JORJItNAL OF BACIIoIOoY, JLIIIC, 1967, 1. 1976--1986Copyright ((j 1967 Americ.an Society tor Microbiolocy

In Vitro ali(I Iii \Viv-0 Characterization of Pv-ociiiTHOMAS B. HIGERD, CHARLES A. BAECHLER, AND RICHARD S. BERK

Dcparozcni of.o\icrobiology, lfo(tYn Stotic ULnivcr.sitY School of' .1Icdicice,D)ctroit, Mlicligan 48207

Recci\edclor publication 4 I-ebruar_\ 1967

Pyocin, a bacteriocin obtained from l1sates of ultraviolet-induced cultures ofPseuldcolionaces aceruiiginosa was clhLrLacterized in vitro and in vivo aLfter I,000-foldpurification by chemical, column, and differential centrifugation procedures. Elec-

tron micrographs of negatively stained pyocin p)repalrations contaLined rod-shapedparticles whichi resembled the contrcactile tcalil pr-otein of the T-even phages of Es-cherichia coli. Although two septLrate anid distinct pyocin fractions were eluted fromdiethylamninoethyl cellulose (p1H 7.5) during the puriticatioll procedure, the pLar-ticles aLppeared identical. In addition, the two fractions exlibited a close correlationbetween their titers and the pacrticle numbers as observed in the electronl mlicro-scope. The particles were approximnately 20 by 90 mily with a core diamneter of 5 mpand a sheath length of 50 mii. Neitlher intaLct phlage nor ghosts were seen in any ofthe preparaltions, alltlhouglh ringlets of two different diameters, which apper-ed tocorrespond to the diameters of the sheath and inner core, were observed. Otherstudies indicated thalt, altlhouglh crude preparations were stable to freezing andthawing, purified preparaLtions lost all of their activity under similalr treaLtment.However, the addition of 50, glycerol to purified prepar-ations completely pro-tected activity. Conversely, aged normal human or rabbit serai enlhanced the Lanti-bacterial activity of pyocin approximately fouIfold, althlouglh seruIm albumnin aLndhemoglobin h1ad no effect. In vivo studies indicated that purified pyocin wcas notlethal for mice when injected intraperitoneally in concenitrations of 28,000 to 1 400,-000 units (5.6 to 276 ,ug of protein), nor was 7,200 to 36,t)00 units dermoniecr-otic forrabbits.

Bacteriocins are antibiotics produced by manybaLcteria that kill sensitive orgalnisms of the sameor closely related species. 'yocin, a baLcteriocillof Pseudonionals aietliginosc, was ii-st desci-;bedby Jacob (17), who showed its synthesis WaISinducible by irradiation witlh ultraviolet (UV)light; later studies by Hanmon (12) indicated theexistence of protease-sensitive and -resistantvarieties. More recently, KageyarnmaL and Egami(19) have purified the protease-stable pyocin 100-fold. Electron microscopic studies indicated a

structure of rod-like paLrticles of uniform-l size (18).More refined electron mict-oscopic studies byIshii et al. (16) indicated thalt the main structureof pyocin closely resembles T-even phage tailswhich consists of a contractile sheath and innercore. At the present time, studies on pyocin areof interest for a variety of reasons, one ofwhich is from a host-paraslite point of viewwith regard to the infectious processes in mcan,since Homma and Suzuki (13, 14) lhave shownthat pyocin and the protein moiety of P. cwerugi-nosa endotoxin are chemicaLlly and imimunolog-ically related, if not identical. Both are capable of

eliciting a pyrogenic arnd Shwartzmaln reactionin rcabbits in equal potenlcies. Similcar studies (1,9, 10) with colicine K fromii Es/ewici(chi coli K235indicate that it is serologically anid chemincallysimilar to the 0 Cantigen in this acterial strain;however, the antibiotic activity resides in theprotein moiety of the comiiplex. A second area ofinterest is the possible relationship of bacterio-phage with batcteriocins since they appear to havecertatin piroper-ties in commllon (8, 23). Conse-cluently, the alim of this study is the in vitro andin vivo chcaraccterization of a bacteriocin fi-omP. (weruginosa in order that its relationship toboth phalge Cand host-par(-Asite studies he clarified.

