: doi:10.1371/journal.pone.0000955.s002 (0.06 MB
Genome dot plots for homologous genome partitions
Found at: doi:10.1371/journal.pone.0000955.s003 (0.03 MB
Guanine quadruplet repeats in D. radiodurans.
in D. geothermalis (DSM11300).
Found at: doi:10.1371/journal.pone.0000955.s004 (0.12 MB
Verification of the presence of megaplasmid DG206
neurin-like phosphoesterase subfamily of COG0639 with proteins
from other organisms represented by this COG.
Found at: doi:10.1371/journal.pone.0000955.s005 (0.06 MB
Phylogenetic relationships of tdCOGs of the calci-
Found at: doi:10.1371/journal.pone.0000955.s006 (0.25 MB
Structure of D. radiodurans homozygous mutants.
formation of covalently closed circular (ccc) derivatives of tandem
duplications in irradiated D. radiodurans.
Found at: doi:10.1371/journal.pone.0000955.s007 (0.08 MB
The ESDSA model does not fully explain the early
the Thermus-Deinococcus group with selected representatives of
Found at: doi:10.1371/journal.pone.0000955.s008 (0.05 MB
Multiple alignment comparisons for RecA proteins of
Found at: doi:10.1371/journal.pone.0000955.s009 (0.13 MB
Chrome azurol S agar plate assay for siderophore
essential, the fur gene is dispensable.
Found at: doi:10.1371/journal.pone.0000955.s001 (0.13 MB
Whereas the nramp gene of D. radiodurans is
Found at: doi:10.1371/journal.pone.0000955.s011 (0.04 MB
Homology between the D. radiodurans and D.
Deinococcus and Thermus (tdCOGs).
Found at: doi:10.1371/journal.pone.0000955.s012 (0.24 MB
Clusters of orthologous groups of proteins for
geothermalis (DG), D. radiodurans (DR), T. thermophilus HB27
(TT27), and T. thermophilus HB8 (TT8).
Found at: doi:10.1371/journal.pone.0000955.s013 (0.05 MB
Lineage specific expansion of selected families in D.
Found at: doi:10.1371/journal.pone.0000955.s014 (0.05 MB
Protein families expanded in D. geothermalis.
Found at: doi:10.1371/journal.pone.0000955.s015 (0.07 MB
Protein families expanded in D. radiodurans.
proteins shared by two Deinococcus species, but for which
homologs outside the lineage do not exist.
Found at: doi:10.1371/journal.pone.0000955.s016 (0.17 MB
Gene context and motifs of predicted cytoplasmic
D. geothermalis (DG) and T. thermophilus (TT).
Found at: doi:10.1371/journal.pone.0000955.s017 (0.23 MB
Stress response-related genes in D. radiodurans (DR),
tion functions in E. coli, D. radiodurans and T. thermophilus.
Found at: doi:10.1371/journal.pone.0000955.s018 (0.15 MB
Genes coding for replication, repair and recombina-
Found at: doi:10.1371/journal.pone.0000955.s019 (0.08 MB
Manganese- and iron-related homeostasis genes.
evolutionary events in the Deinococcus/Thermus lineage.
Found at: doi:10.1371/journal.pone.0000955.s011 (0.11 MB
Parsimony pattern rules for reconstruction of
We are grateful to Deb Ghosal at Uniformed Services University of the
Health Sciences (USUHS) for conducting the chrome azurol S agar plate
assay for siderophore production. We are also grateful to Susan Lucas and
Tijana Glavina del Rio of the DOE-Joint Genome Institute for support in
genome sequence quality control, production and assembly
Conceived and designed the experiments: MD KM AL PR KK EG VM.
Performed the experiments: BL KK CD ES EG VM AV MZ BR TB.
Analyzed the data: EK MD MG YW KM MO AL AC ML BL KK JF CD
ES EG VM BR TB AS AG SP EK KM. Contributed reagents/materials/
analysis tools: EK MG. Wrote the paper: MD KM. Other: JGI Genome-
sequencing project coordinator: AL. Genome annotation QC: EK.
Genome annotation: ML KM. Data management/transfer, NCBI sub-
mission: SP. Genome Library construction: CD. Genome finishing: TB ES.
Programmers: AS AG JF. Contributed to writing the paper: EK.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org18September 2007 | Issue 9 | e955
1. Gupta RS (1998) Protein phylogenies and signature sequences: A reappraisal of
evolutionary relationships among archaebacteria, eubacteria, and eukaryotes.
Microbiol Mol Biol Rev 62: 1435–1491.
2. Wolf YI, Rogozin IB, Grishin NV, Tatusov RL, Koonin EV (2001) Genome
trees constructed using five different approaches suggest new major bacterial
clades. BMC Evol Biol 1: 8.
3. Lai WA, Kampfer P, Arun AB, Shen FT, Huber B, et al. (2006) Deinococcus ficus
sp. nov., isolated from the rhizosphere of Ficus religiosa L. Int J Syst Evol
Microbiol 56: 787–791.
