UraniumBioaccumulation by a Citrcbactersp. as a Resultof Enzymically MediatedGrowthof Polycrystalline HUO2PO4 (
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LynneE. Macaskie,r RuthM. Empson, AnthonyK. Cheetham,t ClareP. Grey,+A. JeromeSkarnuliss A Cr,trobaclor sp. accumulatesh6avydepositsof metal phosphat€,d6riv6dfrom an enphosphate zymicallyliberated ligand.Th€cettsarenotsubjscno saturation constraints and can accumulateseveraltimeslheir own weightol precipitatodm€tal.This high capacity is attribuliablelo biomineralizationiuranyl phosphateaccumulatesas polycrystajline HUOTPO. at lhe cellsurfac€-Th€ prscipitated metalis indistinguishabte fromcrystattine HUOrPO..aH,O grownby chemacal methods.
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A Cftooare'sp. a*umulareshea!"yme(al. asa rcsuhofprecipirarionwith en;ymically l i b . 6 r c d , n o r s " n np h n , p h a r c( 1 , 2 1 . T h h r€actionh.s beFn hamesrJ ro a bturcchnologicalprocessior the reatnenr of mct al'b€a ns streams(J, 4)r ir alm hx poten (5). ual fornRle& wdrede(on'amhat'on facrlrtared bv rhe high €droacnverole€ncc of rhc c.ll-bound phosph.rerelealinsen ryme (phosphara*) (6). The nmob'lkJ whole.cellbrqaralv't rolenrcs load5of up I o 9 8 o f a c c u m u l z t eudE n , u mp c r g r a mo r bionass(900% of the cellular dry weish0 over several qeek withour apparcnr ill eflect (4). A patkagingmechanBmfor rhc U mrghr b. pnnulared to fcmr( cnrymc , u m o v e ry, e rt h eh r 8 hr ^ x ( r . r r i u r a n r l r " r . (7) makesa bichemicat comparrnentati,a. rion on rhis scalcunlikely- Wirh a conrur
R M Er4M, Sai.r Ma,ry'sHcpitar Med@r Schoor. Pra.d grer, Lddm. wzr|.IY. u.fta K,nodorn A K CrEelnam.C. P Grey,A J Sk3nlls.-Lrbor.rory or chni@r cry{anogGphy, unive&v ot oxtord PadsFoad,Oxlord,oxl 3PD lnired Knodom J. be 6ddrss.d "rb@sp.'i."." Malenats "r*rd lPr€s.ntadd.ess Oepanred.lJn'@rsryor calitomia.Sa.la aadaa. c,q 93i06 tPGsr add.ess Mol6utar Spectosc.cv Llborato. .y, Faculryot Physicarsffies. T@,n@rd. unrye. et ol N,!r€96, N'tEq6. Dp Nerhedaids6525€D sPresr 4rdress JeolsFrffi U h Lrd J€d Frtus $Lq
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uous supply of organic phospharedonor molecrlesto cclk immobili?ediD a now' rh.oush carftidge ("bioreacro!"),a sready stareis opidly achieved{hcrcby oeral is rcnoved conrinuouslyind cficicndy 6om rheflos with a coEcsponding accuhuiation ofmctal wirhin thc biorcacror(J). In this s.!dy wc lsed elecfoD microro, py to examinc thc m(hanistu of meial accumularionand rhe farc of individlal cells subir.tcd $ u6nyl ion loadins.The cellswerenor 6xedor srainedin any way, and, assuch,beforcU challengethey *ere indisrinct,and there was no accumularion ot elecrrondensemarerial(Fic. lA). Cells challengedin suspcnsionwith uranyl niftareand phosphalase subsrrate for 12 horr accumularedU to about 250% of rhe bac terial dry weishi (8) and *de higbly clecrrcn opaquewilh I'ttle eisiblesu.faced€tail (Fig. lB). Dlied, Found samplesof rhe loadedcelk wefclcmon yello* in color and exhibitedfluorcscence characrcrisric of u.a nyl compounds.A video image ot a cell rcvcal.d ateasoi€nhanced elecrronopacr rr, primarilyar rhe cell periphery{Fis. lC), which wc an.lyredfu her by imaBincfor IJ (Fi8. lD) and phosphorus(Fis. lE), usins the i ray enissionscharacrc.isricof rhese elemenrs(U l-a and P K). Clear evidcnccfor .odcposnionsusse\ir thar a fom ofuranium phosphat€had ben prodoced.X'r.y micoanaly$sof spr.rmen microarca (Fie- lF) indicarcs thar cclk