NIATLRIAiS.S\N1) MN L-i11()1)S

Orgtloism. P. ('eruiioos strainl \W'1, served as a

senisitive indicator strain or the titraition of pyocinproduced by strain C,

.M!cdiumo. The compositioin of the growtih aind titra-tioIn mediulll consistecd of '2, trypton-e, I'' glucose,0.5', SOdiumni clhloridc Id 1.5' agar.

P!ocili prodhctioii. Ani 1 8-lhr cultuLre of C was SuS-pended in sterile salinc 0(.8V5) and WaIS diluted so asto have ani absorbanc\ of ().10) Cat 660 m1. Of thiS SLS-

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CHARACTERIZATION OF PYOCIN

pensioll, 1I) ml was added to I liter of 2' tryptonebroth, anid togetlher theyvwere in11cIbated at 37 C for 3hr under conistant agitation. Samples of 50 ml of' thebroth cuLltuLre were tlhenl exposed to UV irradiationi 'or

40 sec in alPyrex bakinig dish (205 by 340 mmll) unlderconistanit agitation and at a distance of' 30 cmil from thelight souLrce \WestingloiLse Sterilampii) 782t.L-2t). Thetreated broth was thieni reincubated inl the dark for 3

hr at 37 C unlder conistaLnt shiakinig, at the end ol whiclltime the birotlh was cenitrif'uged at 13,200 X g for 30min at 4 C ini the Sorvall RC-2 Cenitrifluge. The suLper-nlatant fiIiCi \vlas desigiiatecd as the crude brotlh lysate.

Pj,ocin assay. A modifiedt assay system of' Jacob(17T) was used to titrate the pyocin activity of lysates.An 18-lhr cullture of the W, strainl \w'as SUispenided inI

sterile saiiie Liid was dilulted so as to have ani absorb-

ancy of It) cn a Colemilani Jullior Spectrophotometerat 660 niip. Tlis stanidard SuspeCInsioni was diluitedwith sterile sailine. The additioni of ()25-ml samples otthe dilLIted SiISpCenSieil was tmade to lpetri plates coIn-taininig 20) ml of the 2', tryptonie agar. anid the orgaLn-isils were spread over the agar sUrlface by tIle ise ofa, bent glass rod. AIterwvards, the plates were in1cubLatcdat 37 C lfor 3 lir, ancd oiie drop (0.05 till) ot eachl seri-

ally diluted pl Ocill sample wvas placed oni the Laar stir-

face. The plates were reilIcLubated at 37 C ulltil tile Sell-sitive lawnlfirst became visible, at vhichl tinme the plates

were reai. The Ullit of' pvocin activity was expressedas 20 tililes the reciprocal of the ligilest dilLutioll whichgave a comiletely clear zone of inhibition ()1()5 il-l ofeacih dilLitioii spotted on the agar).

Proteiil. Deteriniaiition- of proteil- iiI p\ ocm saiil-flies was blased oii the Zak anc C(oelii (31) imiodifica-tion of the Lowry iiietiiod. Boviine seritil alilu-iilln('Ariiioir Ilharmiaceuitical Co., KaLiikakee, 111.) was

used in tile coiistruiction of the staiciirii cuirve.PYoCcill o)11l ficMtion. PIurificatioii ot pyocin iii l-

sates was performed accorcding to a modificatioii ol' tiemethodt of KageyalaL aLid Egaimi (19'). Of tile cell-free lysate, I liter was treated with 61) ml of I NI MnCI2and was adjusted to pH 7,5 under vigoroLls acgitaitioliprior to ceiltrifLigatioii at 13,200 X g f'or 30 mii. Totile sLiperiitaiit fiLlid, 500 g of aimimiioniLmlll sLilICite xvas

added, aiiii the mixture was refrigeratedflor at least 2hr. The pirecifpitate was then centrif'uged at 30,0()0 X g

for 15 niiii at 4 C and was dissolved in 50 ml of 0.0)2 Ni

tris (lhlydrox\ methi ly) aili noiiietiliaiie (Tris cil ioridebLufIer (pH 7.5), conltaiiling 0.01 NI mlagilesiulum chilorideand 0.01 mi magnesiumn sulfate. Al'ter dialysis lor atleast 24 hir aigainst 2 liters of the same buffer, the pyo-

cin soiLitioll was cleared by centrif'ligation at 8,1)00 Xg for 15 miiii at 4 C. The soliitioii containinig fpvocinwas thell cenitrifuged in the Beckmaina Model L P're-piarative Ultracentrif'Llge for 60 niiii at 100,001) X g

