Sequence of Kineococcus radiotolerans SRS30216

16 Pages·2009·0.8 MB·English

Survival in Nuclear Waste, Extreme Resistance, and
Potential Applications Gleaned from the Genome
Sequence of KineococcusradiotoleransSRS30216
ChristopherE. Bagwell1, Swapna Bhat3, Gary M. Hawkins3, Bryan W. Smith1, Tapan Biswas2, Timothy R.
Hoover3, Elizabeth Saunders4, Cliff S. Han4, Oleg V. Tsodikov2, Lawrence J. Shimkets3*
1SavannahRiverNationalLaboratory,EnvironmentalSciencesandBiotechnology,Aiken,SouthCarolina,UnitedStatesofAmerica,2DepartmentofMedicinalChemistry,
CollegeofPharmacy,UniversityofMichigan,AnnArbor,Michigan,UnitedStatesofAmerica,3DepartmentofMicrobiology,UniversityofGeorgia,Athens,Georgia,United
StatesofAmerica,4DOEJointGenomeInstitute,BioscienceDivision,LosAlamosNationalLaboratory,LosAlamos,NewMexico,UnitedStatesofAmerica
Abstract
KineococcusradiotoleransSRS30216wasisolatedfromahigh-levelradioactiveenvironmentattheSavannahRiverSite(SRS)
and exhibits c-radiationresistance approaching thatofDeinococcus radiodurans.Thegenome wassequencedby the U.S.
Department of Energy’s Joint Genome Institute which suggested the existence of three replicons, a 4.76 Mb linear
chromosome, a 0.18 Mb linear plasmid, and a 12.92 Kb circular plasmid. Southern hybridization confirmed that the
chromosomeislinear.TheK.radiotoleransgenomesequencewasexaminedtolearnaboutthephysiologyoftheorganism
withregardtoionizingradiationresistance,thepotentialforbioremediationofnuclearwaste,andthedimorphiclifecycle.
K.radiotoleransmayhaveauniquegenetictoolboxforradiationprotectionasitlacksmanyofthegenesknowntoconfer
radiationresistanceinD.radiodurans.Additionally,genesinvolvedinthedetoxificationofreactiveoxygenspeciesandthe
excisionrepairpathwayareoverrepresented.K.radiotoleransappearstolackdegradationpathwaysforpervasivesoiland
groundwaterpollutants.However,itcanrespireontwoorganicacidsfoundinSRShigh-levelnuclearwaste,formateand
oxalate,whichpromote the survivalof cellsduring prolonged periodsofstarvation. Thedimorphic lifecycle involvesthe
productionofmotilezoospores.Theflagellarbiosynthesisgenesarelocatedonamotilityisland,thoughitsregulationcould
notbefullydiscerned.TheseresultshighlighttheremarkableabilityofKradiotoleranstowithstandenvironmentalextremes
and suggest thatinsitubioremediationoforganiccomplexants from highlevelradioactive wastemay befeasible.
Citation:BagwellCE,BhatS,HawkinsGM,SmithBW,BiswasT,etal.(2008)SurvivalinNuclearWaste,ExtremeResistance,andPotentialApplicationsGleaned
fromtheGenomeSequenceofKineococcusradiotoleransSRS30216.PLoSONE3(12):e3878.doi:10.1371/journal.pone.0003878
Editor:NiyazAhmed,CentreforDNAFingerprintingandDiagnostics,India
ReceivedOctober6,2008;AcceptedNovember4,2008;PublishedDecember5,2008
Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsPublicDomaindeclarationwhichstipulatesthat,onceplacedinthepublic
domain,thisworkmaybefreelyreproduced,distributed,transmitted,modified,builtupon,orotherwiseusedbyanyoneforanylawfulpurpose.
Funding:CEBwouldliketoacknowledgesupportfromtheU.S.DepartmentofEnergy,OfficeofEnvironmentalManagementasadministeredthroughthe
LaboratoryDirectedResearchandDevelopmentProgram,andtheU.S.DepartmentofEnergy,OfficeofScience,OfficeofBiologicalandEnvironmentalResearch,
EnvironmentalRemediationSciencesProgram(Contract#KP1302000).LJSwouldliketoacknowledgesupportfromtheOfficeoftheVicePresidentforResearch,
UniversityofGeorgia.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction decontamination reagents is oxalic acid, which can create
problems for storage and final disposal due to its solubility
High-level radioactive waste (HLW) is an anthropogenic
propertiesasasodiumsalt.AdditionalreagentsinuseatSRSand
disturbance to which few organisms are resistant. During the
Cold War, Pu239 production for national defense began by Hanford include glycolic acid, citric acid, and formic acid.
Removal of organic constituents directly in HLW tanks could
irradiatinguraniumorothertargetelementsinanuclearreactor.
At Hanford, WA and the Savannah River Site (SRS), SC, greatly improve processing efficiencyof HLW.
irradiated fuel and targets were reprocessed to reclaim approxi- In 2001, the Committee on Long-Term Research Needs for
mately 99% of the U235 and Pu239 isotopes. All remaining Radioactive High-Level Waste at Department of Energy Sites
radionuclides,fissionproducts,fuelcomponents,andnonradioac- recommended the investigation of radioactive waste to identify
tivechemicalsusedduringreclamationmakeuptheHLW,which promising new radiation resistant microorganisms that might be
currently resides at over 100 different sites across the contiguous used to degrade some of the organic constituents in the HLW.
U.S.andexceeds1billioncuries.ThemajorityofthislastingCold Radiation dosage is measured in gray (Gy); 1 Gy causes the first
WarlegacyislocatedatHanford(roughly65milliongallons)and signsofradiationsicknessinhumans.Halfofallpeopleexposedto
SRS (roughly 35milliongallons). 4.5 Gy die, and doses of 8 Gy or more are invariably fatal to
The SRS waste contains Fe, Al, Si, Ca, F, K, alkali cations, humans. Bacteria are the most extreme examples of radiation
organic solvents, radionuclides, and other fission products. resistantorganisms.TheparadigmisDeinococcusradiodurans,which
Organicconstituentsincludecomplexantsusedduringseparations, wasisolatedfromcannedmeatthatreceived4,000Gyofionizing-
radiolysisproductsfromdegradationofcomplexantsandsolvents, radiation[1],butmanyotherradiationresistantbacteriahavealso
and waste tank decontamination reagents. One of the preferred been identified.
