CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Bacterial,

Fungal

and

Virus

Molecular

Biology

CspB

of

an

arctic

bacterium,

Polaribacter

irgensii

KOPRI

22228,

confers

extraordinary

freeze-tolerance

Youn

Hong

Jung

_,

_Yoo

_Kyung

_Lee

_,

_Hong

_Kum

_Lee

_,

_Kyunghee

_Lee

_,

_Hana

_Im

a_{SejongUniversity,DepartmentofMolecularBiology,Seoul,RepublicofKorea}

b_{KoreaOceanResearchandDevelopmentInstitute,DivisionofLifeSciences,KoreaPolarResearchInstitute,Incheon,RepublicofKorea} c_{SejongUniversity,DepartmentofChemistry,Seoul,RepublicofKorea}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26January2016 Accepted11April2017 Availableonline31July2017 AssociateEditor:ValeriaOliveira

Keywords:

Cold-shockprotein(Csp) Psychrophile

Cold-resistance

a

b

s

t

r

a

c

t

Freezingtemperaturesareamajorchallengeforlifeatthepoles.Decreasedmembrane fluid-ity,uninvitedsecondarystructureformationinnucleicacids,andproteincold-denaturation alloccuratcoldtemperatures.Organismsadaptedtopolarregionspossessdistinct mecha-nismsthatenablethemtosurviveinextremelycoldenvironments.Amongthecold-induced proteins,coldshockprotein(Csp)familyproteinsarethemostprominent.Agenecodingfor aCsp-familyprotein,cspB,wasclonedfromanarcticbacterium,PolaribacterirgensiiKOPRI 22228,andoverexpressionofcspBgreatlyincreasedthefreeze-survivalratesofEscherichiacoli

hosts,to agreaterlevel thananypreviouslyreportedCsp.Italso suppressedthe cold-sensitivity ofan E.colicsp-quadruple deletion strain,BX04. Sequenceanalysis showed thatthisproteinconsistsofauniquedomainatitsN-terminalendandawellconserved cold shockdomain atits C-terminalend. Themostcommon mechanismofCsp func-tionincoldadaptionismeltingofthesecondarystructuresinRNAandDNAmolecules, thusfacilitatingtranscriptionandtranslationatlowtemperatures.P.irgensiiCspBbound tooligo(dT)-celluloseresins,suggestingsingle-strandednucleicacid-bindingactivity.The unprecedentedleveloffreeze-toleranceconferredbyP.irgensiiCspBsuggestsacrucialrole forthisproteininsurvivalinpolarenvironments.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Livingorganismsencounterseveralseriouschallengeswhen theyare exposed to coldenvironments, including reduced

∗ _{Correspondingauthorat:DepartmentofMolecularBiology,SejongUniversity,209Neungdong-ro,Gunja-dong,Gwangjin-gu,Seoul05006,}

RepublicofKorea.

E-mail:hanaim@sejong.ac.kr(H.Im).

enzymeactivity,decreasedmembranefluidity,reduced trans-portofnutrientsandwasteproducts,reducedprotein move-ment,decreasedratesoftranscriptionandtranslation,slow DNAreplication,retardedproteinfolding,cold-denaturation of proteins, and intracellular ice formation.1–3 _Although

https://doi.org/10.1016/j.bjm.2017.04.006

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

freezing temperaturesare amajor environmentalthreatat theNorthandSouthPoles,relativelyhighnumbersofdiverse microorganismsthriveinthepolarregions.Inparticular,␣-, ␤-, and ␥-proteobacteria and the Cytophaga-Flavobacterium-Bacteroidesphylaarethemostcommonlyfoundbacteria,and eukaryotes, such as yeasts and microalgae, are frequently foundinpolarenvironments.4_{Organismslivinginthepolar}

regionsmusthavetheabilitytoadapttoextremelycold tem-peratures.Toavoidthetransitionfromafluid-togel-phase cell membrane,the proportionofunsaturatedand methyl-branchedfattyacids inlipid membranesisincreased.5 _The

formation of stable secondary structures in nucleic acids atlow temperaturesinhibits replication, transcription,and translation. Thus, nucleic acid-binding proteins, such as Csp-familyproteinsandRNAhelicases, areinduced during temperaturedownshifts and functionasRNA chaperones,6

melting the stablesecondarystructures inRNAmolecules, whichfacilitatestranslationandribosomebiogenesis.7–9