4. Gutman PD, Fuchs P, Minton KW (1994) Restoration of the DNA damage
resistance of Deinococcus radiodurans DNA polymerase mutants by Escherichia coli
DNA polymerase I and Klenow fragment. Mutat Res 314: 87–97.
5. Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, et al. (2004)
Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation
resistance. Science 306: 1025–1028.
6. Daly MJ (2000) Engineering radiation-resistant bacteria for environmental
biotechnology. Curr Opin Biotechnol 11: 280–285.
7. Ferreira AC, Nobre MF, Rainey FA, Silva MT, Wait R, et al. (1997) Deinococcus
geothermalis sp. nov. and Deinococcus murrayi sp. nov., two extremely radiation-
resistant and slightly thermophilic species from hot springs. Int J Syst Bacteriol
8. Saarimaa C, Peltola M, Raulio M, Neu TR, Salkinoja-Salonen MS, et al.
(2006) Characterization of adhesion threads of Deinococcus geothermalis as type IV
pili. J Bacteriol 188: 7016–7021.
9. Kimura H, Asada R, Masta A, Naganuma T (2003) Distribution of
microorganisms in the subsurface of the manus basin hydrothermal vent field
in Papua New Guinea. Appl Environ Microbiol 69: 644–648.
10. Marteinsson VT, Hauksdottir S, Hobel CF, Kristmannsdottir H,
Hreggvidsson GO, et al. (2001) Phylogenetic diversity analysis of subterranean
hot springs in Iceland. Appl Environ Microbiol 67: 4242–4248.
11. Brim H, Venkateswaran A, Kostandarithes HM, Fredrickson JK, Daly MJ
(2003) Engineering Deinococcus geothermalis for bioremediation of high-temper-
ature radioactive waste environments. Appl Environ Microbiol 69: 4575–4582.
12. Ghosal D, Omelchenko MV, Gaidamakova EK, Matrosova VY, Vasilenko A,
et al. (2005) How radiation kills cells: survival of Deinococcus radiodurans and
Shewanella oneidensis under oxidative stress. FEMS Microbiol Rev 29: 361–375.
13. Fredrickson JK, Kostandarithes HM, Li SW, Plymale AE, Daly MJ (2000)
Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by Deinococcus radiodurans R1.
Appl Environ Microbiol 66: 2006–2011.
14. Brim H, McFarlan SC, Fredrickson JK, Minton KW, Zhai M, et al. (2000)
Engineering Deinococcus radiodurans for metal remediation in radioactive mixed
waste environments. Nat Biotechnol 18: 85–90.
15. Brim H, Osborne JP, Kostandarithes HM, Fredrickson JK, Wackett LP, et al.
(2006) Deinococcus radiodurans engineered for complete toluene degradation
facilitates Cr(VI) reduction. Microbiology 152: 2469–2477.
16. White O, Eisen JA, Heidelberg JF, Hickey EK, Peterson JD, et al. (1999)
Genome Sequence of the Radioresistant Bacterium Deinococcus radiodurans R1.
Science 286: 1571–1577.
17. Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M (2007)
Protein Oxidation Implicated as the Primary Determinant of Bacterial
Radioresistance. PLoS Biol 5: 769–779.
18. Daly MJ, Ouyang L, Fuchs P, Minton KW (1994) In vivo damage and recA-
dependent repair of plasmid and chromosomal DNA in the radiation-resistant
bacterium Deinococcus radiodurans. J Bacteriol 176: 3508–3517.
19. Gerard E, Jolivet E, Prieur D, Forterre P (2001) DNA protection mechanisms
are not involved in the radioresistance of the hyperthermophilic archaea
Pyrococcus abyssi and P. furiosus. Mol Genet Genomics 266: 72–78.
20. Cox MM, Battista JR (2005) Deinococcus radiodurans-the consummate survivor.
Nat Rev Microbiol 3: 882–892.
21. Makarova KS, Aravind L, Wolf YI, Tatusov RL, Minton KW, et al. (2001)
Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans
viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev
22. Zahradka K, Slade D, Bailone A, Sommer S, Averbeck D, et al. (2006)
Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature 443:
23. Daly MJ (2006) Modulating radiation resistance: Insights based on defenses
against reactive oxygen species in the radioresistant bacterium Deinococcus
radiodurans. Clin Lab Med 26: 491–504, x.
24. Zimmerman JM, Battista JR (2005) A ring-like nucleoid is not necessary for
radioresistance in the Deinococcaceae. BMC Microbiol 5: 17.
25. Pavlov AK, Kalinin VL, Konstantinov AN, Shelegedin VN, Pavlov AA (2006)
Was Earth ever infected by martian biota? Clues from radioresistant bacteria.
Astrobiology 6: 911–918.
26. Lin J, Qi R, Aston C, Jing J, Anantharaman TS, et al. (1999) Whole-genome
shotgun optical mapping of Deinococcus radiodurans. Science 285: 1558–1562.