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ResistanceofK.radiotolerans
KineococcusradiotoleransSRS30216wasisolatedfromHLWwithin thoughttobeduetoaccumulatedmutationalbiasesduringleading
a shielded cell work area at the SRS [2]. K. radiotolerans is an vs lagging strand synthesis. There is no obvious GC skew at the
orange-pigmented, aerobic bacterium belonging to the Actino- center of the chromosome (Figure 1, compare green vs magenta
bacteria phylum that is capable of withstanding relatively high peaksininnercircle).ThereishoweveraremarkableGCskewat
concentrationsofmetalsandalkalications,aswellasexposureto the telomers but it seems unlikely that replication proceeds from
extreme doses of ionizing radiation. K. radiotolerans exhibits thetelomerstowardthecenterbasedontheBorreliaandStreptomyces
radiation resistance approaching that of D. radiodurans [2]. D. systems.AsecondmethodusedtolocateoriCisthepresenceofthe
radiodurans and K. radiotolerans belong to different phyla and it dnaA gene and DnaA binding sites. dnaA (Krad0001) is near the
remains unknown whether both organisms attain radiation center of the chromosome, as with Streptomyces and Borrelia.
resistance through a common set of gene products. Because K. Putative, imperfect blocks of DnaA binding sites, observed using
radiotoleranssurvivedaHLWenvironment,itisexpectedtopossess DoriC [6], are widely scattered in the center of the chromosome
potent cellular defense and repair mechanisms for radiation with theclosest beingabout 94Kb fromthe dnaAgene.
exposure,osmoticstressandchemicaltoxicity.Theoccurrenceof ThetopologyofthechromosomewasinvestigatedbySouthern
all these features in a naturally occurring bacterium may have hybridization using an end-specific probe homologous with
direct applications for thebioremediation of nuclearwaste. Krad2223. Three restriction enzymes were predicted to generate
In this work the genome sequence of K. radiotolerans SRS30216 singlefragmentswiththisprobe.Ifthechromosomeislinearthen
was examined in three different contexts. First, the presence of products with sizes predicted from the DNA sequence should be
genes known to confer radiation resistance in D. radiodurans was visibleafterhybridization.Ifthechromosomeiscircularthenthe
examined in the K. radiotolerans genome. Second, the capacity for hybridization products with the probe should be the sum of the
bioremediation was assessed by comparative genomics as well as sizesofthepredictedrestrictionfragmentsfromeachendplusany
growthandrespirationstudies.Finally,thedimorphiclifecycleof missing sequence. Restriction fragments homologous with the
theorganism,inparticulartheproductionofmotilezoospores,was Krad2223 probe were similar in size to those predicted by the
examined by identifying genes involved in flagellar motility and DNA sequence (Fig. 2). These hybridization results confirm the
chemotaxis. lineartopologypredictedbytheinabilityofJGItoclosetheDNA
sequence.
Results and Discussion The ends of linear DNA replicons have special features that
preserve their integrity. B. burgdorferi linear replicons contain
Phylogeny covalently closed hairpin ends [4]. ResT, telomere resolvase,
K. radiotolerans belongs to the suborder Frankineae in the order hydrolyzesaphosphodiesterbondoneachDNAstrandthenjoins
Actinomycetales and the phylum Actinobacteria. Complete the opposite strands to form a covalently closed telomere. The
genome sequences are available for only two other members of process is reversed during chromosome replication and ResT is
the Frankineae suborder, Frankia alni and Acidothermus cellulolyticus. essentialforB.burgdorferigrowth.AResThomologisnotencoded
F.alniisdistinguishedbyitsabilitytofixnitrogeninsymbiosiswith bytheK.radiotoleransgenome.Streptomycescoelicolorrepliconendsare
alder (Alnus spp.) and myrtle (Myrica spp.), two pioneer plants in composedofsingle-strandedsequencesthatcanannealtoforma
temperateregions.TheK.radiotoleransgenomeshowsnopotential noncovalent circular molecule [3]. Streptomyces telomeres bind a
for nitrogen fixation. A. cellulolyticus is a thermotolerant organism family of conserved terminal binding proteins that have no
isolated from the hot spring in Yellowstone National Park that orthologsintheK.radiotoleransgenome.Thus,uniquemechanisms
degrades cellulose. While the K. radiotolerans genome may encode must protect theK.radiotolerans telomers.
proteins with the potential for degrading complex carbohydrates
likecellulose(Krad4622,3823),cellobiose(Krad0408,2526,2530, Ionizing radiation resistance
2531, 2539, 3436, 3480, 3961), glycogen, and starch (Krad1294, Gamma radiation is one of the most energetic forms of
1298), growth on these substrates has not been demonstrated electromagnetic radiation. Gamma rays penetrate tissues and
experimentally. cells, causing direct damage to DNA (namely double strand
More distant relatives in the same phylum include Streptomyces breaks),proteins,andmembranes.Gammaradiationalsoinduces
and Mycobacterium, whose physiology has been extensively indirect cellular damage through the ionization of water with
examined, and which serve as reference points for comparative formation of free radical species, primarily NOH. Oxygen free
genomics. radicals are extremely reactive, compounding cellular and DNA
damage.DNAdamageblockstranscriptionandreplication,andif
Genome size and organization not correctly repaired, could introduce detrimental mutations or
TheK.radiotoleransSRS30216genomewassequencedbytheUS causecelldeath.RelativelyfewDNAdoublestrandbreaks(DSB)
DOEJointGenomeInstitutewiththediscoveryofthreereplicons. arelethalformostbacteria.Escherichiacolicellssuccumbtoaround
The bulk of the DNA is contained on a 4,761,183 bp linear 10 DSB and Shewanella oneidensis cells die after 1 DSB (based on
chromosome(CP000750).Inadditionthereisa182,572bplinear calculations of0.0114 DSBs/Gy/Genome; Daly etal.,2004).
