Paradoxical DNA Repair and Peroxide Resistance Gene
Conservation in Bacillus pumilus SAFR-032
Jason Gioia1., Shailaja Yerrapragada1., Xiang Qin1, Huaiyang Jiang1, Okezie C. Igboeli1, Donna Muzny1, Shannon Dugan-Rocha1, Yan Ding1,
Alicia Hawes1, Wen Liu1, Lesette Perez1, Christie Kovar1, Huyen Dinh1, Sandra Lee1, Lynne Nazareth1, Peter Blyth1, Michael Holder1, Christian
Buhay1, Madhan R. Tirumalai4, Yamei Liu4, Indrani Dasgupta4, Lina Bokhetache4, Masaya Fujita4, Fathi Karouia4, Prahathees Eswara Moorthy4,
Johnathan Siefert4, Akif Uzman5, Prince Buzumbo5, Avani Verma5, Hiba Zwiya5, Brian D. McWilliams3, Adeola Olowu6, Kenneth D.
Clinkenbeard7, David Newcombe8,9, Lisa Golebiewski3, Joseph F. Petrosino3, Wayne L. Nicholson10, George E. Fox4, Kasthuri Venkateswaran9,
Sarah K. Highlander1,3, George M. Weinstock1,2,3*
1Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, United States of America, 2Department of Molecular and Human
Genetics, Baylor College of Medicine, Houston, Texas, United States of America, 3Department of Molecular Virology and Microbiology, Baylor College
of Medicine, Houston, Texas, United States of America, 4Department of Biology and Biochemistry, University of Houston, Houston, Texas, United
States of America, 5Department of Natural Sciences, University of Houston-Downtown, Houston, Texas, United States of America, 6University of
St. Thomas, Houston Texas, United States of America, 7Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma
State University, Stillwater, Oklahoma, United States of America, 8University of Idaho Coeur d’Alene, Coeur d’Alene, Idaho, United States of America,
9Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California, United States of America, 10Department of Microbiology and
Cell Science, University of Florida Space Life Sciences Laboratory, Kennedy Space Center, Florida, United States of America
Background. Bacillus spores are notoriously resistant to unfavorable conditions such as UV radiation, c-radiation, H2O2,
desiccation, chemical disinfection, or starvation. Bacillus pumilus SAFR-032 survives standard decontamination procedures of
the Jet Propulsion Lab spacecraft assembly facility, and both spores and vegetative cells of this strain exhibit elevated
resistance to UV radiation and H2O2compared to other Bacillus species. Principal Findings. The genome of B. pumilus SAFR-
032 was sequenced and annotated. Lists of genes relevant to DNA repair and the oxidative stress response were generated and
compared to B. subtilis and B. licheniformis. Differences in conservation of genes, gene order, and protein sequences are
highlighted because they potentially explain the extreme resistance phenotype of B. pumilus. The B. pumilus genome includes
genes not found in B. subtilis or B. licheniformis and conserved genes with sequence divergence, but paradoxically lacks
several genes that function in UV or H2O2resistance in other Bacillus species. Significance. This study identifies several
candidate genes for further research into UV and H2O2resistance. These findings will help explain the resistance of B. pumilus
and are applicable to understanding sterilization survival strategies of microbes.
Citation: Gioia J, Yerrapragada S, Qin X, Jiang H, Igboeli OC, et al (2007) Paradoxical DNA Repair and Peroxide Resistance Gene Conservation in
Bacillus pumilus SAFR-032. PLoS ONE 2(9): e928. doi:10.1371/journal.pone.0000928
Bacillus pumilus is a Gram-positive, aerobic, rod-shaped, soil-dwelling
bacterium . Like other Bacillus species, B. pumilus produces spores
that are more resistant than vegetative cells to heat, desiccation, UV
radiation, c-radiation, H2O2, and starvation. B. pumilus has been
found in extremeenvironments such as the interior of Sonoran desert
basalt and the Mars Odyssey spacecraft [2,3]. Spores and vegetative
Propulsion Lab (Pasadena, CA) spacecraft assembly facility, are
endowed with UV radiation and H2O2resistance capabilities that
significantly exceed other Bacillus species and allow survival of
standard sterilization practices [3–5]. Sterilization is significant not
only for prevention of contamination of extraterrestrial environments
via spacecraft, but also for fundamental processes in bacteriology,
medicine, the pharmaceutical industry, and counter-bioterrorism
measures, and hence such resistance is cause for concern.
UV radiation induces the formation of deleterious DNA lesions
such as pyrimidine dimers [5,6]. Bacillus spores are more resistant to
UV radiation than vegetative cells because desiccation and the
presence of small acid soluble spore proteins (SASP) mitigate DNA
damage. A variety of DNA repair mechanisms that become active
upon germination also permit survival of UV radiation. H2O2kills
damage is combated by a variety of reducing agents that react with
oxidative agents or oxidized cellular components.
Here we present an analysis of the B. pumilus SAFR-032 genome.
In comparing this genome to less UV- and H2O2-resistant Bacillus
species (B. subtilis and B. licheniformis) we identify genomic differences
that provide important insights into the DNA repair pathways and
oxidative stress response pathways of B. pumilus. The genes identified
in this study are candidates for further experimental research.