27. Omelchenko MV, Wolf YI, Gaidamakova EK, Matrosova VY, Vasilenko A, et
al. (2005) Comparative genomics of Thermus thermophilus and Deinococcus
radiodurans: divergent routes of adaptation to thermophily and radiation
resistance. BMC Evol Biol 5: 57.
28. Eisen JA, Heidelberg JF, White O, Salzberg SL (2000) Evidence for symmetric
chromosomal inversions around the replication origin in bacteria. Genome Biol
29. Makarova KS, Wolf YI, White O, Minton K, Daly MJ (1999) Short repeats
and IS elements in the extremely radiation-resistant bacterium Deinococcus
radiodurans and comparison to other bacterial species. Res Microbiol 150:
30. Makarova KS, Aravind L, Galperin MY, Grishin NV, Tatusov RL, et al.
(1999) Comparative genomics of the Archaea (Euryarchaeota): evolution of
conserved protein families, the stable core, and the variable shell. Genome Res
31. Sen D, Gilbert W (1988) Formation of parallel four-stranded complexes by
guanine-rich motifs in DNA and its implications for meiosis. Nature 334:
32. Mojica FJ, Diez-Villasenor C, Soria E, Juez G (2000) Biological significance of
a family of regularly spaced repeats in the genomes of Archaea, Bacteria and
mitochondria. Mol Microbiol 36: 244–246.
33. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, et al. (2007)
CRISPR provides acquired resistance against viruses in prokaryotes. Science
34. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV (2006) A
putative RNA-interference-based immune system in prokaryotes: computa-
tional analysis of the predicted enzymatic machinery, functional analogies with
eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1: 7.
35. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, et al. (2003)
The COG database: an updated version includes eukaryotes. BMC Bioinfor-
matics 4: 41.
36. Tatusov RL, Koonin EV, Lipman DJ (1997) A genomic perspective on protein
families. Science 278: 631–637.
37. Yokoyama K, Ishijima SA, Clowney L, Koike H, Aramaki H, et al. (2006)
Feast/famine regulatory proteins (FFRPs): Escherichia coli Lrp, AsnC and related
archaeal transcription factors. FEMS Microbiol Rev 30: 89–108.
38. Gerischer U (2002) Specific and global regulation of genes associated with the
degradation of aromatic compounds in bacteria. J Mol Microbiol Biotechnol 4:
39. Molina-Henares AJ, Krell T, Eugenia Guazzaroni M, Segura A, Ramos JL
(2006) Members of the IclR family of bacterial transcriptional regulators
function as activators and/or repressors. FEMS Microbiol Rev 30: 157–186.
40. Ramos JL, Martinez-Bueno M, Molina-Henares AJ, Teran W, Watanabe K, et
al. (2005) The TetR family of transcriptional repressors. Microbiol Mol Biol
Rev 69: 326–356.
41. Hobman JL, Wilkie J, Brown NL (2005) A design for life: prokaryotic metal-
binding MerR family regulators. Biometals 18: 429–436.
42. Bearson SM, Albrecht JA, Gunsalus RP (2002) Oxygen and nitrate-dependent
regulation of dmsABC operon expression in Escherichia coli: sites for Fnr and
NarL protein interactions. BMC Microbiol 2: 13.
43. Sandrini MP, Clausen AR, Munch-Petersen B, Piskur J (2006) Thymidine
kinase diversity in bacteria. Nucleosides Nucleotides Nucleic Acids 25:
44. Xi H, Schneider BL, Reitzer L (2000) Purine catabolism in Escherichia coli and
function of xanthine dehydrogenase in purine salvage. J Bacteriol 182:
45. Knofel T, Strater N (1999) X-ray structure of the Escherichia coli periplasmic 59-
nucleotidase containing a dimetal catalytic site. Nat Struct Biol 6: 448–453.
46. Liu Y, Zhou J, Omelchenko MV, Beliaev AS, Venkateswaran A, et al. (2003)
Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing
radiation. Proc Natl Acad Sci U S A 100: 4191–4196.
47. Galperin MY, Moroz OV, Wilson KS, Murzin AG (2006) House cleaning,
a part of good housekeeping. Mol Microbiol 59: 5–19.
48. Shallom D, Shoham Y (2003) Microbial hemicellulases. Curr Opin Microbiol
49. Kolari M, Nuutinen J, Rainey FA, Salkinoja-Salonen MS (2003) Colored
moderately thermophilic bacteria in paper-machine biofilms. J Ind Microbiol
Biotechnol 30: 225–238.
50. Vaisanen OM, Weber A, Bennasar A, Rainey FA, Busse HJ, et al. (1998)
Microbial communities of printing paper machines. J Appl Microbiol 84:
51. Venkateswaran A, McFarlan SC, Ghostal D, Minton KW, Vasilenko A, et al.
(2000) Physiologic determinants of radiation resistance in Deinococcus radiodurans.