with the type 50 rotor. The jelly-like iellet obtainiedwas gently cifssolveid in the saiiie Tris chloride buffterancd was chromatograpiled on1 a dilethiylaniiiioethyil(DEAE)-celuilose (Calbiochleml, Los Angeles, Calif.)coluiin which had previously lieen waslied with 2liters of the Tris chloride buffer (pH 7.5). After thepyocin samilple was applied to the column (25 by 150mm) for I hr, 200 ml ot' the Tris chloride buffer was

passed to elimilinate an-y substance not readily adlier-ing to the column niaterial. SubseCIuIently, 800 ml of a

sodiuIll chloride gradient ()0to 1.0 .N SOCdiLiIm chloridlein the 0.02 mi Tris chloride hliller) was applied in aliniear mainiier. The resLIltilng chromatographic frac-tioIn cointaininig pyocin activity was theni dialyzedagainist thte samiie bluffer to remilove the sodiILiI chloride.The dialN'sate was dtesiginatetd as the purified p!ocillsolutioll. Conicentrated solutiolis nlecessary for animalillOCulations ancid clcctroni microscopy studies wereobtained bV iltr icenitrifLugatioil as previouLsly de-scribed.

Ini vim stiulics. [)eterminaction of dermiioiiecrotic ac-ti\itv of' pLirihiUfed pyocin preplarations was performedin triplicate by inljcctiig 0.2 ml of varioLls dilutiolis(7,200 to 36,000 1un1itS) SulbCutanlCOuSly inlto rabbits(New Zealanid strali ). Conitrol rabbits received anCiLial amllOulnt of tIlC suslpending Tris chiloride buLffer.The inijectcd areas w\ere observed daill for derimio-necrosis over a 3-day period. The possible lethal eftectof puLirified pyocin on1 Imlice was deterinilCed in ClUad-rLllicatC by inijectiig the mnaterial intrarperitoneally(28.500 to 1.440.000 uLnitS. Inljected Imlice were ob-served at several time intervals over a 72-lhr lperiod. Allill ViVo stLudies employed pyocin which lhacd beelnsterilizecd by treatment with UV for 2 mill.

Elecctron lnicroscopY. Negative-contrast metIioiso. 4, 11 25) which haive beci described for the plir-pose of1 StUdyiing the ultrCastructUre ol' virions w\rereCempl)loyeCd ill tilh stuldy of py)ocill. Uranyl acetcate(0.5', or silico-tunllgstate ( 1.5' w) as uIsed as thenecgative staill upo)n1 WhiCh tihe collodon pSeuLdorepli-cas were floated from agar blocks. Thesc pscudo-replicas were placcd1 upon 400-miieshi copper grids, anida thilin film of carboni was evaporated over the sUrftciceto stabilize the membranes. Grids were e\xalined i amodifiecd RCA EMU-3E electronl microscope at aninistrLtillelital magnification of 32,000 diameters aiidt ataii operating voltage of 50 kv. The electroni microscopeWas m11odlifiCed to iiicltde a specimen chliamber puILmpIII)ouit, ' a heaited placitinlumil 9%)-u objective aperture, andthe stanldard high-voltage stabilization Lulnit.

E/kct of normiuf scraetiil seriom comipolite/ts o0l (c-tivit v. Supplenients of 0.5 ml of varioLus sera wereaddecd to 0.5-mIl samples ol a p)Urified yi)OCin1 SOILutionlto effect a finial conicenitrationi ol () 1, 1.0, 5.0, 10, anicl25'. The normal sera testecl i1cludecd bovine, calf',chicken, horse, huiman, anicd ralbbit sera. Alter 1 hr ofiLIcubation at 37 C, the mixtures were assayed lorpyoci n activity. Hemolyzed h liiman whole blood anidhemolyzed sheep) blood were also tested in the samemaniner. In addition, puirified humanl hemoglobinanid humlilan albuminlll (Nutritionial Biochemilcals Corp.,Clevelaiid, Ohio) were addled to sampiles of pyocin ina tinal concentration of 2.0 aLd 20.0 pg/ml. Thesemixtures were inIcubated at 37 C for hir anit wereassayed for pyocin activity.