Bacterial strain growth and DNA isolation
A single B. pumilus SAFR-032 colony exhibiting circular, crateri-
form morphology and raised ridges on its surface, was used to
Academic Editor: Abraham Sonenshein, Tufts University, United States of America
Received May 18, 2007; Accepted August 31, 2007; Published September 26,
Copyright: ? 2007 Gioia et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: This research was supported in part by grants to GMW (NSF-414410),
GEF (NASA-Exobiology #NNG05GN75G) and KV (contract to JPL/Caltech from
NASA and funded by NRA ROSS 2005). The sponsors had no role in the conduct of
Competing Interests: The authors have declared that no competing interests
* To whom correspondence should be addressed. E-mail: email@example.com.
. These authors contributed equally to this work.
PLoS ONE | www.plosone.org1 September 2007 | Issue 9 | e928
inoculate trypticase soy yeast (TSY) broth. The culture was grown
overnight at 37uC with vigorous shaking. Genomic DNA was
purified from CsCl gradients of whole cell lysates .
DNA sequencing and genome assembly
DNA sequencing was performed by a combined approach using
traditional Sanger dideoxy whole genome shotgun (WGS)
sequencing and 454 Life Sciences pyrosequencing strategies
. Genomic DNA was nebulized into 5 kb fragments, and
cloned into a derivative of pUC18 . The clones were used for
WGS DNA sequencing using ABI 3700 sequencers, and reads
were assembled using the ATLAS assembler . Read-pair
information was used to create higher order scaffolds. WGS reads
were sequenced to ten-fold coverage. The WGS plasmid libraries
were not random, but had cloning bias of unknown cause.
Consequently, the WGS sequence was supplemented with short
reads generated on a 454 Life Sciences GS20 sequencer and
lacking cloning bias. Here the coverage was thirteen fold.
Gene identification and annotation
Previously described gene prediction and manual annotation
protocols were followed . Glimmer  and GeneMark 
were used independently to predict open reading frames (ORFs).
Visualization of gene predictions was performed using the
Genboree system (www.genboree.org) and the CONAN database
. DNA comparisons were performed with BLASTN and
BLASTZ. Protein sequences were analyzed by BLASTP vs. the nr
database at NCBI . When appropriate, other predictive tools
such as InterProScan , PFP , PSORTb , ExPASy
ENZYME , Helix-Turn-Helix Predictor , MEROPs ,
and the Transport Classification Database  were used. The B.
pumilus SAFR-032 genome is 3.7 Mb and 3848 features (3687
ORFs, 12 frameshifts, 38 pseudogenes, 7 rRNA operons, 69
tRNAs, and 21 ncRNAs) were annotated. The B. pumilus genome
has been deposited in GenBank under the accession number
CP000813. Locus tags of genes discussed in this paper are listed in
Supplementary Table S1.
Comparative Genomic Analysis
The database of annotated genes was searched for genes relevant
to DNA repair and H2O2 resistance. B. pumilus genes were
considered homologs of B. subtilis and B. licheniformis genes if their
translated sequences aligned with $50% identity to the homolog
of either species. Exceptions were made in deference to conserved
gene order and local alignments to functional domains character-
istic of specific proteins. We examined the B. subtilis and B.
licheniformis genomes and available literature to find DNA repair
and H2O2resistance genes not found in our B. pumilus gene list.
Relevant genes absent from the B. pumilus gene list were confirmed
as absent using B. subtilis and B. licheniformis sequences as queries
for local BLAST against the B. pumilus genome.
Spore survivability to UV radiation and H2O2
Methods of measuring survival of spores exposed to UV radiation
and H2O2have been previously described [3,4]. Data presented
here include but are not limited to measurements previously
reported in those studies.
RESULTS AND DISCUSSION
B. pumilus SAFR-032 was selected for genome sequencing and
analysis because its spores exhibited unusually high resistance to
UV radiation and H2O2compared to the standard dosimetric
strains B. subtilis 168 and B. licheniformis. Whereas .90% lethality
of B. subtilis and B. lichenifiormis spores is achieved by exposure to
200 J/m2UV254, 1500 J/m2are required to kill 90% of B.
pumilus SAFR-032 spores (Figure 1a). Twelve percent of B. pumilus
SAFR-032 spores survive 5% liquid H2O2, which is nearly thrice
the survival rate of B. subtilis spores (Figure 1b).
The B. pumilus SAFR-032 genome was annotated and analyzed
for features relevant to UV radiation resistance and H2O2
resistance. Mechanisms of DNA repair and the oxidative stress
response were compared among B. pumilus, B. subtilis, and B.
licheniformis to generate lists of genes common to all three species,
genes unique to B. pumilus, and genes absent in B. pumilus (Table 1).