ApplEnvironMicrobiol 66: 2620–2626.
52. Holland AD, Rothfuss HM, Lidstrom ME (2006) Development of a defined
medium supporting rapid growth for Deinococcus radiodurans and analysis of
metabolic capacities. Appl Microbiol Biotechnol 72: 1074–1082.
53. Frankenberg N, Moser J, Jahn D (2003) Bacterial heme biosynthesis and its
biotechnological application. Appl Microbiol Biotechnol 63: 115–127.
54. Pereira MM, Santana M, Teixeira M (2001) A novel scenario for the evolution
of haem-copper oxygen reductases. Biochim Biophys Acta 1505: 185–208.
55. Junemann S (1997) Cytochrome bd terminal oxidase. Biochim Biophys Acta
56. Harborne NR, Griffiths L, Busby SJ, Cole JA (1992) Transcriptional control,
translation and function of the products of the five open reading frames of the
Escherichia coli nir operon. Mol Microbiol 6: 2805–2813.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org19 September 2007 | Issue 9 | e955
57. Cabello P, Pino C, Olmo-Mira MF, Castillo F, Roldan MD, et al. (2004)
Hydroxylamine assimilation by Rhodobacter capsulatus E1F1. requirement of the
hcp gene (hybrid cluster protein) located in the nitrate assimilation nas gene
region for hydroxylamine reduction. J Biol Chem 279: 45485–45494.
58. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol
Mol Biol Rev 61: 533–616.
59. Nakamura K, Kawabata T, Yura K, Go N (2003) Novel types of two-domain
multi-copper oxidases: possible missing links in the evolution. FEBS Lett 553:
60. Hille R (2002) Molybdenum and tungsten in biology. Trends Biochem Sci 27:
61. Makarova KS, Aravind L, Daly MJ, Koonin EV (2000) Specific expansion of
protein families in the radioresistant bacterium Deinococcus radiodurans. Genetica
62. Fisher DI, Cartwright JL, McLennan AG (2006) Characterization of the
Mn(2+)-stimulated (di)adenosine polyphosphate hydrolase encoded by the
Deinococcus radiodurans DR2356 nudix gene. Arch Microbiol 186: 415–424.
63. Hou B, Xu ZW, Yang CW, Gao Y, Zhao SF, et al. (2007) Protective effects of
inosine on mice subjected to lethal total-body ionizing irradiation. J Radiat Res
(Tokyo) 48: 57–62.
64. Azeddoug H, Reysset G (1994) Cloning and sequencing of a chromosomal
fragment from Clostridium acetobutylicum strain ABKn8 conferring chemical-
damaging agents and UV resistance to E. coli recA strains. Curr Microbiol 29:
65. Jobling MG, Ritchie DA (1988) The nucleotide sequence of a plasmid
determinant for resistance to tellurium anions. Gene 66: 245–258.
66. Kitajima S, Sato F (1999) Plant pathogenesis-related proteins: molecular
mechanisms of gene expression and protein function. J Biochem (Tokyo) 125:
67. Miura S, Zou W, Ueda M, Tanaka A (2000) Screening of genes involved in
isooctane tolerance in Saccharomyces cerevisiae by using mRNA differential
display. Appl Environ Microbiol 66: 4883–4889.
68. Brinkrolf K, Brune I, Tauch A (2006) Transcriptional regulation of catabolic
pathways for aromatic compounds in Corynebacterium glutamicum. Genet Mol Res
69. Gury J, Barthelmebs L, Tran NP, Divies C, Cavin JF (2004) Cloning, deletion,
and characterization of PadR, the transcriptional repressor of the phenolic acid
decarboxylase-encoding padA gene of Lactobacillus plantarum. Appl Environ
Microbiol 70: 2146–2153.
70. Huillet E, Velge P, Vallaeys T, Pardon P (2006) LadR, a new PadR-related
transcriptional regulator from Listeria monocytogenes, negatively regulates the
expression of the multidrug efflux pump MdrL. FEMS Microbiol Lett 254:
71. Kobayashi I, Tamura T, Sghaier H, Narumi I, Yamaguchi S, et al. (2006)
Characterization of monofunctional catalase KatA from radioresistant
bacterium Deinococcus radiodurans. J Biosci Bioeng 101: 315–321.
72. Yun EJ, Lee YN (2000) Production of two different catalase-peroxidases by
Deinococcus radiophilus. FEMS Microbiol Lett 184: 155–159.
73. Imlay JA (2003) Pathways of oxidative damage. Annu Rev Microbiol 57:
74. Nurizzo D, Shewry SC, Perlin MH, Brown SA, Dholakia JN, et al. (2003) The
crystal structure of aminoglycoside-39-phosphotransferase-IIa, an enzyme
responsible for antibiotic resistance. J Mol Biol 327: 491–506.