RESULTS

Initial studies were designed to determinewhetlher an uninduced pyocinogenic strain ofP. (cei-leginosca (C,) spontaneously produces de-tectable pyocin titers during growtll. As shownin Fig. 1, pyocin- was first detectable in the growthmediumii at about 8 hr. Fromii 8 to 24 hl-, the titer

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HIGERD, BAECHLER, AND_BERK

rose from approximately 40 to 1,200 units/ml,and its production generally corresponded to thegrowth response of the pyocinogenic strain.However, when a 3-hr broth culture was treatedwith 40 sec of UV irradiation or 1 ,ug of mito-mycin C per ml, then pyocin titers of 40,000 to60,000 units/ml were obtained within 60 to 120min after induction. Consequently, all subse-quent studies were initiated with UV-inducedcultures.

Prior to characterization of pyocin, brothlysates were purified by the modified methoddescribed in Materials and Methods. Pyocin was

purified 1,030-fold with a recovery of 22.5%- oftotal activity by use of chemical, differentialcentrifugation, and column procedures (Table 1).

0.8 r-

0.7 *- * q"I!l ""l!e ..Eo- , p in c lll c lx /

0.6

0.4 0-

0.3 /

0.2 /

/~~~~~~~~~~

0.1 _ j //

r, aQ 1) IA1 A ON ,4 8 1 2

H 0 U R S

FIG. 1. Sponitanieolus release ofpyocini dutrinlg growth

of a toninidulced ciultutre of Pseuidomonias aerluginiosa

(C9) .

The elution profile of pyocin from DEAE cellu-lose (pH 7.5) is presented in Fig. 2 and indicatesthe presence of two protein peaks which exhibitpyocin activity. The majority of activity was inthe first peak after initiation of the sodiumchloride gradient; it exhibited a 280 to 260 myuratio of 1.35, and the second gradient peak had a

280 to 260 m,u ratio of 0.7. Electron microscopic

examination of negatively stained material sedi-mented from each peak at 100,000 X g for 60min indicated the presence of rod-shaped parti-cles which were identical in appearance. Thepyocin titer of the second peak was approximately1,000-fold less than that of the first gradient peak,and the relative pyocin particle numbers of bothpreparations also differed approximately 1,000-fold. Consequently, there was good correlationbetween pyocin activity and the visually observedparticle numbers of the two preparations, sug-

gesting that the observed particles were respon-

sible for pyocin activity.Electron micrographs of the structure of

pyocin prior to column chromatography andafter final purification can be seen in Fig. 3 and4, respectively. The purity of the precolumnpyocin preparation was approximately 500-fold,and the appearance of these particles appearedto resemble the tail protein of T4 coliphage in

size and shape (30). In addition, they were

morphologically identical to, but varied somewhatin size with, a pyocin preparation previouslydescribed (16). The majority of the particles inFig. 3 measured approximately 20 by 90 mji witha core diameter of 5 my, and the length of thesheath was about 50 m,u. Two strands of what isbelieved to be flagella from P. aeruginosa arealso present in Fig. 3, in addition to a few sheathswhich appear devoid of cores and seem to bepaired together end-to-end. None of the electronmicrographs of precolumn and postcolumnpyocin preparations exhibited the presence of

TABLE 1. Slummniary of puirificationi data

Vol (ml) Pyocin activityTreatment _ Protein Specific Recoxvery Purification(mg/mi) activity" (' (fold)

Initial Recovered Units/mml Total units

lnduced broth lysate 1,800 1,745 14,000 2.44 X 107 14.4 972 -MnCl2 1,745 1,810 -(NH4)2SO4 1,810 105 120,000 1.26 X 107 12.5 9,600 51 9.8Dialysis 105 141 80,000 1.13 X 107 3.7 21,620 46 22.0Centrifugation at

100,000 X g 141 15 700,000 1.05 X 107 1.4 500,000 43 514.0Chromatography,

centrifugation at100,000 X g 15 64 80,000 5.12 X 106 0.08 1,000,000 22.5 1,030.0

a Expressed in units per milligram of protein.