The presence or absence of genes is indicative of unique functions
that may explain phenotypic differences. Despite gene conserva-
tion, the possibility of altered functions of homologous genes due
to sequence divergence cannot be excluded. Therefore, the
translated sequences of common genes were also compared
(Tables 2 and 3). In addition to gene conservation and sequence
similarity it is also important to understand gene functions in
context of the organism’s growth phase. Although the temporal
activity of only some proteins discussed here are known, two recent
studies describe transcription of many B. subtilis DNA repair and
H2O2resistance genes. Keijser et al. identified transcripts more
abundant in spores and germinating cells than in vegetative cells
. Moeller et al. identified transcripts induced after exposure of
vegetative cells to UVC radiation (200–280 nm) . We cross-
referenced our gene lists with these temporal transcription data to
augment our genomic comparisons (Table 1). However, it should
be understood that because spore survivability assays entail
growing surviving spores to countable levels in liquid or solid
media, resistance mechanisms at any stage of growth may be
important to survivability.
Previous analyses of the resistance properties of Bacillus spores
centered on small acid-soluble spore proteins (SASP) and the spore
photoproduct lyase DNA repair system [26,27]. SASP are spore
core proteins that play a crucial role in resistance to UV radiation,
heat, desiccation, and oxidative damage by binding DNA and
altering its reactivity . When exposed to UV radiation, SASP-
bound DNA more readily forms the spore photoproduct (SP), 5-
thyminyl-5,6-dihydrothymine, rather than cyclobutane dimers or
(6-4)-photoproducts, which are formed in the absence of SASP.
Unlike these other DNA lesions, SP is easily repaired by the spore
photoproduct lyase (SP lyase), which is encoded by splB gene and is
negatively regulated by the splA gene product . B. pumilus has an
intact splAB operon. The translated SplB (BPUM_1283) sequence
is highly conserved in B. pumilus, but SplA (BPUM_1282) shows
much more sequence diversity among B. pumilus, B. subtilis, and B.
licheniformis (Table 2), indicating possible differences in SP lyase
Bacillus subtilis produces 18 SASPs, whose sequences are short
(40–100 amino acids) and highly conserved. The a/b-type SspA
and SspB predominate, and there are also minor a/b-type SASPs,
a c–type, and novel SASPs . Fifteen SASP genes were
annotated in B. pumilus (Table 1); homologs of SspC, SspG, and
SspH were not found. SspC is a minor a/b-type SASP that
contributes to UV radiation resistance , hence its absence from
B. pumilus is paradoxical. SspH and SspG are novel type SASPs,
that have no effect on B. subtilis UV radiation resistance [28,30].
The B. pumilus and B. licheniformis homologs of the c–type SASP
appear to be amino-terminal truncations of the 84 amino acid
SspE of B. subtilis. The significance of such a truncation is unclear,
as the only known function of SspE is as an amino acid source for
germinating spores . Although these differences in gene
content and sequence conservation may contribute to the
B.pumilus UV& H2O2 Resistance
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enhanced UV and oxidation resistance of B. pumilus, other
important factors are likely to be found among DNA repair and
oxidative stress response genes
DNA Repair Mechanisms–Single Strand Repair
Base Excision Repair (BER)
repaired by BER, which is performed by DNA glycosylases and AP
(apurinic/apyrimidinic) endonucleases [32,33]. DNA glycosylases
remove damaged bases from the DNA backbone to create an AP
site. AP endonucleases bind to this site and cleave the DNA 59 to the
abasic site, forming a free 39-hydroxyl which is repaired by DNA
glycosylase activity, whereas bifunctional DNA glycosylases have
both glycosylase and lyase activities as well as the ability to cleave the
phosphodiester backbone 39 to the AP site. B. pumilus encodes both
monofunctional [AlkA (BPUM_0752), Ung (BPUM_03444)] and
Oxidative damage to DNA is
bifunctional [MutM (BPUM_2550), Nth (BPUM_1966)] DNA
glycosylases in addition to the AP endonuclease IV, Nfo
(BPUM_2246). Nth and Nfo are highly conserved among B.
pumilus, B. subtilis, and B. licheniformis, but AlkA, Ung, and MutM
have greater sequence divergence (Table 2).
B. pumilus lacks a homolog of the AP endonuclease ExoA and
the DNA glycosylase YxlJ, both of which are present in B. subtilis
and B. licheniformis. The lack of ExoA is not surprising, as B. subtilis
exoA mutants do not exhibit enhanced sensitivity to H2O2.
YxlJ functions in the repair of DNA alkylation damage and
removal of deaminated purines and cyclic etheno adducts ,
and it is transcribed during spore germination and outgrowth ;
its absence suggests that another protein compensates for its loss.
Nucleotide Excision Repair (NER)
and repairs individual bases by specific DNA glycosylases, NER
identifies multi-base distortions in the double helix and removes
bulky single-stranded lesions, which are repaired by DNA
polymerase I . The E. coli NER pathway consists of UvrA
While BER recognizes
Figure 1. Resistance of B. pumilus SAFR-032 spores to UV radiation and H2O2. a) Survivability of spores exposed to varying doses of UV254
(100 uW sec21cm22). Key: B. pumilus SAFR-032, circles; B. subtilis 168, squares; B. licheniformis ME-13-1, triangles. b) Survivability of spores exposed to
5% H2O2liquid for one hour.
B.pumilus UV& H2O2 Resistance
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Table 1. List of Bacillus genes involved in DNA repair and oxidative stress resistance.