Sev-eral chaperones,such asGroEL10 _{and DnaK,}11 _{are induced}

uponcoldshock,possiblytocope withcold-denatured pro-teins. Protein folding, especially cis/trans isomerization of peptidyl prolyl bonds, occurs very slowly at low tempera-tures,andpeptidylprolylisomerasesplayanimportantrole in cold adaptation by facilitating the folding of function-allysignificantproteins.12_{Cryoprotectants,suchasantifreeze}

proteins,13 _trehalose,2 _{and exopolysaccharides,}14 _{have also}

beenimplicatedinprotectionofpolarorganisms, eitherby preventingicecrystalformationorbyavoidingdehydration. Genomic sequencingresultssuggest someother character-istics ofpsychrophiles, like enhanced antioxidant capacity tocope withincreasedproductionoftoxicreactive oxygen species.15

IncreasingnumbersofCsphomologsarebeingreported frompsychrophiles,mostlythroughgenomicDNA sequenc-ing projects,but onlylimited functionaldata are available. Inanefforttoelucidatethedetailedmechanismsthatallow polarorganismstosurviveatlowtemperatures,westudied thefunctionalrolesofCsp-familyproteinsinpolarbacteria. Apsychrophilic bacterium, Polaribacter irgensiiKOPRI22228, was isolated from Arctic Sea sediment.16 _{Two csp genes,}

cspAPi and cspCPi, were previously cloned from this bac-terium and exhibited considerable homology to canonical

Escherichia coli cspA (CspAEc). Overexpression of either of

thesetwogenesconferredsignificantcold-resistance pheno-types totheir recombinant hosts, and also complemented the cold-sensitivity of a quadruple csp deletion mutant BX04 (cspA, cspB, cspG, and cspE) strain,17

suggest-ingfunctionalhomologyamongthoseCsp-familyproteins.16

In this study, we report another Csp-homologous protein from P. irgensii KOPRI 22228, CspBPi, which contains an

extra domain on its N-terminal region, in addition to the well conserved cold-shock domain (CSD) atits C-terminal

region. This belongs to a subfamily of CSD-fold proteins, recently identified through metagenomic studies of psy-chrophilic bacteria, and no functional data isavailable for thiskind ofproteinsatourbestknowledge.Therefore, the function of this unique Csp protein from an arctic bac-terium in conferring cold tolerance was analyzed in this study.

Materials

and

methods

BacterialcultureandcloningofthecspBPigene

P.irgensiiKOPRI22228wasisolatedfromArcticSeasediments nearDasanKoreanArcticStation(Ny-Alesund,Norway),and culturedaspreviouslyreported.16 _ThecspB

Pi gene(GenBank WP 004570868) was obtained by PCR amplification. The template DNA was extracted from P. irgensii KOPRI 22228 cells using G-spinTM _{bacteria genomic DNA extraction kit}

(Intron Co., Korea), according to the protocol suggested by the manufacturer. The forward primer sequence was CspBPiF1H,5-AGTAAGCTTATGGCAAAATCGCAGCAGACCT-3, and the reverse primer was CspBPi B150*B, 5 -CCGGATCCTTATATTTTGGTAACTTTAACTGCATTCATT-3 (manufacturedbyBioneerCo.,Daejion,Korea).ThePCR mix-tureconsistedof5␮lof10×PCRbuffer(finalconcentrations: 50mM KCl,0.01% gelatin, 10mM Tris–HCl, pH 9.0),2.5mM MgCl2,0.2mMofeachdNTP,200nMofeachprimer,1␮lof