plasmid(pKRAD01;CP000751)anda12,917bpcircularplasmid Radioresistance has been partially characterized for K. radio-
(pKRAD02; CP000752). The three contigs were derived from at tolerans(Figure3).Acutelyirradiated,exponentiallygrowncultures
least 20 reads and average 74.2mol % G+C. The genome is have a broad shoulder of death, which contrasts with the
predicted tocontain4,715 genes. exponential death of E. coli. This shoulder is due in part to
Linear chromosomes are rare among prokaryotes. Aside from efficient repair systems and in part to the multicellular nature of
one atypical Agrobacterium isolate, the two principle examples are the organism. In rich medium K. radiotolerans grows in cubical
Streptomycesspecies[3]andBorreliaburgdorferi[4].InbothStreptomyces packetsthatformbyalternatingcelldivisionplanes(Phillipsetal.
andBorreliabidirectionalreplicationoccursfromasingleoriginof 2002). Thus, the colony forming unit method used to estimate
replication(oriC)locatednearthemiddleofthereplicon.Aplotof survivorship likely overestimates culture viability. Nevertheless,
the GC skew=(C2G)/(C+G) along the chromosome sometimes these results may portray a more ecologically relevant context as
inverts at the replication origin [5]. The shift in GC skew is cell clustering is common for certain species or developmental
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ResistanceofK.radiotolerans
Figure1.Genedistributionofthe4.76MbKineococcusradiotoleransSRS30216linearchromosomedepictedincircularform.The
breakisindicatedbyawavylineat12o’clock.Fromtheoutertotheinnerconcentriccircle:circles1and2,predictedproteincodingsequences(CDS)
indicatedbybluearrowsontheforward(outerwheel)andreverse(innerwheel)strands.RedindicatestRNAgenesandpurpleindicatesrRNAgenes;
circle3,GCcontentshowingdeviationfromaverage(74.2%);circle4,GCskew(+isgreenand2ispurple);circle5,genomicpositioninkbbeginning
withKrad2223andproceedingclockwise.ThechromosomemapwasgeneratedusingCGview[56].
doi:10.1371/journal.pone.0003878.g001
stages of Actinobacteria (eg., Frankia, Geodermatophilus, Actinoplanes, resistantbacterialspeciesarefoundintheActinobacteriathanany
Micrococcus) or other extremophiles (Kocuria, Deinococcus, Chroococci- otherphylum,radiationresistanceisnotawidespreadtraitinthis
diopsis). Remarkably though, Kineococcus can withstand the phylum and may have multiple evolutionary origins. The
damagingeffectsof20kGyofc-radiation(theoreticallygenerating mechanisms that render this trait remain poorly understood.
more than200DSB/genome; Dalyetal.,2004)andcelldivision Radiationresistantbacteriasufferseveredamagefromc-radiation
resumes within4days (Figure4). [8], which implies that molecular repair processes function with
Several other Actinobacteria species exhibit extraordinary high efficiency.
radiation resistance including Rubrobacter radiotolerans, Rubrobacter Analysis of genes common to four ionizing radiation resistant
xylanophilus, and Kocuria rosea [7]. While more ionizing radiation bacteriawithfullysequencedgenomesindicatedthatDNArepair
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ResistanceofK.radiotolerans
homologs of the D. radiodurans pprA, ddrA, ddrB, ddrC, ddrD genes
[11].
Mutations in recA render D. radiodurans as sensitive to ionizing
radiation as E. coli illustrating the importance of recombinational
repairinrepairingDSB(table1).D.radioduranscellsexposedto10
kGyc-radiationaccumulateabout100DSBpergenomethatare
repaired over the course of several hours. D. radiodurans DSB are
repairedbyhomologousrecombinationintheextendedsynthesis-
dependentstrandannealing(ESDSA)process[12].ESDSArepair
is carried out by RecA and PolA, enzymes found in the K.
radiotoleransgenome(table2).Basedontheirgenomicanalysis,recA
andpolAexhibitsignsofcoevolutioninionizingradiation-resistant
bacteria [9].
Genes involved in carotenoid biogenesis have been shown to
conferamodestlevelofradiationresistance[13–15]byscavenging
electrons from reactive oxygen species (table 1). K. radiotolerans
Figure2.Southernhybridizationrevealsalinearchromosome.
producescarotenoids[2]andthecarotenoidbiosyntheticpathway
PCRwasusedtogenerateprobesforthefirstendinthepresumptive
issimilartothatfoundinotherorganisms.Inadditiontothetwo
linear chromosome homologous with a portion of the first gene
Krad2223. The genome was digested with one of three restriction geneslistedintableIencodingphytoenesynthetaseandphytoene
enzymespredictedfromtheDNAsequencetogenerateahybridization desaturase, K. radiotolerans produces polyprenyl synthase
product with each probe. Lane 1, NcoI; Lane 2, BamHI; Lane 3, BglII. (Krad3227),lycopenecyclase(crtY,Krad0091),andneurosporene
Sizes of molecular markers in base pairs are given in the right hand dehydrogenase(crtD,Krad3225).Ahydroxlasegene(crtZ)wasnot
column. The predicted sizes of the homologous restriction fragments
discovered suggesting that the K. radiotolerans carotenoids are not
fromtheDNAsequenceareNcoI,865bp,BamHI,3692bp,andBglII,
hydroxylated.
1619bpwiththeKrad2223probe.Thepredictedsizesoftherestriction
fragments from the other end of the chromosome are NcoI, 775 bp, Higher eukaryotes repair DSB using nonhomologous end
BamHI,1363bp,andBglII,4018bp.Theobservedsizeswerecalculated joining(NHEJ)([16,17]Rihaetal.2006).NHEJrepairismediated
usingbacteriophagelambdaDNAstandardsdigestedwithHindIII.Inall by the Ku complex and the Ligase IV/XRCC4 complex along
cases the observed sizes were approximately equal to the sizes
withotherproteinswhoseprecisebiochemicalfunctionsremainto
predictedfromalinearchromosomeratherthanthesumofthesizes
be elucidated. While E. coli lacks the NHEJ pathway, some
frombothendssuggestingalineartopology.
doi:10.1371/journal.pone.0003878.g002 Actinobacteria genera such as Mycobacterium have this repair
pathway. Mycobacterial Ku binds DNA ends and recruits a
must have played a major role in evolutionary adaptation to polyfunctional DNA ligase/polymerase(LigD) invitro(Della etal.