75. Markillie LM, Varnum SM, Hradecky P, Wong KK (1999) Targeted
mutagenesis by duplication insertion in the radioresistant bacterium Deinococcus
radiodurans: radiation sensitivities of catalase (katA) and superoxide dismutase
(sodA) mutants. J Bacteriol 181: 666–669.
76. Daly MJ, Ling O, Minton KW (1994) Interplasmidic recombination following
irradiation of the radioresistant bacterium Deinococcus radiodurans. J Bacteriol
77. Daly MJ, Minton KW (1995) Interchromosomal recombination in the
extremely radioresistant bacterium Deinococcus radiodurans. J Bacteriol 177:
78. Daly MJ, Minton KW (1996) An alternative pathway of recombination of
chromosomal fragments precedes recA-dependent recombination in the radio-
resistant bacterium Deinococcus radiodurans. J Bacteriol 178: 4461–4471.
79. Daly MJ, Minton KW (1997) Recombination between a resident plasmid and
the chromosome following irradiation of the radioresistant bacterium
Deinococcus radiodurans. Gene 187: 225–229.
80. Gallegos MT, Schleif R, Bairoch A, Hofmann K, Ramos JL (1997) Arac/XylS
family of transcriptional regulators. Microbiol Mol Biol Rev 61: 393–410.
81. Sorensen KI, Hove-Jensen B (1996) Ribose catabolism of Escherichia coli:
characterization of the rpiB gene encoding ribose phosphate isomerase B and of
the rpiR gene, which is involved in regulation of rpiB expression. J Bacteriol 178:
82. Labie C, Bouche F, Bouche JP (1989) Isolation and mapping of Escherichia coli
mutations conferring resistance to division inhibition protein DicB. J Bacteriol
83. Pennella MA, Giedroc DP (2005) Structural determinants of metal selectivity in
prokaryotic metal-responsive transcriptional regulators. Biometals 18: 413–428.
84. Buts L, Lah J, Dao-Thi MH, Wyns L, Loris R (2005) Toxin-antitoxin modules
as bacterial metabolic stress managers. Trends Biochem Sci 30: 672–679.
85. Gerdes K, Christensen SK, Lobner-Olesen A (2005) Prokaryotic toxin-
antitoxin stress response loci. Nat Rev Microbiol 3: 371–382.
86. Bhoo SH, Davis SJ, Walker J, Karniol B, Vierstra RD (2001) Bacteriophy-
tochromes are photochromic histidine kinases using a biliverdin chromophore.
Nature 414: 776–779.
87. Davis SJ, Vener AV, Vierstra RD (1999) Bacteriophytochromes: phytochrome-
like photoreceptors from nonphotosynthetic eubacteria. Science 286:
88. Boyd A, Chakrabarty AM (1995) Pseudomonas aeruginosa biofilms: role of the
alginate exopolysaccharide. J Ind Microbiol 15: 162–168.
89. Gacesa P (1998) Bacterial alginate biosynthesis–recent progress and future
prospects. Microbiology 144 ( Pt 5): 1133–1143.
90. Agostini HJ, Carroll JD, Minton KW (1996) Identification and characterization
of uvrA, a DNA repair gene of Deinococcus radiodurans. J Bacteriol 178:
91. Lipton MS, Pasa-Tolic L, Anderson GA, Anderson DJ, Auberry DL, et al.
(2002) Global analysis of the Deinococcus radiodurans proteome by using accurate
mass tags. Proc Natl Acad Sci U S A 99: 11049–11054.
92. Tanaka M, Earl AM, Howell HA, Park MJ, Eisen JA, et al. (2004) Analysis of
Deinococcus radiodurans’s transcriptional response to ionizing radiation and
desiccation reveals novel proteins that contribute to extreme radioresistance.
Genetics 168: 21–33.
93. Gutman PD, Carroll JD, Masters CI, Minton KW (1994) Sequencing, targeted
mutagenesis and expression of a recA gene required for the extreme
radioresistance of Deinococcus radiodurans. Gene 141: 31–37.
94. Altschul SF, Koonin EV (1998) Iterated profile searches with PSI-BLAST–
a tool for discovery in protein databases. Trends Biochem Sci 23: 444–447.
95. Schultz J, Copley RR, Doerks T, Ponting CP, Bork P (2000) SMART: a web-
based tool for the study of genetically mobile domains. Nucleic Acids Res 28:
96. Marchler-Bauer A, Panchenko AR, Shoemaker BA, Thiessen PA, Geer LY, et
al. (2002) CDD: a database of conserved domain alignments with links to
domain three-dimensional structure. Nucleic Acids Res 30: 281–283.
97. Iyer LM, Koonin EV, Aravind L (2002) Classification and evolutionary history
of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52.
BMC Genomics 3: 8.
98. Harris DR, Tanaka M, Saveliev SV, Jolivet E, Earl AM, et al. (2004) Preserving
genome integrity: the DdrA protein of Deinococcus radiodurans R1. PLoS Biol 2:
99. Mironov AA, Koonin EV, Roytberg MA, Gelfand MS (1999) Computer
analysis of transcription regulatory patterns in completely sequenced bacterial
genomes. Nucleic Acids Res 27: 2981–2989.