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L € r,tac€s&€.scjml ol Bior.g@r sci4es, unre. $ry ol Aim.ErEn. Edqbasrd, Biminghah. S152Tr
unchallengedwith the uranyl ion sho*ed xray emissionsarriburable ro rhe low level wirh biologicalmarenal, as of P associatcd well as the copper pres€nr in rhc sdd. Qualuatively, the cellular deposiris indistinguithableftom a hydrogenuranyl phcphatc standard (HUP,HUOTPO{.4HrO) (Fig. lF). Quanrierive cohpa.i$ns w(e m.de by integEtion of rhe areasundcr the U Lo and P K peak and wer€in(erpr.ted by the mtio method (9). Xtfl't = kllr, whdc k is a constantadd X,, Xr, I,, and I, rcpresent,respecnveltr and the emission intensLriesof the iwo clcments.Thesedarado nor providc infor mation on the absoluteamountsofU and P accumolared. bur th€ peakotiocan be uscd for companon wirh the corespondingErio obraincdusinsrhe known aromic..rio ofU (o P in rhe HUP srandard(U:P mrio = l,l). The r.rio of rhe peak areasfor the standardwas l.9l I 0-048, while rhar for r h c a c c u h u h t e dm a r e n asl a s l . ? t t 0 . 1 5 (ncan a srandardeftors ftom 15 and l0 deteminations,respecrivelv). Thcsevab* arc not siSnilicandydiferenr at P = 0.95i taking inro accounrrhar rhe cell also contains "biolosical P (which would rend ro reduce rhe peak area moo), we conclude that the ltomic U:P m.io in rhe cellular dcB,ir G close$ 1:1. Hor.cvcr, th6 Rrio could alsobc s1v€n othcr unniun phG, ")L phare!,and, asconnmarion rhar HUP has indccrlbeenfomed, sc eraminedrhe sran dard and accumulatedmaielial br solid5tate inta.ed spccro*opy, x.ray di&acthn, and maEic ansle spinninS nuclear masnericreenance (MASNMR). The HUP standardsas prcparedby rhe neth( of weis€l and Hodilan (J0), and its x{ay difli.ction paftem w6 found ro be !n e(\rl asr€em€ntwirh rhe plblished d.t f(r HUP (i0, lr). hFfied 5pecr@opt was pcrfomed in a caftier of cesiumchle ide at d weight:weighr rario of Found standardor sround U Ioaded bbro$ ro CsCI of 1,30. The spectrawerc ven similar, *irh srronsabsoQrionpeaksar 9l0 nm chalacterGti. of the uranyl ion (J2)- \(/e concludethat the accunularcdmaterialF a fom of uranyl phospMre. Funher analys sirh ltP MASNMR tndicated that p.oton-decouplcdspecrE from {he Uloaded.elk and the ur.n}i phosphalestandardexhibit t.ronances ar similar isorropic chemical shifrs {o,- = 2 2 . ?a n d 1 9 . 5p p m ,r e p c r i v e l y ) a n l h a v e similar side band inretunies. &)th rhe is tropic shifr and rhe ch€mical shift diso! ropy can be lsed to idenrifo thc phophate speciespreseot.In geneEl, pyophcphate! r.sonate upfcld liom rhe concsponding onhophosphate compounds (ll), and hcncc rhe similar isor.opic chcmrei shtfr (ir rht uranylpho5phar.srandardacj loadcd cells con6med rhar rh. phare qoup.