templateDNA,and2.5unitsofTaqDNApolymerase(Takara, Japan)inthe final50␮lvolume.ThePCRwasperformedin aDNAEnginethermalcycler(Bio-RadLaboratoriesInc.,USA) usingacyclingconditionthatconsistedofaninitial denatur-ationat95◦Cfor5minandthen30cycleswithdenaturation at94◦Cfor1min,annealingat55◦Cfor30s,andextension at72◦Cfor1min.Afinalextensionwasperformedat72◦C for 5min. The PCRproducts that were 0.5kb in size were double-digested withHindIII and BamHI, and cloned using pAED4,18_{anE.coliexpressionvector,digestedwiththesame}

enzymes. Toavoid any mutationsarising from error-prone

Taq DNA polymerasereactions, several cloneswere picked forsequencinganalysis.Theresultingplasmidswasnamed pAED-cspBPi.

ExpressionofCspBPiinE.coli

The recombinant plasmid for expression ofCspBPi,

pAED-cspBPi,wastransformedintocompetentE.coliBL21(DE3)cells (Invitrogen Co., CA, USA) using the method described by Sambrook etal.19 For overproductionofCspBPi,1mlofthe

overnight liquidculturewas transferredtoaflask contain-ing 50mlfreshLBmediumcontaining100␮g/mlampicillin. When cell cultureshad reached anOD600 of0.4, IPTG was

addedtothefinalconcentrationof0.1mM.Thecultureswere incubatedfurtherat37◦Cfor2hwithvigorousshaking. Over-expressionoftheCspBPiwasanalyzedby20%SDS-PAGE.The

proteinbandswerevisualizedbyCoomassiebrilliantblueR250 staining.

Resistancetofreezingandthawing

CspBPiproteinwasoverexpressedinE.coliasdescribedabove.

Astheexperimentalcontrol,E.colitransformedwithapAED4 plasmid lackingthe cspBPi insertwas used.One mlaliquot of liquid culture was placed at −20◦_{C for 2h. Thefrozen}

cellsweretakenoutofthefreezerandputonicefor1hto be thawed. This process was performed in duplicates and repeated up to three cycles. Aliquots were taken at each

cycleoffreeze-and-thaw,andcolony-formingunits(CFU)were countedafterincubationonLBplatesat37◦Cfor24h.Thedata werecollectedfromfiveindependentexperimentsandshown asanaverageforeachpoint.

PurificationofCspBPi

CspBPi were overexpressed at 37◦C in E. coli BL21(DE3) as

describedabove,exceptthatthecultureswerescaled-upto 1LliquidLBmedia.Cellswere harvestedbycentrifugation, and resuspended in 40ml of 10mM phosphate buffer, pH 6.5.Cells were lyzed bysonication using a Bandelin Sono-plus HD2200 ultrasonic homogenizer (Berlin, Germany) as describedforCspAPi.16Thesupernatantfractionwasloaded

onaQ-sepharoseTM_{(AmershamBioscienceCo.)fastflowion}

exchangecolumnequilibratedwiththesame buffer.CspBPi

proteinwaselutedwitha0–0.5MNaClgradient. Concentra-tionsofproteinsweredeterminedusingBio-RadDC(detergent compatible)proteinassaykit,andthepurityofproteinswas analyzedby20%SDS-PAGE.

Oligo(dT)-cellulosebindingassays

PurifiedCspBPi proteinwasdialyzed againstbindingbuffer

(10mMTris–HCl,pH8.0,1mMEDTA,50mMKCl,and7.4% glyc-erol).Fifty␮lofoligo(dT)-cellulosetype7beads(Amersham BioscienceCo.)were incubatedwith50␮gofCspBPi protein

at4◦Cfor4h.Theresinsandboundproteinswerecollected bybrief centrifugation,andwashed twicewiththe binding buffer.Inparallel,50␮gofbovineserumalbumin(BSA) pro-tein,insteadofCspBPiprotein,wasusedastheexperimental

control.Co-precipitatedproteinswereanalyzedby20% SDS-PAGEandCoomassiebrilliantblueR250staining.