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CHARACTERIZATION OF PYOCIN

intact phage, phage heads, or ghothe appearance of the particles in thpyocin preparation resembles thacolumn pyocin particle in that twoplus empty sheaths joined end-to-eOf all particles seen in the precolhcolumn preparations, 90K'' consisttile rod-shaped forms, and the r

DEAl CELLUIOSE. pH

AASORBANCY Al 280ABSORBANCY A' 260 m

PYOCIN ACTIVIT Y

IU E NI "

FIG. 2. Coluimn chromatography (

piarified pyocin prepar atioli oli diethlXlclose, pH 7.5.

sts. In Fig. 4, sisted of partially degrcaded rods and emptye final purified sheaths. In addition to the rod-shaped forms,it of the pre- ringlets of two different diameters (5 and 20 m,)intact particles were frequently observed in most purified prepa-nd are visible. rations and corresponded in size to the diametersimn and post- of the cores and sheaths, respectively. Figure 5ed of contrac- shows the presence of four small ringlets.emainder con- In Fig. 6, some of the fine structure of purified

pyocin can be observed. It is suggested that theparticle consists of double hollow cylinders withcross striations. In addition, the unusual lengthof some of the particles seen appears to be due

______ to two cores situated in close proximity to oneanother.

Because of the close immunological relation-ship between the protein moiety of P. clerll,ginosaendotoxin and pyocin (I13, 14), both crude lysatesand purified preparations of pyocin were exam-ined in vivo. Mouse toxicity studies employingintraperitoneal injections of purified pyocin in-dicated no lethality over a 72-hr period whenconcentrations of 28,000 to 1,400,000 units (5.6

of the partiallY to 276 ,ug of protein) were administered. No

,ninoetlYl ceIlIu- lethality was noted with crude broth prepara-tions consisting of equal protein concentra-

FIG. 3. Llectmo0lim 7iCrogKraphi of a pasmtiallv phl)ifiedl pyocinl prepar(ationlStlainled sWith ilic tllngKst(ate. T'1 o f.lagellammIYhe /el traversimig lic field. The numaneroius comitractile r-odi-like particles aref believed to h)e resfo)lsihle fin pvocimactivity. X 195,000.

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HIGERD, HAECHLER, AND BERK

F-ii. 4. ELeu/cro, i:ic(let :1.aph(/i a purl/ifdpy/io prepuira/IiOl s/(iIiC(d ti/li Iultl'v/ Tce/o/.l inlc/a )I'Oci/lpa/-ti(cl'S citicitl () wtip/v A/lea//ijOuitdo cad-io-cndti!ar visihle. X 340,000.

tioIns. In addition, nlo dermInoniecIrosis Was ob-seerved with purified preparations in rclbbitswhichi had received 7,200 to 36,000 units (1.44to 7.2 ,g of proteini) subcutaneously.

Purified pyocin was first characterized in vitroby ther-mlal labilitv studies by use of a standardsolution conisisting of 4,000 units iml for this aLndCall subsequent studies. Pyocin was found to bestaLble at r0oom1 temperature and at 4 C over a15-day incubation period. However, when pyocinwas frozen for any length of time, aL complete lossof activity was noted. No morphological altera-tions were detected in fr-ozen and thalwed prep-arations when examined by electr-on microscopy.On the other hand, the activity of unpur-ifiedbroth lysates was unaffected by freezing and thalw-ing. Supplemenits of 50, glycerol completelyprotected purified prepl-rations against freezingand tlawing, whereas 10()- NI cysteine had noprotective effect. Since 1pyocin was sensitive tofreezing or thawing, an attempt wcas also maLde todetermine whether- activity would be lost afterpassage througlh the Freniclh pr-essur-e cell. Treat-ment of purified pvocin in this manner reducedthe activity 10-fold. A shearing mechiLanisill may'

ltve beeni responsible for loss of activity in bothprocedures.

Another- study concer-ning ther-mal lability in-dicated pyocin activity was completely retainedafter 10 min of incubation at 40, 50, and 60 C;however, no activity was retained at 70 C orhigher. In addition, treatment of pyocin witlhUV for 2 min did not aflect activity. Lipase (2 mgiml), palpa,in (2 mg/ml), trypsin (2 mg ml), orP. wCie) lgifoasC purified protease (2 ig/ml; G.Johnson et al., Bacteriol. PIoc., p. 40, 1966) alsodid not affect the activity of pyocin over a 3-hrincubation period.To determine the effect of potenitial inhlibitors

on pyocin activity, supplements of various com-pounds were added to salmples of purified pyocinalnd wer-e incubated at 37 C for 1 hr at whichitime the activity was meacsured. No inhibitionof activity was obtained with 10-1 Ni cyanide,10-1 NI ethylenediamniinetetralacetatte, 10-' NI CyS-teine, cnd 0(.1 to 0.7 NI sodium clloride. However,8 NI urea and 10-4 NI )-chloromercuribenzoate'PCMNB) abolished all activity, and 10- Ni mIer-cuiric clhlor-ide reduced the activity 95(,. However,supplements of I NI urea, 10- .NI PCMNB, and

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CHARACTERIZATION OF l'CYOCIN

;i1':K"

*V .,e ..

iStH 'SC' ":.J :;: :, .. .:

a:. ,S,,l, sw .. ;.