. flltfifitE ,rre pr.senr rn klth mrtcnals- Spurung (l{) di )pecrracollc(tcd r banJ rnr|r hpinn'ns ipLcdsof lim H: savc lrlucs , t r t h c c h e m i c i ls h i f tr . n n ) r so , L , o I , r h J ' , , ( 1 4 )o f ) 2 . 5 i 2 , 2 0 1 4 , a n d6 : l 'tn for H[JO,PO] 4H,O ah,t l5 a l, 18 ! 4, and 5 1 2 as th. major rewr.ncs in rhc loaded cells. Thc* are rn rhe tneeexpccrcdfor phosphetc,PO.' , rnlts u o p p o s eod H P O . r u n u s ( J 5 ) a . J n t r c .ndensedphospharesp.cies(tb' .\imrl.,
fro?){16.r/). The siructureof HUP may be Jescribed ,r comprisinghyers of UO,PO, k-twe.n dhich rhc tl' ions .re hcld in r ($! .limcnsional nerwort o{ water mol..ulcs { 1 8 ) .T h e H ' i o n s a r eh i s h l ym o b i l cw n h i n rhe watcr hy$s (19). Wc susse$rt,trr rhc
o ctnr PO,' li.n dn' cn:!ni. ,crc(nnr srlt'rscrnblts qrh UO:j' (' l;m a .o\(,rl rhrt r ertrudr,l rrom rlt l,'.r oi !h.sph.,r i s cr c r i v i r l . n J r h r t u i r ( J h y d b e c nb o n d inr .r$st\ in rh. formrnon ol an o *cJ
qc!. nor l]lrl.ntrcd. Morr h..tr1lr ll,id.d cclls tcnJcJ t,, Jrlntcsntc in tltr clc.roo b.rh ondcr hrsh va.uu , hr!inu p3rticlc\ ol dcpositeLl matcnal as a c . l l ! h o ' ( ( 7 ) ( F i e . l B ) H t r c n c c , l l e so f
Elcdron dilih.rion parr.ms oi (hc c(ll bdun,l mrteli.l (Fis. l) rrc ruesestivcol i two-dimcnsionrlhbrcusstrlcturr, *hich is .onn$cnt *irh the known structure oi HtJP. Evrdenccfor thc JeFution ofnbrillar rtructureswasrcught- Eximrnationof r lightly loaded ccll reverlelt tuint 'bnrh likc cxrrusionsfrom the ccll surtuce(Fig. lA). Thcsc appeareds cltctron'denscli' brik lcs than Il:D A in Jirnricr tllt iJ ot dt.oFect wnlth rccoirlin8 ro rhr publ N i l e ds ( r u d m cf d H U P ( l E ) , b u t l r c c i s c
Flg.2. El6lron drfiraclon paller. ol lhe acc! mllatcd materaL.acqured usrngrhe J&rebc ron mrc.oscope(see leqend ro F,9 r)
x+yono.sy(req Flg. 1. Acclmualon ol !'an'rn by Cnrcbactct.p and characrerzar o. ol Ine accum! alcd melal
[email protected] (2r) ura.'!m- lnslpp ene.red c€rrs (a) are ndsrr.cr whereas . ll chalcnged cclls rhe {l rs vrsb e as an eleclron opaquc slatr obsclr ng celluLardela I rB) A complle. vdco niage (C) revealsdeposrronparrernsnol vBb e n rhe nal!€ specmen. a.d rurlher'mag'.g ror lr (O)and P (E) shows codecDs('onol lhe two.ehe.rs Th. dara were obra'nedwlh rrie Jco nslrumed 'f conlunctronwrh an en.'9y-drspe'srve x.ay sp€ctomere' (I/aco0. whch alows elemenlary l n a y s s o r s p c c r m e .m c r o a r e a s( 2 ) l . l h c { r l a n l 1 3 l ' v a c n a y s s o l s p e c , m e .m c . o a . e a s( < l F m r ) ne used lhe x rav emsso.s chzracrerslc ol l-l.nd P loalow cacuLalronor relalve peak areasror