Rescueofcold-sensitiveE.coliBX04strain

E. coli quadruple csp deletion strain BX04 cells harboring pAED,18 _pAED-cspA

Pi,16 pAED-cspBPi, or pAED-cspCPi16 were grownintheliquidculturetoanOD600of0.4.Thecellswere

then streaked onto LB plates containing 0.1mM IPTG,and incubatedattemperaturesrangingfrom16to37◦C.After15h at37◦C,24hat25◦C,or60hat16◦C,thegrowthofBX04cells ontheplateswasobserved.

Results

CloningandsequenceanalysisofthecspBgenefromP. irgensiiKOPRI22228

P.irgensiiKOPRI22228wasisolatedfromArcticSeasediments andgrownaspreviouslyreported.16_{Thisbacteriumisa}

psy-chrophile,withanoptimumgrowthtemperatureof10◦C.In anefforttoelucidatetherolesplayedbyCspsinpsychrophilic bacteria,DNAfragmentsfromthisbacterium,whichcontain aregionencodingaconservedCSD,werecloned.Anew

csp-homologous gene 0.45kb insize was obtainedand named

cspBPi.

ABLAST nucleotidehomology search ofcspBPi was per-formed:cspBPiexhibitedlowhomologytothecanonicalcspAEc

(15.8%) and E. coli cspD (12.4%). The deduced amino acid sequenceofcspBPiencodedaprotein150residuesinlength, and theCspBPi sequence wasalignedtopreviouslystudied

Cspsequences frompolar bacteria: CspAfromStreptomyces

sp.AA8321(CspASt),20CspAfromPsychromonasarcticaKOPRI

22215(CspAPa)21andCspAPiandCspCPifromP.irgensiiKOPRI

22228.16 _{All Cspshave atypical ␤-barrelCSD composedof}

five␤-strands, butCspBPi hasaunique extradomainatits

N-terminalend(Fig.1).CSDishighlyconservedamongmany bacterialCsps,andthethree-dimensionalstructuresofsome Csps,including CspAEc andBacillussubtilisCspB, havebeen

reported.22,23_{CSDisbelievedtomediatenucleicacidbinding.}

Inparticular,twoRNA-bindingmotifs,RNP1(withconsensus sequenceK-G-F-G-F-I)andRNP2(withconsensussequence V-F-V-H-F) arecrucialforbindingtoRNAor ssDNA(boxedin

Fig.1).24_InRNP2ofCspB

Pi,thefirstandthesecondValresidues

werereplacedwithTyrandThr,respectively,andthelastPhe residuewasreplacedwithVal.However,allthreePheresidues (Phe-15,Phe-17,andPhe-28;residuenumbersaccordingtothe canonicalCspAEc)intheRNA-bindingmotifs,whichare

con-sideredtobenecessaryfornucleicacid-bindingactivity,24,25 wereconservedinCspBPi.SequenceanalysisofCspBPi

sug-gestedthattheproteinmaybindtoRNAorssDNAthrough acanonicalcoldshockdomain␤-barrelstructure.Meanwhile, theadditionalN-terminaldomainofthisproteindidnotshow anynoticeablehomologytootherproteins.

Cold-resistanceofthehostoverexpressingCspBPiwas

greatlyincreased

TostudytherolesofCspsfrompsychrophilicbacteria,cold resistance of the hosts harboring csp genes, was exam-ined. Csp-overexpression was induced by the addition of IPTG to mid-log phase liquid cultures of cells carrying a

csp-expression vector. When E. coli cells harboring pAED4 were frozen and thawed once, less than 1% of the origi-nalcellssurvived.Followingrepeatedcyclesoffreezingand thawing,thenumberofsurvivingcellsdecreasedalmost expo-nentially. Overexpression of previously reported csp genes from polar bacteria increased freeze-survival rates of the hosts only moderately: the Ps. arctica CspAPa-expressing

cells exhibited a slightly increased survival rate and the CspAPi or CspCPi-overexpressing cells showed more than

five-fold increase in the survival rates in the first freeze-thawcycle(Fig.2).16,21_{Surprisingly,theCspB}

Pi-overexpressing

cells showed anextraordinary increaseinfreeze-tolerance: more than fifty fold the number ofcells survived the first freeze–thawcycleandthenumberofsurvivingcellsincreased togreaterthan100000-foldafterthreecyclesoffreezingand thawingcomparedtothenumberofsurvivingpAED4-carrying cells(Fig.2).