Function (No. of genes)
Bp & Bl [1,2]
Bl & Bs [2,3]
DNA repair (88)
ada2,3; dinG; disA; dnaE; end1; gyrB; hbs; kapD; mfd; mutL,M,S2(yshD); ogt; pcrB;
phrB2,3; polA; polY2(yqjW); priA; recD, J, N,R,Q(recS),X; sbcC,D; scpA,B; sms; uvrX1;
xseA,B; yjhB1;ykoW; ylbH; yocI; yobH1,2; yorK1,2; yozK1,2; yqfN; yrrK; yvcI; ywbD
alkA; dinB1; lexA; mutS1; polY1(yqjH); radC; recF,G,O; ruvA,B; ssb; yjcD; yneB;
addA,B; exoA1,2; gyrA; mutT1,Y; nth; pcrA; recU; topA; ung; uvrC; ydiP2; yhaZ2;
ypcP; yprA; ypvA; yrrT; yrvN; ywqA, yxlJ1
recA; uvrA,B; ykoU(lig),V(ku)
Oxidative stress resistance (35)
bcrC; cotJC1; katX22,3; msrA; ohrA,B,R; sigM; sodF; trxA; ycgT; ygaF; yjqC; ykuU;
ahpC1,F1; bsaA; perR; sigB; sodA; spx; tpx; trxB; ydbD1; yqjM
csgA1; sspA,B,C1,2,D,E,F,G1,2,H1,I,J,K,L,M,N,O (cotK),P(cotL);tlp
1=absent in B. pumilus SAFR-032 (Bp). 2=absent in B. licheniformis (Bl). 3=absent in B. subtilis 168 (Bs).
V=genes transcribed in B. subtilis vegetative cells .
G&O=genes transcribed during B. subtilis spore germination & outgrowth [24,42,70].
S=gene products present in B. subtilis spores [26,34,42].
B.pumilus UV& H2O2 Resistance
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and UvrB, which recognize DNA lesions, the UvrC nuclease, and
the UvrD helicase. The NER machinery can be recruited to DNA
damage by the Mfd protein in a process called transcription-
coupled NER. In B. subtilis, NER is associated with UV radiation
resistance in vegetative cells , and uvrA and uvrB are
transcribed in germinating/outgrowing spores . B. subtilis
lacks UvrD, but likely uses PcrA to perform the UvrD helicase
function [32,37]. B. pumilus encodes homologs of UvrABC
(BPUM_3147, 3148, & 2506), PcrA (BPUM_0625), and Mfd
(BPUM_0039), the amino acid sequences of which are conserved
with respect to B. subtilis and B. licheniformis (Table 2).
Mismatch Repair (MMR)
mismatched bases in newly synthesized DNA daughter strands,
and although not associated with DNA repair related to UV
radiation or oxidative damage, it is important in maintaining
genomic integrity . In E. coli, MMR involves MutS and MutL,
which recognize mismatches, and endonuclease MutH. Bacillus
species lack MutH and must use another, unidentified mechanism
. B. pumilus MutS (BPUM_1608) and MutL (BPUM_1609)
homologs are moderately well-conserved compared to those of B.
subtilis and B. licheniformis (Table 2). Homologs of XseA
(BPUM_2162) and XseB (BPUM_2161), subunits of a MMR
exonuclease, were also annotated in B. pumilus.
MMR recognizes and repairs
DNA Repair Mechanisms–Double Strand Repair
Non-Homologous End-Joining (NHEJ)
repairs double-strand DNA (DSB) breaks by directly joining DNA
ends without requiring a homologous template to guide the repair
. Prokaryotic homologs of the eukaryotic DNA-end-binding
protein, Ku, and DNA ligase IV were recently identified in several
bacteria . In B. subtilis, the NHEJ proteins are encoded on the
ykoUVW operon, and ykoU and ykoV are transcribed both in
vegetative cells and germinating/outgrowing spores . B. subtilis
YkoV (Ku) specifically recruits YkoU (DNA ligase IV) to DNA
ends to stimulate DNA ligation, and loss of these proteins leads to
hypersensitivity to UV radiation in B. subtilis . YkoW is
hypothesized to interact with dsDNA ends.
There is significant amino acid sequence variation in NHEJ
proteins among B. pumilus, B. subtilis, and B. licheniformis (Table 2).
The YkoV and YkoU sequences of B. subtilis and B. licheniformis are
more closely related to each other than to their B. pumilus
The NHEJ pathway
Table 2. Sequence conservation of DNA repair proteins
among Bacillus species.