.z.: .. ' - B .: !... ..... X, wC ,R, ... :!

, .... ., ,.

.. _f, st. :. tt1 S.. ... . r #! .... # s>fz* .... %. 1. . i;:. :. :, 9

..

W

:., -' :: :.:. :

.. ::

* 44

V .'i74'4,.;,

:.

FIG. 5. Eleciro/In71icrgrp1(h1fp)11( lp-iified /vc0(1i1l/prC(lpraioll siiedi /llt'ilY/'Ilra v(a(ca(1e. f(0' ssma/ll ringleris11(11'1)b observed (indic/aed hY)1iep1/01/t)iswc//(151/ (s Cmpi)/sheXIhsl//iX.' 1(1i/11ta(ct /par/ticles.. X /62,000.

10- xi mercuric clhloride exerted little or no effectonl activity.

Electron microgl-rLplhs of urea and PCMNB-treated pyocin prepar'ations can be seen in Fig.7 and 8. The studies with 8 xi urea demonstraLtedaL cleavage of sheLtlhs into mnany lairge ringletforms contLining subunit structures whiciprobably r-epresent the smaLller ringlet form (core).Concomitantly, with the increase in numbers ofsheaths and large ringlets, ther-e wals a mIlar-kedreduction in the numbers of intact pairticles andnaLked inner cores. On the other- hand, PCMNBtr-eatment did not seem to produce ringlets, butaLppeared to halve aL degraLdaLtive effect upon thesheaths. In addition, these prepairaLtions occa-sionially exhibited long tubular str-uctur-es severaltlhunidred millimicrons in length whichi diffeiredmarL-kedly from the structure of the individuallparticles. This ditference in chemical reactivityof pyocin to urea and to PCMB suggests that thepr-otein content of the sheaths and cor-es isdissimilar.

Previous studies halve indicated tlat ser-a froma variety of animals enhanced the activity ofcolicin (I , 28, 29). Therefore, similla- studies wereperfor-med with pyocin. Serumii was added tosamples of purified pyocin to obtain a final coin-centr-ation of 0.1, 1.0, 5.0, 10.0, and 25.0('>. After1 hr of incubation of the mixtur-es at 37 C, pyocinCactivity was measur-ed. Bovine, calf, clicken, Candhorse sera lhad no effect on activity, whereas agedfrozen 3 years) human sera enhanced activity

threefold at 10.0(', and fivefold at 25','. Inaddition, aged 25' rcabbit serum (frozen 1 year-)enhalnced pyocin activity fourfold, wher-eas freslhhunan serumii hlad no effect on activity. Supple-ments of either purified hemiioglobin (2 to 20ug/ml) or serum calbumin (2 to 20 yg/mll) didnot enhalnce activity.

DisC'USSION

Despite the fact that bacteriocins have notbeen thor-ouglhly investigated for their possiblerole in infectious processes, recent studies with

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HIGERD, BAECHLER, AND BERK

FIG. 6. Electron micrograploP o p ri/ifiel 1)o0(in1 /)pre/palrtion/i stlield wit/h uranylI! a(cetate. SegmenitationI an/pe-riodIicitv oJ tl/ie particlesrci.e rec-d1lY discerIhce. Long fi/claients appear tO he i/l/lie (Oores distal/Y clttauclied. X320,000.