Ll a.d P lor lnc u roaded cers lF, botiom) and cohpa.ed lhem ro a hydroQc. uranyl ohospMle rardard IHUP HUO,POI 4H-O) (F, rop) prepafcd accord'ngro pubr shed rnerho.lsL t ) ano wrrrr l: denliry conlrmed by r ray powder d'nraclio. anaysrs reler€.cea lo plbr'shed slandards (', J?) x ray sDcdra werc ac.urnulalcd ld. 100s a.d dtl, -nalyse! wcre done w(h a Tracor I N 5500 3ysrem loral dala acclmllat on was l28by l2ap,xr's lola: rln r,ne was severalho'rs Scal€b.rs n
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Frg. 3. Derar or rhe accumlrared L]@yl phos phare under rreavy meral loao'ng lhe cells onen bsr inlegrry nr lhe elccton b€am u.der hgh laculm (2) blllhc accun! alell marefa '5 evde.l as qranuar o. needle [ke detns rs IB ar@s) A l€hrly loadedcelreEined inreq rry wrh exocellular vrsllrle as brush 'ke e{rls'ors a znowsr .dMdla lbrls &e esl'maled r. ce .1r lha_ rOOA rt,ck (bar = I Fmi CLLrn:.r. e,l,!s'o.:
precipitare were visible, intebpesed wnhin a more pa.riculare manix. Possiblythe particles representagge8atesof precipitate; rhe brushlike exnusiotu in the lightly load' ed cel Gle- 3A) appa.entlyGcur in discreteclumps,gesumablycorespondiogro lci of phosphatdeactivity. V,hether the example of biocatalytr cally mediatedcrystalsrowth usedby this Grrobacrersp. hascvolved asa gencralized mechanisn of defenseagainstmetal roxiciry by exocellularcompanmentaliration remainsto be connmed. Similarly, ir 6 not known what rolc, lfany, rhe archirec' rure or integ.icy of the outer *ail layers plays, or, indecd, whethcr thc phosphatas€rtseLfislocaredin,uxraposirion to rhe precipitareo! distanced6om ir, eithin rhe
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REFEFENCES AND NOTES 1 R M Aickn.A C F Oem,A K Cheerham.A J Ska.nulis, Microbios Len 9. 711979) R 2 L E Ma@kie A C R DeanA K Ch@rham. J S Jakemn A J Skarnlls J Ge" MJc@b/o/ 3 LE Ma€skeandA C R Dean adv A,orsh nor P@esses12 159(1949) 4 L E Ma€sk'e J Chem Iect'rct B@technot49 357(199O) s crlr Fev ator$h'ol r1 4r {1991) 6 L g.a.han, L E Macask'eB C Jm.A. n Ptrcedngs af tE mlst A,renan Chentcal Saiety M@tin? {Amenaf Crrem€l S@i€ly wasrrLnlion. DC,lssl). vol 31. p 123 7 E J Plummermd L E Ma.askie. Bol Enion c@tan ror@t a,173 (19901 3 L.E Ma€ske J D Bra.knore.R M Empson. FEMSMicrcbialLe .55 157tlgaa) s G cifiandc w r$net, r Mtcr6c londd)
6sh wrth Hg wnt n!'ey'at'J Lheionrnbuliom thc'iatchnenc surround
2). Hci
(_r, e r r b l er e s e a r cohn H c r n Hg liabic
10 F Weg€l md G HoflrnanJ Less Comrcn Mel rr H W Ounn, taak Rrdge Na
Lab Pubt 2Oe2
lake (15).
12 C G.eares,A K CheelhamB E F Fe.de. /ro.g Chen 12 3003(1973) 13 A K Chelham.N J Cayden C M DobsonR J B Jar