CspBPibindsoligo(dT)-cellulose

SinceCspBPicontainsCSDandtheconservedPheresiduesin

theRNP1andRNP2sequencemotifs,whichsuggest single-stranded RNA or DNA binding activity, the functionality of the single-stranded nucleic acid-binding motifs of this proteinwasexamined.UponincubationofE.coliBL21 cells harboringpAED-cspBPiwith0.1mMIPTG,aproteinbandwith

RNP2

RNP1

S. sp. CspA

P. irgensii CspA

P. irgensii CspA

P. irgensii CspA

P. irgensii CspA

P. irgensii CspC

P. irgensii CspB

Ps. arctica CspA

P. irgensii CspC

P. irgensii CspB

Ps. arctica CspA

P. irgensii CspC

P. irgensii CspB

P. irgensii CspC

P. irgensii CspB

Ps. arctica CspA

Ps. arctica CspA

S. sp. CspA

S. sp. CspA

S. sp. CspA

Fig.1–SequencealignmentofvariousCspproteinsfrompolarorganisms.AbbreviationsforbacterialCspswhoseamino acidsequenceswereanalyzedhere:S.sp.CspA,Streptomycessp.AA8321CspASt20;P.irgensiiCspAandCspC,Polaribacter

irgensiiKOPRI22228CspAPiandCspCPi,respectively16;P.irgensiiCspB,P.irgensiiKOPRI22228CspBPifromthisstudy;Ps.

arcticaCspA,PsychromonasarcticaKOPRI22215CspAPa.21TheaminoacidsequencesofCspproteinswerealignedusingthe

defaultsettingsofCLUSTALW.27_{Belowtheproteinsequencesisakeydenotingconservedsequence(*),conservative} mutations(:),semi-conservativemutations(.),andnon-conservativemutations().Gapsindicatedbyhyphens(-)were introducedtoimprovealignment.TheRNA-bindingmotifsRNP1andRNP2areboxed.

an apparent molecular mass of 23kDa on SDS-PAGE was observed.CspBPiwasexpressedinsolubleformandpurified

byanion-exchangecolumnchromatography.Partiallypurified CspBPi wasincubated witholigo(dT)-cellulosebeads at4◦C

for 4h and subjected to a brief centrifugation. When the reaction products were analyzed by20% SDS-PAGE, CspBPi

wasboundtotheoligo(dT)-celluloseandco-precipitatedwith theresins,whileneitherbovineserumalbuminusedatthe same concentration nor contaminating proteins from the CspBPi preparationwere co-precipitated (Fig. 3). The result

suggeststhatCspBPibindsssDNA.

CspBPisuppressesthecold-sensitivephenotypeofthecsp

quadruple-deletionE.colistrain

Since overexpression of CspBPi greatly increased the cold

resistance ofwild-type E. coli (Fig. 2), the ability of CspBPi

tosuppress thecold-sensitivity ofthe E.coliquadruple csp

deletionstrain BX04 (cspA,cspB, cspG,and cspE)was alsoexamined.Mid-logphaseculturesofBX04cells harbor-ing pAED4, pAED-cspAPi, pAED-cspBPi, or pAED-cspCPi were streaked onto LB plates containing 0.1mM IPTG and incu-batedattemperaturesrangingfrom16to37◦C.Asdescribed previously,17_{thegrowthofBX04cellswascomparabletoother}

clonesat37◦C,butthegrowthwasextremelyretardedat16◦C (Fig.4).Meanwhile,overexpressionofanyCspsfromP. irgen-siicomplementedthecold-sensitivityofBX04at25◦Candat

16◦C(Fig.4).WhenthegrowthofBX04wasfollowedbyOD600,

overexpressionofCspBPipromotedthegrowthatlow

temper-aturesatslightlyhigherlevelsthanCspAPiorCspCPidid(data

notshown).