ionizingradiation[9].D.radioduranshasservedastheparadigmfor 2004). Though repair is mutagenic, it does help maintain cell
radiation resistant organisms and both genetic and biochemical viability and loss of Ku and LigD increases sensitivity to ionizing
approachesareconvergingtorevealacomplexnetworkofrepair radiation(Stephanouetal.2007).K.radiotoleransappearstolacka
and protection processes [7,10]. Many D. radiodurans genes Ku-like DNA binding protein. While it does encode an ATP-
required for ionizingradiation resistance havebeen identified. In dependentDNAligase(Cdc9,orLigB;table2)NHEJrepairisnot
table1theyareorderedaccordingtothec-radiationsensitivityof likelywithout Ku.
a D. radiodurans strain lacking that gene based on published kill Ionizing radiation also causes many other types of DNA
curves.Whiletherolesofonlyafewofthesegenesareknownwith damage.TheK.radiotoleransDNAreplication,recombinationand
certainty, K. radiotolerans apparently employs a different genetic repair gene set is overlapping with [9], but generally different
toolboxfromthatofD.radiodurans.AbsentfromK.radiotoleransare from those of D. radiodurans [18] and E. coli [19] which may be
expectedsincetheseorganismsbelongtodifferentphyla(table2).
As one illustration of this difference, K. radiotolerans but not D.
radiodurans contains RecB and RecC which are involved in
recombinational repair in E. coli (reviewed in [20]). DNA
replication, repair and recombination systems in K. radiotolerans
are more similar to those of Mycobacterium tuberculosis [21],
possibly a reflection of the phylogenetic proximity of the two
organisms. In addition, M. tuberculosis is at least 10-fod more
resistant to ionizing radiation than E. coli [22]. Because M.
tuberculosis is an actively studied pathogen its genome is well-
annotated. Because it has coevolved the majority of its DNA
replication, repair, and recombination mechanisms with K.
radiotoleranswechoseitasareferenceorganism,togetherwithE.
coli and .
BothK.radiotoleransandM.tuberculosislacktheclassicalbacterial
mismatchrepairgenesMutS,MutH,MutL,RecJ,ExoVIII,ExoI
and Mug (table 2). A compensating factor may be production of
Figure 3. Resistance of K.radiotoleransto acute c-radiation
DNA polymerases with increased fidelity and proofreading
exposure. E. coli was used as a reference strain. Prior to irradiation,
efficiency as K. radiotolerans contains four exonuclease (e; DnaQ)
both strains were grown to exponential phase in TGY and LB,
respectively. Colony forming units determinations were conducted in subunits,three polymerase (a; DnaE)subunits andtwob(DnaN)
triplicate. subunits of thereplicative DNA polymerase III.Mismatch repair
doi:10.1371/journal.pone.0003878.g003 in K. radiotolerans may also be handled by proteins unrelated to
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ResistanceofK.radiotolerans
recombinational repair, or maintain multiple chromosomes. The
directreversalpathwayusedtorepairpyrimidinedimersincludes
two copies of the phrB photolyase gene and an additional, splB
photolyase gene, which is absent in E. coli, D. radiodurans and M.
tuberculosis. Other differences from M. tuberculosis include the
presenceinK.radiotoleransofPolA,TopoIV,HolIII,YejH,which
are absent in M. tuberculosis. The translesion DNA repair system
UmuC/UmuD, present in E. coli but not D. radiodurans or M.
tuberculosis,isalso a part oftheK. radiotolerans genome.
Similar tomycobacteria, the K. radiotolerans replicationgene set
lacks a well-defined homolog of the helicase loader (DnaC), but
contains the other main replication genes (DnaA, DnaB, DnaG).
K. radiotolerans and M. tuberculosis both contain a eukaryotic-like
DNA primase gene.
Figure 4. Post-irradiation recovery and growth of K.radio-
In summary, many of the genes known to confer radiation
tolerans. The irradiated cultures were exposed to 20 kGy c-radiation
resistanceinD.radioduransaremissingfromK.radiotoleranssuggesting
(open circles), and control cultures were incubated under laboratory
conditions(closedcircles). novel components to the repair and protection toolbox. Two
doi:10.1371/journal.pone.0003878.g004 pathways are involved in DSB repair in other organisms, ESDSA
repairmediatedbyRecAandPolAandNHEJrepairmediatedby
KuandLigD.RecAandPolAarepresentandmaybeaidedbythe
classical bacterial mismatch repair proteins. Some base excision presence of RecB and RecC. K. radiotolerans lacks the Ku protein
repair genes that may take on such roles are also uniquely makingthepresenceoftheNHEJpathwayunlikely.Baseexcision
overrepresented. There are three Fpg and four Nei base andnucleotideexcisionrepairpathwaygenesareoverrepresentedin
excisionases in K. radiotolerans, compared with one Fpg and no K.radiotoleransrelativetootherbacteria.
Nei homologs in D. radiodurans. As an additional example of
divergencewithD.radiodurans,3-methyladenineDNAglycosylaseI Reactive oxygen species detoxification
(Tag) ispresent in K.radiotolerans,but absent fromD. radiodurans. Most organisms have both RecA and PolA without exhibiting
Thenucleotideexcisionrepairpathwayisalsooverrepresented extreme radiation resistance. Remarkably, D. radiodurans cells
in K. radiotolerans by three uvrA orthologs and by five genes rendered ionizing radiation-sensitive by a polA mutation are fully
encodingUvrD-likehelicases.Inadditiontothesehelicases,theK. complemented by expression of the polA gene from ionizing
radiotolerans genome, similarly to M. tuberculosis, contains an radiation-sensitive E. coli [24]. Repair proteins, either native or
ERCC3 (XPB)-like superfamily II helicase, whose eukaryotic cloned,mayfunctionbetterafterirradiationinD.radioduranscells
homologperformsessentialfunctionsinnucleotideexcisionrepair due to protection from protein oxidation [25]. The genetic
andtranscription[23].TheK.radiotoleransandM.tuberculosisXPB components and molecular mechanisms of protein repair/
helicaseshavebeenrecentlydemonstratedtobefunctionalinvitro protection remain unknown but appear to be correlated with a
(Biswas andTsodikov, unpublished). Mn/Feratiointherangeof0.12–0.37[26].TheMn/Feratioin
Fivehomologsofhistone-likeproteins(IHForHupB-like)may K. radiotolerans is 0.09, slightly lower than Deinococcus but much
package DNA in order to protect it from damage, aid higherthan that of radiation sensitiveorganisms [27].