100. Mironov AA, Vinokurova NP, Gel’fand MS (2000) [Software for analyzing
bacterial genomes]. Mol Biol (Mosk) 34: 253–262.
101. Kunkel TA, Erie DA (2005) DNA mismatch repair. Annu Rev Biochem 74:
102. Kuzminov A (1999) Recombinational repair of DNA damage in Escherichia coli
and bacteriophage lambda. Microbiol Mol Biol Rev 63: 751–813, table of
103. Touati E, Laurent-Winter C, Quillardet P, Hofnung M (1996) Global response
of Escherichia coli cells to a treatment with 7-methoxy-2-nitronaphtho[2,1-
b]furan (R7000), an extremely potent mutagen. Mutat Res 349: 193–200.
104. Zhang YM, Liu JK, Wong TY (2003) The DNA excision repair system of the
highly radioresistant bacterium Deinococcus radiodurans is facilitated by the
pentose phosphate pathway. Mol Microbiol 48: 1317–1323.
105. Chasin LA, Magasanik B (1968) Induction and repression of the histidine-
degrading enzymes of Bacillus subtilis. J Biol Chem 243: 5165–5178.
106. Kannan K, Janiyani KL, Shivaji S, Ray MK (1998) Histidine utilisation operon
(hut) is upregulated at low temperature in the antarctic psychrotrophic
bacterium Pseudomonas syringae. FEMS Microbiol Lett 161: 7–14.
107. Bruce AK, Berner JD (1976) Respiratory activity as a determinant of radiation
survival response. Can J Microbiol 22: 1336–1344.
108. Earl AM, Mohundro MM, Mian IS, Battista JR (2002) The IrrE protein of
Deinococcus radiodurans R1 is a novel regulator of recA expression. J Bacteriol 184:
109. Hua Y, Narumi I, Gao G, Tian B, Satoh K, et al. (2003) PprI: a general switch
responsible for extreme radioresistance of Deinococcus radiodurans. Biochem
Biophys Res Commun 306: 354–360.
110. Gao G, Le D, Huang L, Lu H, Narumi I, et al. (2006) Internal promoter
characterization and expression of the Deinococcus radiodurans pprI-folP gene
cluster. FEMS Microbiol Lett 257: 195–201.
111. Carroll JD, Daly MJ, Minton KW (1996) Expression of recA in Deinococcus
radiodurans. J Bacteriol 178: 130–135.
112. Cheo DL, Bayles KW, Yasbin RE (1991) Cloning and characterization of DNA
damage-inducible promoter regions from Bacillus subtilis. J Bacteriol 173:
113. Yasbin RE, Cheo D, Bayles KW (1991) The SOB system of Bacillus subtilis:
a global regulon involved in DNA repair and differentiation. Res Microbiol
114. Courcelle J, Khodursky A, Peter B, Brown PO, Hanawalt PC (2001)
Comparative gene expression profiles following UV exposure in wild-type
and SOS-deficient Escherichia coli. Genetics 158: 41–64.
115. Fernandez De Henestrosa AR, Ogi T, Aoyagi S, Chafin D, Hayes JJ, et al.
(2000) Identification of additional genes belonging to the LexA regulon in
Escherichia coli. Mol Microbiol 35: 1560–1572.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org20 September 2007 | Issue 9 | e955
116. Narumi I, Satoh K, Kikuchi M, Funayama T, Yanagisawa T, et al. (2001) The Download full-text
LexA protein from Deinococcus radiodurans is not involved in RecA induction
following gamma irradiation. J Bacteriol 183: 6951–6956.
117. Little JW, Mount DW, Yanisch-Perron CR (1981) Purified LexA protein is
a repressor of the recA and lexA genes. Proc Natl Acad Sci U S A 78:
118. Moseley BE, Mattingly A (1971) Repair of irradiation transforming de-
oxyribonucleic acid in wild type and a radiation-sensitive mutant of Micrococcus
radiodurans. J Bacteriol 105: 976–983.
119. Gutman PD, Yao HL, Minton KW (1991) Partial complementation of the UV
sensitivity of Deinococcus radiodurans excision repair mutants by the cloned denV
gene of bacteriophage T4. Mutat Res 254: 207–215.
120. Minton KW (1996) Repair of ionizing-radiation damage in the radiation
resistant bacterium Deinococcus radiodurans. Mutat Res 363: 1–7.
121. Schlesinger DJ (2007) Role of RecA in DNA damage repair in Deinococcus
radiodurans. FEMS Microbiol Lett 274: 342–347.
122. Daly MJ, Minton KW (1995) Resistance to radiation. Science 270: 1318.
123. Eltsov M, Dubochet J (2005) Fine structure of the Deinococcus radiodurans
nucleoid revealed by cryoelectron microscopy of vitreous sections. J Bacteriol
124. Eltsov M, Dubochet J (2006) Study of the Deinococcus radiodurans nucleoid by
cryoelectron microscopy of vitreous sections: Supplementary comments.