Bp vs. Bs
Bp vs. Bl
Bs vs. Bl
Base excision repair
AlkA (YfiP) 60 6878
MutM (Fpg) 6463 77
MutY (YfhQ) 68 6675
Nth 88 8889
Nfo (YqfS) 86 87 91
Nucleotide Excision Repair
PcrA83 85 87
UvrB 89 8992
UvrC 82 8484
XseA (YqiB) 737175
XseB (YqiC) 74 7773
YkoU 4041 58
YkoW 49 3430
AddA 66 6772
AddB64 65 74
LexA 8787 91
RecD (YrrC) 7982 79
RecF 8484 89
RecG (YlpB) 8180 81
RecJ (YrvE)67 6268
RecQ (RecS)5353 58
RecU70 73 80
RecX (YfhG) 63 6470
RuvB85 85 88
SbcC (YirY)51 49 56
SbcD 74 74 78
Spore Photoproduct Lyase
SplA 6365 73
SplB90 86 88
UVDE-dependent excision repair
PolY1 (YqjH)70 7175
PolY2 (YqjW) 64––
Bp vs. Bs
Bp vs. Bl
Bs vs. Bl
DinG 53 5259
End1 (YurI)60 6061
YpcP 70 6976
YvcI 7881 77
Table 2. cont.
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homologs (BPUM_1667 & BPUM_1666). Additionally, B. pumilus
YkoW (BPUM_1234), at 807 amino acids in length, is much
longer than B. subtilis YkoW (749 amino acids) and B. licheniformis
(549 amino acids). Beyond these differences in amino acid
sequences, which may affect protein function, the regulation of
NHEJ genes appears to be different in B. pumilus. The ykoUVW
operon structure of B. subtilis and B. licheniformis is not conserved in
B. pumilus. In B. pumilus, ykoU and ykoV are adjacent and
divergently transcribed, while ykoW is located on a separate locus.
Homologous recombination (HR)
is a ubiquitous process that is crucial for DNA repair and
maintenance. It is a multi-step pathway involving several proteins
that facilitate the invasion of dsDNA by a ssDNA substrate. As
DNA is unwound by a helicase, the migrating strand replaces
damaged DNA and the intermediate structure is resolved by an
endonuclease . HR in Bacillus species can be initiated by the
AddAB pathway, which is analogous to E. coli RecBCD , or
the RecFOR pathway . Strand invasion and exchange is
catalyzed by RecA, and recA mutations increase sensitivity to UV
radiation . Branch migration is performed by the RuvAB
proteins, and resolution is performed by the RecU (RuvC in E. coli),
and RecG proteins . B. pumilus encodes homologs of all HR
proteins common to B. subtilis and B. licheniformis (Tables 1 & 2).
Control of HR is closely related to the SOS repair system. In B.
subtilis, the SOS regulon is similar to that in E. coli but it is also
induced in competent cells in the absence of any DNA-damaging
treatment . RecA and the SOS transcriptional repressor LexA
are the two main proteins involved in this coordinated cellular
response to UV-light and DNA-damaging agents . RecA is
activated by ssDNA and promotes LexA self-cleavage, causing it to
lose affinity to DNA and allowing expression of the SOS-response
genes. The B. pumilus LexA (BPUM_1686) sequence is 87% similar
to the B. subtilis and B. licheniformis LexA homologs, and their
DNA-binding motifs are identical , suggesting that their
activities are similar in these three species. Several SOS proteins
have been identified in E. coli and B. subtilis, but the identification
of B. pumilus SOS proteins will require experimental verification of
regulation by RecA and LexA . HR is also under the influence
of RecX, a repressor of recA . B. pumilus RecX (BPUM_0795)
has moderate sequence conservation with its B. subtilis and B.
Other DNA repair systems
UVDE-dependent excision repair
homolog of UVDE, a eukaryotic protein that repairs UV
photoproducts . B. pumilus encodes a YwjD (BPUM_3376)
homolog that shares only moderate sequence identity with B.
subtilis YwjD, which is produced in vegetative cells, and there is no
B. licheniformis homolog. Because the sequence conservation is
poor, it is possible that B. pumilus YwjD functions in a way that
enhances its DNA repair activity.
The Y family polymerases are error-
prone, translesional DNA polymerases that are processive through
DNA lesions that block the replicative polymerase . Two Y-
family polymerases were annotated in B. pumilus and named for
their B. subtilus homologs, PolY1 (YqjH; BPUM_2125) and PolY2
(YqjW; BPUM_2102). In B. subtilis PolY2 is an SOS inducible
polymerase that functions in UV damage repair and is necessary
for UV-induced mutagenesis . PolY2 is missing in B.
licheniformis, which may contribute to its relative UV sensitivity.
The fact that PolY2 is present in both B. pumilus and B. subtilis
means that it alone cannot account for the UV resistance of B.
pumilus. However, sequence variation (Table 2) and differences in
expression may influence its activity. In contrast, PolY1 is a DinB
subfamily polymerase that is constitutively expressed and functions
in untargeted mutagenesis rather than UV-induced mutagenesis
. PolY1 is common to B. pumilus, B. subtilis, and B. licheniformis,
and, therefore, unlikely to be responsible for UV resistance. Two
other B. subtilis Y-family polymerases, UvrX and YozK-YobH are
encoded on integrated prophages that are not present in B. pumilus
Alkylating chemicals can mutate DNA
bases or the phosphodiester backbone by adding an alkyl group to
the nitrogen or oxygen atoms. Ogt is a methyltransferase that
removes the alkyl group from O6-alkyl guanine or, preferentially,
O4-alkyl thymine . Ogt also exhibits suicide inactivation by
transferring an alkyl group to a cysteine residue in its own
structure. Ogt (BPUM_1248) is found in B. pumilus, B. subtilis and
B. licheniformis, although the protein sequence is not well-conserved
The B. pumilus genome encodes a second alkyltransferase, Ada
(BPUM_1200), which, like Ogt, removes alkyl moities from DNA
YwjD is a B. subtilis
pyrimidine dimersand 6-4
Table 3. Sequence conservation of H2O2resistance proteins
among Bacillus species
Bp vs. Bs
Bp vs. Bl
Bs vs. Bl
Tpx85 84 86
TrxA92 93 97
YcgT 5146 39
YkuU 9697 98
OhrA73 61 72
SigM (YhdM) 91 9196
SigB (RpoF) 878486
DpsA (YktB) 8282 78
BcrC (YwoA)50 4760
YqjL 42 5657
1blastp vs. KatX.