Table1. Genesconferring ionizing radiation resistance inDeinococcus radiodurans andtheirhomologs inKineococcus
radiotolerans.
D.radioduransgene Function D (kGy)1 Reference Kradlocustag
10
recA Homologousrecombination 0.1 [11] 1492
polA DNApolymerase 1.0 [24] 2951
pprA StimulatesDNAligase 2.0 [57] none
recQ DNAhelicase 6.0 [58] 0829
recD Helicase/exonuclease 6.0 [59] 0992
ddrB Unknown 8.0 [11] none
crtB Phytoenesynthase 9.0 [13] 3229
crtI Phytoenedesaturase 9.0 [13] 3228
ddrA ssDNAbindingprotein 12.0 [11] none
ddrC Unknown .14.0 [11] none
ddrD Unknown .14.0 [11] none
sbcCsbcD ssendonucleases39-59dsexonuclease 15.0 [60] 25532554
polX ssendonucleases39-59dsexonuclease 15.0 [61] 4036
1Doseofcradiationrequiredfora90%reductionincellviabilityestimatedfromdatasuppliedintherelevantreference.Forcomparison,theD10forwildD.
radiotoleransstrainsrangesfrom10–20kGydependingonthestrainandtheassayconditions.
doi:10.1371/journal.pone.0003878.t001
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ResistanceofK.radiotolerans
1 3 1
7 0 5
7 5 1
0 4 0
KineococcusradiotoleransSRS30216(locus) Krad_2866 Krad_4325Krad_3854 Nohomologs Krad_4316 Krad_3435 Nohomologs Krad_4243 Krad_0734(putativeDcm) Krad_4326Krad_3213 Krad_4334 Krad_1504 Krad_0001 Krad_4333 Krad_3187Krad_3215Krad_Krad_4598(onpKRAD01) Krad_3361 Krad_1769Krad_0002 Krad_4419Krad_3247Krad_Krad_1768 Krad_0466 Krad_1557 Krad_3612 Krad_2983 Krad_1377Krad_0158Krad_ Krad_1482 Krad_0007 Krad_0006 Krad_3762 Krad_0757(putativeUvrD) Krad_1885Krad_0173 Krad_1013 Nohomologs
osis
ndK.radiotolerans. MycobacteriumtuberculH37Rv(locus) Nohomologs Rv1317c TIGRlocus:NT02MT1098 Rv3062 Rv2415c Nohomologs Rv0321 Rv3037c(putativeDcm) Rv3056Rv1537(DinX) Rv2836c Rv1329c Rv0001c Rv0058c Rv1547cRv3370c Rv2343c Rv0002 Rv3711c Rv3721c Rv2697c Rv0861c Rv1407 Rv2924cRv0944 Rv2748c Rv0006 Rv0005 Rv1341 Nohomologs Rv2092c(HelY) Rv2101(HelZ) Rv2756c
a
urans,M.tuberculosis DeinococcusradioduransR1(locustag) Nohomologs DR_2584,DR_2074 Nohomologs Nohomologs DR_1855 Nohomologs Nohomologs Nohomologs Nohomologs DR_0792 Nohomologs DR_0002 DR_0549 DR_0507 DR_0601 DR_0001 DR_0856 DR_2410 Nohomologs DR_A0131 DR_2168 DR_0493 DR_0400 DR_1913 DR_0906 DR_0179 DR_1775(putativeUvrD) Nohomologs DR_1259 Nohomologs
d
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adi oli
E.coli,D.r Escherichiac Ada AlkA AlkB Nohomologs ComEA Dam Dcd Dcm DinB DinF DinG DnaA DnaB DnaE DnaG DnaN DnaQ DnaZ/X Dut Nohomologs Fmu Fpg/MutM FtsK GyrA GyrB HAM1/YggV HelD Nohomologs Nohomologs HsdM
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n
fu pe d
escodingforreplication,repairandrecombination BProteindescriptionandcomments O-6-methylguanine/O-4-methylthymineDNAmethyltransferase 3-methyladenineDNAglycosylaseII;DR_2584isofeukaryoticty Alkylationrepairprotein ATP-dependentDNAligase DNAuptakeprotein GATCspecificN6-adeninemethylase dCTPdeaminase Site-specificC-5cytosinemethlytransferase;VSPrepairistargetetowardhotspotscreatedbyDcm DNAdamageinducibleproteinP(DNApolymeraseIV) PossibleDNA-damage-inducibleproteinF;integralmembrane+-drivenmultidrugeffluxpumpprotein;Na ATP-dependentDNAhelicase;SOSinducer Chromosomalreplicationinitiatorprotein ReplicativeDNAhelicase aDNApolymeraseIII(holoenzyme),subunit DNAPrimase bDNApolymeraseIII(holoenzyme),subunit e99DNApolymeraseIII(holoenzyme),subunit-3-5exonuclease ctDNApolymeraseIII(holoenzyme),/subunit dUTPase XPB/ERCC3helicase rRNASAM-dependentmethyltransferase Formamidopyrimidineand8-oxoguanineDNAglycosylase(HomologofNei;seebelow) Chromosomeresolutionandpositioning DNAgyrase,subunitA DNAgyrase,subunitB Xantosinetriphosphatepyrophosphatase,prevents6-N-hydroxylaminopurinmutagenesis 99HelicaseIV(ATP-dependent3-to-5DNAhelicase)involvedintheRecFpathwayofrecombination Probablehelicase,Ski2subfamily(ATP-dependentRNAhelicase) ProbablehelicasewithaZincfingerdomain,Snf2/Rad54family TypeIrestriction/modificationsystemDNAmethylase
n
Ge me
Table2. Proteinna Ada AlkA AlkB Cdc9(LigB) ComEA Dam Dcd Dcm DinB/DinP DinF DinG DnaA DnaB DnaE DnaG DnaN DnaQ DnaZ/X Dut ERCC3 Fmu Fpg/MutM FtsK GyrA GyrB HAM1/YggV HelD HelY HelZ HsdM
PLoSONE | www.plosone.org 6 December2008 | Volume 3 | Issue 12 | e3878
ResistanceofK.radiotolerans
A)
ol
H 5 0 4
KineococcusradiotoleransSRS30216(locus) Nohomologs Nohomologs Krad_3422(unrelatedtoE.coli Krad_0490 Nohomologs Nohomologs Krad_2840 Krad_3104Krad_1244 Krad_2337Krad_1360Krad_280Krad_2005Krad_3371 Krad_1506 Krad_1489 Krad_1315 Nohomologs Krad_0653 Krad_1067 Krad_3154 Nohomologs Nohomologs Nohomologs Nohomologs Nohomologs Krad_1131Krad_2346Krad_314Krad_2697Krad_0113 Krad_0599 Krad_1488Krad_3521Krad_029Krad_3396 Nohomologs Nohomologs Krad_0422 Krad_3712