J Bacteriol 188: 6053–6058; discussion 6059.
125. Gao G, Lu H, Yin L, Hua Y (2007) Ring-like nucleoid does not play a key role
in radioresistance of Deinococcus radiodurans. Sci China C Life Sci 50: 525–529.
126. Setlow DM, Duggan DE (1964) The resistance of Micrococcus radiodurans to
ultraviolet radiation: Ultraviolet-induced lesions in the cell’s DNA. Biochim
Biophys Acta 87: 664–668.
127. Huang L, Hua X, Lu H, Gao G, Tian B, et al. (2007) Three tandem HRDC
domains have synergistic effect on the RecQ functions in Deinococcus radiodurans.
DNA Repair (Amst) 6: 167–176.
128. Kim JI, Sharma AK, Abbott SN, Wood EA, Dwyer DW, et al. (2002) RecA
Protein from the extremely radioresistant bacterium Deinococcus radiodurans:
expression, purification, and characterization. J Bacteriol 184: 1649–1660.
129. Narumi I, Satoh K, Kikuchi M, Funayama T, Kitayama S, et al. (1999)
Molecular analysis of the Deinococcus radiodurans recA locus and identification of
a mutation site in a DNA repair-deficient mutant, rec30. Mutat Res 435:
130. Bernstein DA, Eggington JM, Killoran MP, Misic AM, Cox MM, et al. (2004)
Crystal structure of the Deinococcus radiodurans single-stranded DNA-binding
protein suggests a mechanism for coping with DNA damage. Proc Natl Acad
Sci U S A 101: 8575–8580.
131. Bowater R, Doherty AJ (2006) Making ends meet: repairing breaks in bacterial
DNA by non-homologous end-joining. PLoS Genet 2: e8.
132. Englander J, Klein E, Brumfeld V, Sharma AK, Doherty AJ, et al. (2004) DNA
toroids: framework for DNA repair in Deinococcus radiodurans and in germinating
bacterial spores. J Bacteriol 186: 5973–5977.
133. Kobayashi Y, Narumi I, Satoh K, Funayama T, Kikuchi M, et al. (2004)
Radiation response mechanisms of the extremely radioresistant bacterium
Deinococcus radiodurans. Biol Sci Space 18: 134–135.
134. Lecointe F, Shevelev IV, Bailone A, Sommer S, Hubscher U (2004)
Involvement of an X family DNA polymerase in double-stranded break repair
in the radioresistant organism Deinococcus radiodurans. Mol Microbiol 53:
135. Levin-Zaidman S, Englander J, Shimoni E, Sharma AK, Minton KW, et al.
(2003) Ringlike structure of the Deinococcus radiodurans genome: a key to
radioresistance? Science 299: 254–256.
136. Narumi I, Satoh K, Cui S, Funayama T, Kitayama S, et al. (2004) PprA:
a novel protein from Deinococcus radiodurans that stimulates DNA ligation. Mol
Microbiol 54: 278–285.
137. Blasius M, Buob R, Shevelev IV, Hubscher U (2007) Enzymes involved in
DNA ligation and end-healing in the radioresistant bacterium Deinococcus
radiodurans. BMC Mol Biol 8: 69.
138. Wright EG, Coates PJ (2006) Untargeted effects of ionizing radiation:
implications for radiation pathology. Mutat Res 597: 119–132.
139. Belyakov OV, Mitchell SA, Parikh D, Randers-Pehrson G, Marino SA, et al.
(2005) Biological effects in unirradiated human tissue induced by radiation
damage up to 1 mm away. Proc Natl Acad Sci U S A 102: 14203–14208.
140. von Sonntag C (1987) The chemical basis of radiation biology. London, United
Kingdom: Taylor & Francis.
141. Du J, Gebicki JM (2004) Proteins are major initial cell targets of hydroxyl free
radicals. Int J Biochem Cell Biol 36: 2334–2343.
142. Kemner KM, Kelly SD, Lai B, Maser J, O’Loughlin E J, et al. (2004)
Elemental and redox analysis of single bacterial cells by x-ray microbeam
analysis. Science 306: 686–687.
143. Heinz K, Marx A (2007) Lesion bypass activity of DNA polymerase A from the
extremely radioresistant organism Deinococcus radiodurans. J Biol Chem 282:
144. Qiu X, Daly MJ, Vasilenko A, Omelchenko MV, Gaidamakova EK, et al.
(2006) Transcriptome analysis applied to survival of Shewanella oneidensis MR-1
exposed to ionizing radiation. J Bacteriol 188: 1199–1204.
145. Fong SS, Joyce AR, Palsson BO (2005) Parallel adaptive evolution cultures of
Escherichia coli lead to convergent growth phenotypes with different gene
expression states. Genome Res 15: 1365–1372.