2blastp vs. YdbD.
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PLoS ONE | www.plosone.org6 September 2007 | Issue 9 | e928
by suicide inactivation . Ada also initiates the adaptive
response, which activates several DNA repair enzymes . There
are notable differences between B. pumilus Ada and the homologs
of B. subtilis and B. licheniformis that may be significant in DNA
repair. B. pumilus Ada, like E. coli Ada, incorporates a regulatory
domain fused to the alkyl glycosylase domain. However, in both B.
subtilis and B. licheniformis, these domains are split into two proteins
, AdaA and AdaB, neither of which align with greater than
50% identity to B. pumilus Ada. The fusion of the two proteins in B.
pumilus raises the possibility that the function and transcriptional
regulation of this alkyltransferase may be different in B. pumilus
compared to B. subtilis and B. licheniformis.
The ATP-dependent DNA helicase DinG
can unwind RNA or DNA, and it is a bacterial homolog of
a human DNA repair helicase [54,55]. Homologs of DinG
(BPUM_1971) are present in B. pumilus, B. subtilis and B.
licheniformis, although their sequences are not well conserved
The Nudix hydrolase superfamily MutT protein hydrolyzes 8-
oxo-dGTP (a reactive oxygen species) and prevents its incorpo-
ration into DNA . B. subtilis has three MutT superfamily genes,
mutT, which is transcribed in germinating/outgrowing spores ,
yjhB, and yvcI. The B. pumilus genome has one yvcI gene
(BPUM_3116), but no mutT; or yjhB homologs.
YshD is a MutS2 family protein that maintains genome integrity
(BPUM_2516) sequence is conserved among B. pumilus, B. subtilis,
and B. licheniformis (Table 2), but it is unclear if it has an effect on
UV or H2O2resistance.
recombination. The YshD
DNA repair proteins unique to B. pumilus
The B. pumilus genome encodes PhrB (BPUM_1378), a DNA
photolyase enzyme that repairs cyclobutane-pyrimidine dimers
. Although no homolog exists in B. subtilis and B. licheniformis,
there are homologs in other Bacillus species such as B. firmus, B.
cereus, B. anthracis, and B. thuringiensis. Nevertheless, none of these
species exhibit UV radiation resistance comparable to B. pumilus.
The B. subtilis photolyase amino acid sequence is diverse with
respect to other photolyases. It shares 32% amino acid identity
with E. coli PhrB and only 46% sequence identity with its closest
homolog from B. firmus. It is logical that the presence of
a photolyase gives B. pumilus UV resistance capabilities that B.
subtilis and B. licheniformis lack. However, because less UV-resistant
Bacillus species also have photolyase enzymes, the relation of
photolyase to enhanced UV resistance is not clear. Although the
sequence divergence in the B. pumilus photolyase may indicate
altered function, B. pumilus may rely on a combination of other
factors for its UV resistance properties.
Genes encoding two DNA repair/modification proteins not
found in B. subtilis and B. licheniformis were also annotated in B.
pumilus. One sequence (BPUM_0608) is similar to a Superfamily II
(SF-2) helicase based upon the presence of a DExD Walker B
motif in conserved motif II . SF-2 helicases are known to
function in NER and recombinational repair in yeast .
Although it cannot be predicted that this helicase functions in
DNA repair, if it does have such a function it would be a feature
that B. subtilis and B. licheniformis lack, possibly contributing to the
enhanced UV radiation resistance. B. pumilus also encodes a C-5
cytosine-specific DNA methyltransferase (BPUM_0656) that has
no homolog in either B. subtilis or B. licheniformis. Though unlikely
to be directly implicated in DNA repair, it is possible that a unique
DNA-modifying protein may contribute to genomic stability in B.
pumilus. Additionally, the B. pumilus genome has 517 coding
sequences that are not common to B. subtilis and B. licheniformis,
including 218 hypothetical proteins that have no sequence
similarity to any known sequence in the nr database. It is possible
that one more of these coding sequences of unknown function may
contribute to UV radiation resistance.
Bacillus species use a variety of proteins to resist the toxic effects of
H2O2, including catalases, and various reducing proteins such as
alkyl hydroperoxide reductase and peroxiredoxins . Analysis
of the B. pumilus genome reveals many striking differences
compared to similar proteins in B. subtilis and B. licheniformis.