Discussion

Cold temperatures affect all physical-chemical parame-ters of living organisms, and effects include decreased solute diffusion rates, ice crystal formation, dehydration, decreased membrane fluidity, slow enzymereaction rates, stable secondary structure formationin nucleic acids, and cold-denaturationofproteins.Psychrophilicorganismsfrom cold ecosystems haveevolved biological means to circum-ventthesechallenges.Althoughtranscriptionandtranslation of most genes are nearly stopped upon sudden tempera-turedropsinmesophiles,expressionofcold-shockgenesare selectivelyinduced.1_{Meanwhile,correspondinggenesin}

psy-chrophilesaremoreconsistentlyexpressed,insteadofbeing transientlyinducedduringthecoldacclimationphase,3

sug-gesting that Cspsplayimportant roles forsurvival incold environments.

Inanefforttounderstandthemechanismsplayedby psy-chrophiles to adapt to cold biosphere, this study focused on the functions of Csps from polar bacteria. Although dozens of csp genes from psychrophiles havebeen identi-fied by genomic/metagenomics approaches, their roles on

Freeze and th

aw cycle

Sur

vi

v

al ra

te (%

)

Fig.2–Greatlyincreasedcold-resistanceof

CspBPi-overexpressingcells.Thesurvivalratesofvarious

Csp-overexpressingcellsfollowingcyclesof

freeze-and-thawareshown.Thenumberofviablecells priortofreezingwassetat100%.䊉,Control

pAED4-carryingcells;,CspAPa-overexpressingcells;,

CspAPi-overexpressingcells;,CspCPi-overexpressing

cells;,CspBPi-overexpressingcells.

cold-adaptationwere elucidatedonlyinverylimitedcases. HeterogeneousexpressionofCspsfrompolarmicroorganisms resultedinvariouseffectsoncoldadaptionoftheir recom-binanthosts.CertainCspsfailedtoincreasecold-resistance oftheirhosts:CspAStfromanAntarcticStreptomycesneither

increased the cold-resistance of wild-type E. coli, nor that ofthe E. coli quadruple csp deletion strain BX04.20 _Instead

overproductionofCspAStinhibitedDNAreplication,as

non-canonicalE.coliCspDdoes,suggestingaroleforCspASt in

haltingDNAreplicationuntilthecelladjustsitselfupon sud-dentemperaturedrops.20_{Similarly,overexpressionofaCsp}

fromanAntarctichaloarchaeonHalorubrumlacusprofundidid notsuppressedthecoldsensitivityofBX04.26Meanwhile,it

MW

Csp

Input

Bindi

ng

25

37

20

50

15

75

BSA

B

bea

d

BS

A

CspB

Fig.3–Oligo(dT)-bindingactivityofCspBPi.CspBPiwas

incubatedwitholigo(dT)-celluloseat4◦Cfor4h.Proteins boundtobeadswerecollectedbycentrifugation.Lanes: MW,precisionplusproteinstandards(Bio-RadLaboratories Inc.;sizeofeachproteinbandisshowninkDaatleftofthe gel);BSA,bovineserumalbumin;CspB,CspBPi;bead,

oligo(dT)-celluloseonly.Themigrationpositionofthe boundCspBPiproteinisindicatedwitharedbox.

hasbeenreportedthattheoverexpressionofCspAPaincreased

thecold-resistanceofwild-typeE.coli,butnotoftheE.coli

quadruplecspdeletionstrainBX04.21_{Therefore,the}

contribu-tionofCspAPatothecoldsurvivaloftheirrecombinanthosts

seemed relatively modest. Other Csps from psychrophilic organisms weremoreeffectiveinincreasingcoldtolerance of their hosts; the overexpressionof CspAPi or CspCPi not

onlyincreasedthecold-survivalratesofwild-typeE.coliby morethanfive-foldfollowingonecycleoffreezingand thaw-ing, but also rescued cold-sensitive phenotype of BX04.16

OverexpressionofCspsfromastenopsychrophilicarchaeon

Methanogenium frigidum or a deep sea planktonic archaeon

CrenarchaeotaalsocomplementedcoldsusceptibilityofE.coli

BX04.26_{Sincenoquantitativedataonfreeze-tolerance}

con-ferredbytheseproteinswereprovided,moredetailedstudies wouldbenecessarytocomparetheireffectwiththatofCspBPi.