146. Koonin EV (2007) Chance and necessity in cellular response to challenge.
Molec Syst Biol: in press.
147. Stern S, Dror T, Stolovicki E, Brenner N, Braun E (2007) Genome-wide
transcriptional plasticity underlies cellular adaptation to novel changes. Molec
Syst Biol: in press.
148. Albuquerque L, Simoes C, Nobre MF, Pino NM, Battista JR, et al. (2005)
Truepera radiovictrix gen. nov., sp. nov., a new radiation resistant species and the
proposal of Trueperaceae fam. nov. FEMS Microbiol Lett 247: 161–169.
149. Detter JC, Jett JM, Lucas SM, Dalin E, Arellano AR, et al. (2002) Isothermal
strand-displacement amplification applications for high-throughput genomics.
Genomics 80: 691–698.
150. Ewing B, Green P (1998) Base-calling of automated sequencer traces using
phred. II. Error probabilities. Genome Res 8: 186–194.
151. Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated
sequencer traces using phred. I. Accuracy assessment. Genome Res 8:
152. Gordon D, Abajian C, Green P (1998) Consed: a graphical tool for sequence
finishing. Genome Res 8: 195–202.
153. Tatusov RL, Natale DA, Garkavtsev IV, Tatusova TA, Shankavaram UT, et
al. (2001) The COG database: new developments in phylogenetic classification
of proteins from complete genomes. Nucleic Acids Res 29: 22–28.
154. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy
and high throughput. Nucleic Acids Res 32: 1792–1797.
155. Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, et al. (2006)
Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci U S A
156. Makarova KS, Wolf YI, Mekhedov SL, Mirkin BG, Koonin EV (2005)
Ancestral paralogs and pseudoparalogs and their role in the emergence of the
eukaryotic cell. Nucleic Acids Res 33: 4626–4638.
157. Bordo D, Djinovic K, Bolognesi M (1994) Conserved patterns in the Cu,Zn
superoxide dismutase family. J Mol Biol 238: 366–386.
158. Singleton MR, Wentzell LM, Liu Y, West SC, Wigley DB (2002) Structure of
the single-strand annealing domain of human RAD52 protein. Proc Natl Acad
Sci U S A 99: 13492–13497.
159. Bouchard JD, Moineau S (2004) Lactococcal phage genes involved in
sensitivity to AbiK and their relation to single-strand annealing proteins.
J Bacteriol 186: 3649–3652.
160. Schneider TD, Stephens RM (1990) Sequence logos: a new way to display
consensus sequences. Nucleic Acids Res 18: 6097–6100.
161. Kitayama S, Kohoroku M, Takagi A, Itoh H (1997) Mutation of D. radiodurans
in a gene homologous to ruvB of E. coli. Mutat Res 385: 151–157.
162. Udupa KS, O’Cain PA, Mattimore V, Battista JR (1994) Novel ionizing
radiation-sensitive mutants of Deinococcus radiodurans. J Bacteriol 176:
163. Battista JR, Park MJ, McLemore AE (2001) Inactivation of Two Homologues
of Proteins Presumed to be Involved in the Desiccation Tolerance of Plants
Sensitizes Deinococcus radiodurans R1 to Desiccation. Cryobiology 43: 133–139.
164. Kitayama S, Narumi I, Kikuchi M, Watanabe H (2000) Mutation in recR gene
of Deinococcus radiodurans and possible involvement of its product in the repair of
DNA interstrand cross-links. Mutat Res 461: 179–187.
165. Funayama T, Narumi I, Kikuchi M, Kitayama S, Watanabe H, et al. (1999)
Identification and disruption analysis of the recN gene in the extremely
radioresistant bacterium Deinococcus radiodurans. Mutat Res 435: 151–161.
166. Gutman PD, Fuchs P, Ouyang L, Minton KW (1993) Identification,
sequencing, and targeted mutagenesis of a DNA polymerase gene required
for the extreme radioresistance of Deinococcus radiodurans. J Bacteriol 175:
167. Mattimore V, Battista JR (1996) Radioresistance of Deinococcus radiodurans:
functions necessary to survive ionizing radiation are also necessary to survive
prolonged desiccation. J Bacteriol 178: 633–637.
168. Satoh K, Ohba H, Sghaier H, Narumi I (2006) Down-regulation of
radioresistance by LexA2 in Deinococcus radiodurans. Microbiology 152:
169. Sheng D, Zheng Z, Tian B, Shen B, Hua Y (2004) LexA analog (dra0074) is
a regulatory protein that is irrelevant to recA induction. J Biochem (Tokyo)
170. Huang LF, Zhang SW, Hua XT, Gao GJ, Hua YJ (2006) [Construction of the
recQ double mutants and analysis of adversity in Deinococcus radiodurans]. Wei
Sheng Wu Xue Bao 46: 205–209.
Deinococcus Genome Analysis
PLoS ONE | www.plosone.org 21September 2007 | Issue 9 | e955