Catalases convert H2O2into water and oxygen in
a highly efficient reaction that requires neither ATP nor an
exogenous reducing agent . B. subtilis and B. licheniformis produce
two vegetative catalases, KatA and KatB (KatE), and one
germination catalase, KatX, which is present in spores and
protects germinating cells from H2O2. All three catalases are
transcribed in germinating/outgrowing spores . B. pumilus has
no homolog to either vegetative catalase, however, it has two
KatX homologs.The sequence
(BPUM_3712) is moderate, but KatX2 (BPUM_0892) is more
diverse, sharing less than 50% identity with B. subtilis and B.
licheniformis KatX. A second germination-specific catalase with
substantial sequence diversity is a candidate protein that may
explain the enhanced peroxide resistance of B. pumilus spores.
YjqC and YdbD are two additional proteins with catalase
domains that are found in B. subtilis and B. licheniformis, although
little is known of their functions. B. pumilus YjqC (BPUM_2346)
shares moderate sequence identity with its Bacillus homologs
(Table 3), but there is no YdbD homolog in B. pumilus. However,
a B. pumilus sequence containing a manganese catalase domain
does exist (YdbD uses Mn2+as a cofactor). It is possible that this
catalase (BPUM_1305), which differs greatly from YdbD, may
have properties that contribute to the H2O2 resistance of B.
The spore coat protein CotJC contains a predicted catalase
domain in its amino acid sequence. Although CotJC is present in
B. subtilis and B. licheniformis, no homolog was identified in B.
pumilus, suggesting that it is not necessary for elevated peroxide
Bacteria use peroxiredoxins to reduce H2O2
to water . Four peroxiredoxins were annotated in B. pumilus.
Three peroxiredoxin protein sequences, YkuU (BPUM_1319),
YgaF (BPUM_0826), and Tpx (BPUM_2581), are highly
conserved with respect to their B. subtilis and B. licheniformis
homologs (Table 3). The fourth peroxiredoxin (BPUM_3690)
annotated in B. pumilus has no obvious homolog in B. subtilis or B.
licheniformis. Instead, thesetwo
hydroperoxide reductase that is induced upon H2O2stress. The
enzyme is a heterodimer of AhpC and AhpF, and it uses NADH
or NADPH as a reducing agent. In B. subtilis and B. licheniformis the
subunits are encoded on the ahpCF operon, and their translated
sequences are highly conserved (.90% identity for AhpC and
AhpF). The B. pumilus genome does not contain a homologous
operon. However, it does it does have a gene encoding an NADH
dehydrogenase (BPUM_2106), which, if coupled with the
peroxiredoxin (BPUM_3690), could hypothetically function as
an alkyl hydroperoxide reductase. Given the lack of sequence and
gene order conservation, the function may be distinct from B.
subtilis and B. licheniformis AphCF, possibly explaining the abnormal
H2O2resistance of B. pumilus.
Peroxidases also reduce H2O2to water using
NADH or NADPH as a cofactor . A glutathione peroxidase,
BsaA (BPUM_1925), was annotated in B. pumilus. BsaA uses
species producean alkyl
B.pumilus UV& H2O2 Resistance
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glutathione as a reducing agent to reduce lipid hydroxyperoxides
formed by peroxide stress, and bsaA is transcribed during spore
germination/outgrowth . There is substantial sequence
diversity among the BsaA homologs of B. pumilus, B. subtilis, and
B. licheniformis (Table 3), raising the possibility that differences in B.
pumilus BsaA may contribute to H2O2resistance.
Other reducing agents
Thioredoxins and glutaredoxins are
instrumental to peroxidestress
peroxiredoxins and peroxidases, facilitating their functions, and
act as hydroxyl radical scavengers . They also maintain
oxidation states of cytoplasmic proteins, preventing illegitimate
disulfide bond formation . Several redox proteins were
annotated in B. pumilus, but only those known to be related to
peroxide stress and those unique compared to B. subtilis and B.
licheniformis are mentioned in this work.
TrxA is the product of the thioredoxin A gene, which is essential
in B. subtilis. The reducing potential of TrxA is recycled by the
thioredoxin reductase, TrxB. B. pumilus TrxA (BPUM_2507) and
TrxB (BPUM_3117) share approximately 90% identity with their
B. subtilis homologs. A second TrxB-like thioredoxin reductase
(BPUM_0664) was annotated in B. pumilus (Table 4). This protein
has no B. subtilis homolog and a poor alignment to a B. licheniformis
reductase, so it may provide peroxide stress resistance capabilities
not available to these species. YcgT (BPUM_0777), another
thioredoxin-disulfide reductase, is present in B. pumilus, but its
sequence is not well-conserved with B. subtilis and B. licheniformis,
raising the possibility that differences in YcgT activity may be
important to B. pumilus oxidative stress resistance.