However,overexpressionofanyE. coliCsps, exceptCspDEc,

alsorescuedcold-sensitiveE.coliBX04,17_{andoverexpression}

ofCspAEcincreasedsurvivalratesofthewild-typeE.coliupon

freezingandthawingatacomparableleveltoCspAPiorCspCPi

(datanotshown).Theseresultsshowthattheabilitytoconfer coldresistancetotheirhostsisnottheuniquecharacteristics

CspAPi BX04 CspC_Pi CspBPi CspAPi BX04 CspC_Pi CspBPi CspA_Pi BX04 CspC_Pi

37℃

25

℃

16

℃

Fig.4–OverexpressionofCspBPirescuedcold-sensitivephenotypeofcspquadruple-deletionE.colistrain,BX04.BX04cells

harboringpAED,pAED-cspA_Pi,pAED-cspB_Pi,orpAED-cspC_PiweregrownintheliquidculturetoanOD600of0.4.Thecells

ofCspsfrompsychrophiles:previousstudiesrathersuggest thatseveralCspsfrompsychrophileshaveretainedsufficient similaritythroughoutevolutiontobeabletofunction effec-tivelyinmesophilestoconfercoldtolerance.

Surprisingly, overexpression of CspBPi greatly increased

the cold toleranceof its recombinant hostto an unprece-dentedlevel(Fig.2).OverexpressionofCspBPinotonlyinduced

amorethan fifty foldincrease infreeze-tolerance in wild-typeE.coli(Fig.2),butalsonoticeablypromotedthegrowth oftheE. coliquadruple cspdeletion strainatlow tempera-tures(Fig.4).Elucidatingthe detailedmechanismsinvolved infreeze-toleranceconferredbyCspBPiwouldbeofgreat

aca-demicinterestandwillbepursuedinafuturestudy.CspBPi

possessescoldshockdomainsattheC-terminalregion(Fig.1) and shares the basic characteristics ofCsps, including the ability to bind single-stranded nucleic acids, as indicated by oligo(dT)-binding assays (Fig. 3). One possibility is that the CSD of CspBPi may have greatly improved activity, for

example asa RNAchaperone,maintaining singlestranded nucleic acid structuresatextremely lowtemperatures and allowingefficienttranscriptionandtranslation.Ontheother hand,CspBPi hasanextradomainonitsN-terminalregion,

and it isalso possiblethat this unique region playsa dis-tinct role in conferring an extraordinary increase in cold survival ability. Sequence homology search has identified severalcspBPi-homologousgenes,encodingbothN-terminal extra domain and CSD, from evolutionarily related marine Flavobacteriaceae,including Polaribacter sp.MED152, Lacinu-trixsp.5H-3-7-4,Winogradskyellasp.PG-2,andMaribactersp. HTCC2170.However,theirfunctionalrolesoncoldtolerance ortranscriptional/translationalregulationupontemperature downshiftshavenotbeenstudiedyet.Itwillbeinteresting totest freeze-tolerance ofhosts overexpressing these dual domainCsp-likeproteins.

IntroductionofCspBPiintootherorganismshaspotential

industrialapplications,includingincreasedcold-resistanceof nitrogen-fixingbacteria, suchasRhizobiumand cyanobacte-ria,andthesurvivalofstarterculturesafterstorageatfreezing temperatures.Thelong-pursueddevelopmentoffrozendough maybeachievablebyimprovingthefreeze-toleranceofbaker’s yeast.ThepossibleapplicationofCspBPidoesnotneedtobe

limitedtoindustriallyimportantmicroorganismsanditmay beintroduced into plantsto enhance theirviability atlow temperaturesandincreasetheireconomicvalue.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Theauthorsgreatly appreciatethegiftofE. coliBX04from Dr.SangitaPhadtareandDr.MassayoriInouye(UMDNJ).This workwassupportedbytheKoreaResearchFoundationGrant fundedbytheKoreanGovernment(KRF-2014-011146and KRF-2015R1D1A1A01058206).

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