A)
ol
obacteriumtuberculosisRv(locus) 55c(HsdS’) 52 13c(unrelatedtoE.coliH 44c omologs omologs omologs omologs 86c(HupB)Rv1388(IHF) 20 96 14c 38 31(LigC) 20 88 28c omologs omologs omologs omologs 85cRv1160cRv0413c 89c 97Rv2464c omologs 70 74c 16c
MycH37 Rv27 Rv38 Rv24 Rv36 Noh Noh Noh Noh Rv29 Rv27 Rv32 Rv30 Rv09 Rv37 Rv10 Rv16 Rv25 Noh Noh Noh Noh Rv29 Rv35 Rv32 Noh Rv06 Rv36 Rv13
DeinococcusradioduransR1(locustag) Nohomologs Nohomologs DR_1244 DR_2332 Nohomologs Nohomologs Nohomologs DR_0420 DR_A0065 DR_A0344DR_A0074 Nohomologs DR_2069 Nohomologs Nohomologs DR_1532 DR_2074(alsoseeAlkA) DR_1877DR_0508DR_0587 DR_0715 Nohomologs DR_1976DR_1039containsaframeshift DR_1696 DR_0261 DR_2285 Nohomologs DR_2162 Nohomologs DR_2438,DR_0289,DR_0928 DR_0428
oli
Escherichiac HsdS HNS HolA HolB HolC HolD HolE HrpA HupB LexA Lhr LigA LigB Nohomologs Mfd MPG Mrr Mug(ygjF) MutH MutS MutL MutT MutY Nei Nfi Nfo Nth Ogt
n
nt. BProteindescriptionandcomments TypeIrestriction/modificationsystemspecificitydeterminant HU-histoneprotein dDNApolymeraseIII(holoenzyme),subunit dDNApolymeraseIII(holoenzyme),’subunit DNApolymeraseIII(holoenzyme),chisubunit DNApolymeraseIII(holoenzyme),psisubunit DNApolymeraseIII(holoenzyme),thetasubunit ATP-dependenthelicase DNAbindingproteinII,Integrationhostfactor(IHF);histone-likeproteins Transcriptionalregulator,repressoroftheSOSregulon,autoprotease ATP-dependenthelicasesuperfamilyII DNAligase,NAD()-dependent+ DNAligase,NAD()-dependent+ ProbableDNAligase Transcriptionrepaircouplingfactor;helicase 3-MethylpurineDNAglycosylase TypeIVrestrictionendonuclease G/Tmismatch-specificthymineDNAglycosylase,distantlyrelatedtoDR_1751;Presentasadomainofmanymultidomainproteinsinmanyeukaryotes Endonuclease,ComponentoftheMutHLScomplexfunctionsinthemethyl-directedmismatchrepairpathway ATPase,ComponentoftheMutHLScomplexfunctionsinthemethyl-directedmismatchrepairpathway PredictedATPase,ComponentoftheMutHLScomplexfunctionsinthemethyl-directedmismatchrepairpathway 8-oxo-dGTPase.D.r.encodesadditional17paralogs;onlysomepredictedtofunctioninrepair 8-oxoguanineDNAglycosylase&AP-lyase,A-GmismatchDNAglycosylase EndonucleaseVIII(alsoseeFpgabove) EndonucleaseV EndonucleaseIV(APendonuclease) EndonucleaseIII&thymineglycolDNAglycosylase;DR_0928andDR_2438areofarchaealtypeandDR_0289isclosetoyeastprotei O-6-methylguanine/O-4-methylthymineDNAmethyltransferase
Co me
a
2. nn HF gjF)
Table Protei HsdS HNS HolA HolB HolC HolD HolE HrpA HupB/I LexA Lhr LigA LigB LigC Mfd MPG Mrr Mug(y MutH MutS MutL MutT MutY Nei Nfi Nfo Nth Ogt
PLoSONE | www.plosone.org 7 December2008 | Volume 3 | Issue 12 | e3878
ResistanceofK.radiotolerans
5
0
3
KineococcusradiotoleransSRS30216(locus) Krad_1546 Krad_1534 Krad_3276Krad_1149 Krad_4707Krad_4374 Krad_2951 Nohomologs Krad_2988 Nohomologs Krad_3554Krad_4047 Krad_4702 Nohomologs Krad_3035 Krad_1492 Krad_0993 Krad_1171 Krad_4407 Krad_1855 Krad_0992 Krad_0994 Nohomologs Krad_0004 Krad_1368 Nohomologs Krad_3147 Krad_3375 Krad_0829RecQ-like:Krad_4Krad_4391 Krad_0465 Krad_1418 Krad_1493 Krad_3054
osis
ul
c
er
b
u
t
obacteriumRv(locus) omologs omologs 13 omologs omologs omologs 02 omologs omologs 85c omologs 59c 37c 30c 02c omologs 19 31c 29c omologs 03c 73c omologs 96c 62c 53 15c omologs 36c 93c
MycH37 Noh Noh Rv22 Noh Noh Noh Rv14 Noh Noh Rv35 Noh Rv25 Rv27 Rv06 Rv32 Noh Rv21 Rv06 Rv06 Noh Rv00 Rv29 Noh Rv16 Rv23 Rv12 Rv37 Noh Rv27 Rv25
ns
a
ur
d
o
radig) 2444
DeinococcusR1(locusta Nohomologs Nohomologs DR_0717 DR_C0005 DR_1707 Nohomologs DR_2606 Nohomologs Nohomologs DR_1105 Nohomologs DR_1898 DR_2340 Nohomologs Nohomologs Nohomologs Nohomologs Nohomologs DR_1902 Nohomologs DR_1089 DR_1916 DR_1126 DR_1477 DR_0819 DR_1289DR_ DR_0198 Nohomologs DR_1310 DR_1274
oli
Escherichiac ParC ParE PepA PinR PolA PolB PriA PriB PhrB RadA(Sms) RadC RarA RecA RecB Nohomologs Nohomologs Nohomologs RecC RecD RecE RecF RecG RecJ RecN RecO RecQ RecR RecT RecX RuvA
er