The ohr operon
In B. subtilis resistance to organic peroxides is
encoded by the ohr locus, which produces the peroxide resistance
proteins OhrA and OhrB, and OhrR, a transcriptional regulator
of ohrA . The B. pumilus homologs (BPUM_1211-1213) of these
proteins share moderate homology with their B. subtilis and B.
licheniformis homologs (Table 3), so they may have altered function
due to sequence diversity.
resistance. They reduce
Regulation of the oxidative stress response
transcriptional regulators of the B. subtilis oxidative stress
response are known, including PerR, Spx, and sigma factors
SigM and SigB. All four of these proteins are conserved in B. pumilus
(Table 3). Dps proteins are DNA- binding proteins that protect
bacteria from oxidative stress by sequestering iron and oxidants and
storing them as benign ferric oxide minerals . Two Dps proteins,
DpsA (YktB) and MrgA are known in B. subtilis and B. licheniformis. B.
pumilus does encode a DpsA homolog (Table 3; BPUM_2703),
however, it has no MrgA homolog. Although MrgA is important for
peroxide resistance proteins in vegetative cells, it has no effect on
peroxide resistance in spores .
Other oxidative stress proteins
in the form of superoxide, O22. Although the O22and H2O2
stress responses are distinct, the conditions are related, via the
chemical conversion of O22to H2O2by superoxide dismutases. B.
pumilus has three superoxide dismutases: SodA (BPUM_2230),
which uses a manganese cofactor, SodF (BPUM_1859), which uses
an iron cofactor, and YojM (BPUM_1865), which uses copper or
zinc as a cofactor . B. pumilus SodA is highly conserved with
respect to the homologs of B. subtilis and B. licheniformis, but there is
much greater sequence diversity in SodF and YojM (Table 3). If
these changes in sequence have any effect on protein function, it is
difficult to speculate what benefit there would be for H2O2
resistance, as any decrease in superoxide reductase-mediated
H2O2production would mean less efficient removal of O22. The
hydrolase YqjL (BPUM_2113) and the efflux protein BcrC
(BPUM_3294) both contribute to O22resistance by unknown
mechanisms . The B. pumilus homologs of these proteins are
not well-conserved with respect to their B. subtilis and B.
licheniformis homologs (Table 3). It is possible that B. pumilus YjqL
and/or BcrC may be more adept at O22detoxification than their
B. subtilis and B. licheniformis homologs, and that their activities may
alleviate the production of H2O2by superoxide dismutases.
Two additional proteins related to oxidative stress resistance in
B. subtilis were not annotated in the B. pumilus genome. YlaC is a B.
subtilis extracytoplasmic sigma factor that is regulated by the anti-
sigma factor YlaD, which contains an oxidative stress-sensing
domain . Transcription of ylaCD was also shown to be Spx-
dependent, further linking it to the oxidative stress response.
Nevertheless, the lack of YlaC and YlaD homologs in B. pumilus
indicates that despite the function of these proteins, they are not
essential to H2O2resistance.
The database of annotated B. pumilus coding sequences was
examined for oxygenases, oxidoreductases, and redoxins without
homologs in B. subtilis or B. licheniformis. The predicted proteins and
functions associated with these regions are listed in Table 4.
Additionally, it is possible that proteins functioning in peroxide
resistance are among the hypothetical proteins and other
undefined ORFs that have no B. subtilis or B. licheniformis homolog.
Oxidative stress also occurs
Given the phenotypic differences between B. pumilus and B. subtilis
and B. licheniformis in terms of UV radiation resistance and H2O2
resistance, it was expected that a comparison of the genomes of
these species would point to unique B. pumilus genes related to these
functions. Most genes related to DNA repair and H2O2resistance
are conserved among these species. Paradoxically, B. pumilus lacks
several DNA repair and oxidative stress response genes found in B.
subtilis and B. licheniformis. Nevetheless, this analysis has identified
several B. pumilus genes worthy of further study because of their
absence in related organisms, differences in amino acid sequence, or
predicted differences in genetic regulation.
Table 4. Unique DNA repair and H2O2resistance proteins of B.
BPU locus tag2
BPUM_1378 photolyase PhrB
BPUM_1200DNA repair methyltransfrease Ada
BPUM_0664TrxB-like thioredoxin-disulfide reductase1
BPUM_1716NADH-dependent flavin oxidoreductase2
BPUM_1153possible FAD dependent oxidoreductase
BPUM_0802 possible monooxygenase
BPUM_0482 probable dioxygenase
1homolog in B. licheniformis, but not B. subtilis.
2only 42% identity with B. licheniformis and B. subtilis YqjM; B. pumilus YqjM
found at BPUM_2112.
B.pumilus UV& H2O2 Resistance
PLoS ONE | www.plosone.org8September 2007 | Issue 9 | e928
Found at: doi:10.1371/journal.pone.0000928.s001 (0.09 MB
Locus tag numbers of genes in Table 1.
We like to especially acknowledge John Rummel, NASA’s Planetary
Protection Officer for his constant encouragement of our efforts in this
Conceived and designed the experiments: GW KV GF WN SH.
Performed the experiments: KV DM SD YD AH WL LP CK HD SL
LN PB CB MH XQ HJ. Analyzed the data: GW AU KV AV GF JG SY
MT YL ID LB MF FK PE JS PB AA HZ BM AO KC DN. Contributed
reagents/materials/analysis tools: GW JP KV DM SD YD AH WL LP CK
HD SL LN PB CB MH SH JG SY XQ HJ OI MF LG. Wrote the paper:
GW JG SY.
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