nt. BProteindescriptionandcomments DNATopoisomeraseIV,subunitA(TypeIItopoisomerase) DNATopoisomeraseIV,subunitB(TypeIItopoisomerase) DNAbinding(independentofAminopeptidaseactivity)proteinrequiredformaintenanceofplasmidmonomers. Putativerecombinase DNApolymeraseI DNApolymeraseII Putativeprimosomalproteinn’(replicationfactorY) Corecomponentoftheprimosome,bindstoPriAandsingle-strandedDNA Photolyase(directmonomerizationcyclobutane-typepyrimidinedimers) PredictedATP-dependentprotease Predictedacyltransferase;predictedDNA-bindingprotein ProteinmayplayaroleinrecombinationassociatedwithDNAreplication;putativeATPaserelatedtothehelicasesubunitoftheHollidayjunctionresolvase Recombinase;ssDNA-dependentATPase,activatorofLexAautoproteolysis Helicase/exonuclease RecBfamilyexonuclease1 RecBfamilyexonuclease2 RecBfamilyexonuclease3 Helicase/exonuclease Helicase/exonuclease;ContainsthreeadditionalN-terminalhelix-hairpin-helixDNA-bindingmodules;closelyrelatedtoRecDfromB.subtilisandChlamydia ExonucleaseVIII PredictedATPase;requiredfordaughter-strandgaprepair Hollidayjunction-specificDNAhelicase;branchmigrationinduc Single-strandedDNA-specificexonuclease PredictedATPase Requiredfordaughter-strandgaprepair Helicase;suppressorofillegitimaterecombination Requiredfordaughter-strandgaprepair DNAannealingprotein RegulatoryproteinforRecA Holliday-junction-bindingsubunitoftheRuvABCresolvasome
Co me
2. na o1) o2) o3)
Table Protein ParC ParE PepA PinR PolA PolB PriA PriB PhrB RadA RadC RarA RecA RecB RecB(ex RecB(ex RecB(ex RecC RecD RecE RecF RecG RecJ RecN RecO RecQ RecR RecT RecX RuvA
PLoSONE | www.plosone.org 8 December2008 | Volume 3 | Issue 12 | e3878
ResistanceofK.radiotolerans
7 2 4
D) 05 17 54
c 0 1 0
KineococcusradiotoleransSRS30216(locus) Krad_3053Krad_3828 Nohomologs Nohomologs Nohomologs Krad_2553 D)Krad_0868Krad_2554(BothunrelatedtoE.coliSb Krad_3772 Krad_1409Krad_4481 Krad_0827Krad_4460 Krad_1242Krad_0858 Krad_4338 Krad_0999 Krad_0487 Krad_3900 Nohomologs Nohomologs Krad_3639 Nohomologs Krad_2940Krad_1787Krad_ Krad_2942 Krad_2935 Krad_0757Krad_1179Krad_Krad_4408 Krad_1179 Nohomologs Krad_1122 Krad_1121 Krad_4198Krad_3979Krad_ Krad_1169 Nohomologs Krad_3612(seealsoERCC3)
c
obacteriumtuberculosisRv(locus) 92c 94c omologs omologs omologs 77(unrelatedtoE.coliSb 17 96c omologs 11Rv1253 54Rv2478c 10 46c 22 omologs omologs 76c omologs 38c 33c 20 49Rv3198c(putative) 98c omologs 08c 07c 27c 04 omologs omologs
MycH37 Rv25 Rv25 Noh Noh Noh Rv12 Rv29 Rv28 Noh Rv32 Rv00 Rv12 Rv36 Rv03 Noh Noh Rv29 Noh Rv16 Rv16 Rv14 Rv09 Rv31 Noh Rv11 Rv11 Rv04 Rv32 Noh Noh
ns 2)
a D
radiodurg) 1624 1663 A0188 ativeUvr 2
DeinococcusR1(locusta DR_0596 DR_0440 Nohomologs Nohomologs DR_1922 DR_1921 Nohomologs DR_0120 Nohomologs DR_0335DR_ DR_0099 Nohomologs DR_1374 DR_1751 Nohomologs Nohomologs DR_0689DR_ DR_1819 DR_1771DR_ DR_2275 DR_1354 DR_1775 DR_1775(put Nohomologs DR_0186 DR_2586 DR_0354 DR_0428 DR_C0020 DR_A0131_1_
Escherichiacoli RuvB RuvC RusA(YbcP) SbcB SbcC SbcD Nohomologs Smf Nohomologs RhlE Ssb Tag TopA UDG UmuC UmuD Ung Uve1/BS_YwjD UvrA UvrB UvrC UvrD UvrD2 Vsr XseA/nec7 XseB XthA YbaZ YhdJ YejH
e
et
nt. BProteindescriptionandcomments HelicasesubunitoftheRuvABCresolvasome EndonucleasesubunitoftheRuvABCresolvasome Endonuclease/Hollidayjunctionresolvase ExodeoxyribonucleaseI Exonucleasesubunit,PredictedATPase Exonuclease DNAorRNAhelicaseofsuperfamilyII PredictedRossmannfoldnucleotide-bindingproteininvolvedinDNAuptake Photolyase SuperfamilyIIATP-dependenthelicase Single-strandbindingprotein;D.radioduransR1hasthreeincomplORFscorrespondingtodifferentfragmentsoftheSSB 3-methyladenineDNAglycosylaseI DNAtopoisomeraseI(TypeIAtopoisomerase)/DNAtopoisomeraseIII(TypeItopoisomerase) UracilDNAglycosylase Error-proneDNApolymerase;inconjunctionwithumuDandrecA,catalyzestranslesionDNAsynthesis InconjunctionwithUmuCandRecA,facilitatestranslesionDNAsynthesis;autoprotease UracilDNAglycosylase;DR_0689isalikelyhorizontaltransferfromaeukaryoteoraeukaryoticvirus UV-endonuclease;activitywascharacterizedinNeurospora ATPase,DNAbinding Helicase Nuclease DNAhelicaseII(DNA-dependenthelicaseactivity)initiatesunwindingfromanick;DR_1572hasaframeshift Putativehelicase Strand-specific,sitespecific,GTmismatchendonuclease;fixesdeaminationresultingfromDcm ExonucleaseVII,largesubunit ExonucleaseVII,smallsubunit ExodeoxyribonucleaseIII(APendonuclease) Possiblemethylated-DNA-[protein]-cysteineS-methyltransferase Adenine-specificDNAmethylase DNAorRNAhelicaseofsuperfamilyII
CoTable2. Proteinname RuvB RuvC RusA(YbcP) SbcB SbcC SbcD SdrA Smf SplB SrmB Ssb Tag TopA/TopB UDG UmuC UmuD Ung Uve1/BS_YwjD UvrA UvrB UvrC UvrD UvrD2 Vsr XseA/nec7 XseB XthA YbaZ YhdJ YejH
PLoSONE | www.plosone.org 9 December2008 | Volume 3 | Issue 12 | e3878
ResistanceofK.radiotolerans
D. radiodurans is adept at detoxifying reactive oxygen species
(ROS) during radiation exposure when many free radicals are
generated from hydrolysis of water [13–15]. Like Deinococcus, K.
ns radiotolerans produces carotenoids as one level of protection. K.
a
er radiotolerans also possesses an impressive suite of genes involved in
ol 1 4
KineococcusradiotSRS30216(locus) Krad_3031 Krad_3139Krad_169 Krad_1887 Krad_0652Krad_415 Krad_0047 RbPtshuaaOegcthgtSeheorsogitadsetalnetipatochasxotbihpfraiooaccguartteetgirnhooisanfRoaaOf(rntemaSbiarnldoemneutamto3itnxe)aielfsliciymcosaummetcixophupnanoaresnasebedrNtleewestipoosstoreokorni.xaseitdh.goaonTtsioevhrreehfsooseetuarerneesd[ss2u8blit]nys.
Bioremediation potential
osis Because K. radiotolerans was isolated from a HLW facility, the
cul genome was examined for gene products that could be used to
er
b generatecarbonorenergyfromorganiccompoundsinthetanks.
u
obacteriumtRv(locus) 54c 01Rv2894c 90 30cRv0269c 50cRv2310 Od(eDtehrtOteyhclDboteel)odnsgzesfeleonbcrete,elUodan.nSpgd.riinoDxgryietltpyeoanprektosmnllou(etwthnanetnstBdsoeTffgoErEraXnidneacrctlgoiuoymdnip(nDpogaOubtnhEedwn)sz)a,aeynncsdeh,wloDteorrielneufeaenntneosedet,
MycH37 Rv25 Rv17 Rv20 Rv37 Rv37 hydrocarbons, polynuclear aromatic hydrocarbons, polychlori-
natedbiphenyls,ketones,alkanes,phenols,phthalates,explosives,
ans andpesticides.Severalhomologuesforatrazinedegradationwere
diodur 0155 npaotthedwa(AytzseAeBmZs,iKncroamd1p9l4et7e,.4281, 0533, respectively), though the
DeinococcusraR1(locustag) Nohomologs DR_C0018DR_ADR_B0104 DR_1707 Nohomologs Nohomologs cttiaoommTnihpnaelaentxdiSoaRnnptSrsroewHcaeghLseiscWnihntsgi.aanlttsCeotrhofmeecroSmenRtowaSniitnlhiysncdulloouswewddenmsoctrooxemalealcamputleelca,xhragenlywmtcseioicglaaahnltteds,toacdrbiegtirclaiaozntnaeic--,
and formate. Growth using each organic acid as a sole carbon
sourcewasevaluatedforK.radiotoleransincomparisonwithglucose
oli asapositivecontroland0.1%yeastextractasanegativecontrol.
Escherichiac YqgF XerC/XerDE/ 53EXOc Nohomologs Nohomologs ewrRaveetoreslepsuitruaiaonntndsiou.nibPtairobewmllieamasgsisnreoaxywraiyetmhldsitnssuuedodbinestsurgarsltiuenevcsgeoas(delOeadwt2aethcrneoaotnthscusiightmhroaepwtrteniot)a.hnnaRdnaengstpdlhyiorcasoCetliOaootne2f
m othercarbonsources(table4).Biomassyieldswith5 mMoxalate
Fi
A, and5mMformateweregenerallyconsistentwiththe0.1%yeast
m
Cont.Table2. BProteinnameProteindescriptionandcomments YqgFPredictedHollidayjunctionresolvase XerC/XerDPutativerecombinaseinsegregationofchromosomes,homologousrecombination;tyrosinerecombinase/inversionofon/offregulatoroffiFimB 999953EXOc5-3exonuclease;T5type5-3exonucleasedomainsmayco-occurwithDNApolymeraseI(PolI)domains,orbepartofPolIcontainingcomplexes Predictedeukaryotic-typeDNAprimase Predictedsite-specificintegrase-resolvase doi:10.1371/journal.pone.0003878.t002 owreOaxxGGDOPFSTEACMrCa2ttaaeueeaynslllyrrankuu/lgppttdpscazayMoattbatesCo-yiaacllticwtonairndttametlyhnhmohhsietetpceyrexcssaoiieooodeuriui,almohdllam3nnerpkenamynpeoseyeae.dererdeptthltrarp,rhrrpEoeRecsoiokonibterpgdxofpexaioprilsoeuehondietoaxed.rAtctrirneeixmrcnhwadtomoiraestdedxoixseeadiiaaivfeidusserdinsounte,meaegnaedrdless,fshuoeouoeGsftrcfCxc,oayAhxSoitomsndrHayiOrentgmesmaPgh,ehcu2xasr,eaeeoaGesantdpendtrehe,uArtpcsG,rcoaCotapSdanontPesuidx-ecocSc,aeniytomelnnisaxos.tstarrneAdalW,atewitaotheeonn.irlxdeseifw230A13A300K1bibYc783057882bberoia515975614aEssrot703187557eetdehm,innotLtttbaonohsexcsetgiutceesp.rrnerTSoseaaipmdgstiuriilalncoattwriiKooley.nnr,
doi:10.1371/journal.pone.0003878.t003
PLoSONE | www.plosone.org 10 December2008 | Volume 3 | Issue 12 | e3878