P. cretltginosci (13, 14) aLnd E. coli (9, 10) indicatetlmLt these agents are closely related to classicalO endotoxins, or fractions thereof. Conseqluently,to characterize pyocin comprehensively, it waLsof interest to determine whether it exerted atoxic effect in vivo. However, the resultaLnt lackof lethaLlity aLnd of dermonecrosis Cappeatred torule it out as a possible toxic faLctor in P. (ielagi-iOSCl infections.At the present time, the in vitro studies requilre

further study atnd clar-ification. Althouglh previousstudies by Ishii et al. (16) suggested that pyocinmiay undergo contr-Caction or reltxation, it isnot known whetlher the uncontrLacted form isresponsible for baLcteriocinogenic aLctivity. Tothe present time, we hlave never- been aLble toobserve uncontracted particles in our prepa-rations, regardless of purity. Furtlhermore, thepresence of hollow shecathls in pur-ified pyocinpreparations suggests that some paLrticles areeither damaged during the purlificcation pro-

cedure or they are incompletely synthesizedduring induction. At present, it is not knowniwlhetlher the hollow sheaths can also exert anti-bacterial activity. Since previous studies indicatedthat freezing and thalwing resulted in the con-traction of phalge T4 tail protein (30), all of ourelectron micrographs were obtained with un-frozeni prepa-trations.

Althouglh some smnall round forms or ringletswere detected in botlh crude and purified prepal-raLtions intalct phage, tail-free heads, or ghosts'were not observed during tlis study, nor wereplaques pr-oduced onl pyocin-senisitive stralins.Tlherefore, our findings difler fromii the observa-tions of Kagey-ama et al. (20), who noted thepresence of an intaLct, but immunn ologicailly dis-tinct, infectious phage fraction wlichi was elutedfrom DEAE cellulose after the main pyocini peLk.The size of the two ringlet form-ls frequently ob-served in our preparations appears to correspondquite closely to the diaLmeter of the tail Cand its

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CHARACTERIZATION OF PYOCIN

.r 4i.-

t~~ ~ ~ ~ ~ ~ U

.i,CE v X~~~~~~~~MM

't::4.i. -l:.I.- .:.!.

i...11. ...V.:

..q.1-.t4 '.

.. ;ii!-::

4" -!,-' -,.-X.. .. A;

f*~.-,"@;Adi

FIG. 7. Electron miicrograp/i of i pvi/i-ci i pjyocin prepcration treccted wsit/i 8-l inrea anti stainied wit/i ianyl ace-taite. Lalrge rilnglets and enipty sheaths are nionerous whereas intact contractile rod-like pataticles atre inifieqioentlobserved. X 260,000.

sheath; their small size, however, rules out thepossibility that they are phage heads. Althoughpyocin has previously been reported to consistentirely of protein (18, 19), it does morphologi-

cally resemble the rod-shcaped, single-strandeddeoxyribonucleic acid (DNA), headless Pf phagerecently isolated fi-om P. aeraginosa (26). Thisphage forms minute plaques on indicator strains

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HIGERD, BAECHLER, AND BERK

FIG. 8. Lectron,, /ilnro'raph/ 01(1 p)tlrifwed /)1('Ocill rlC/)r(11io01 It/elitel it'hl/ 10- I'PC\1 B and siined wiihilll v/ia(ct(aiIl. Ililei ('0/CS CIlt'h)/)C(/h'/(trslelnvelotp's/yaIpp(tpar/)('(tOlotthe I(ftei/tr /'oi/UsIrtl7ea/nwllm. X 195/000.

and resemilbles pyocin cores in tht it appears as gener-ail appeairance aind size of the conitractilehollow rods once it is without its DNA. tail-like pyocin for-ms certainly suggest a closeAt present, the relationship between pyocin structural similarity to the E. coli Tj phage tail

aind baIcteriophage remains unclecar; however-, the moiety (5), ailthoughi no base plate, spikes, or

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CHARACTERIZATION OF l'YOCIN

talil fibers were ever observed in these prepara-tions. It hals been suggested that pyocin particlesmay possess sucli structures (16); however, wewere unable to observe these structures in ourprreparations. In addition, the pyocin structuresailso resemble those seen in the induced lysatesof defective lysogenic strains of E. coli (2) andother systems (6, 22, 25, 27). Despite the struc-tural similarities between pyocin and phalge,Ikeda et al. (15) showed that a P. aerulginosaphage isolated from pyocin lysates was notneutralized by aintipyocin sera, thereby suggestingthlt they mlay be chemically unrelated. On theother hand, Homma and Suzuki (13) demon-strcated that antisera to the protein moiety ofP. (lerllginosa endotoxin neutralized the activityof pyocin from the same organism (strain P1-111). Therefore, it is primiarily this problem ofrelatedness between the two systems thlt re-quires further elucidation.Another problem which remains to be re-

solved is the mechanism by whiclh pyocin activityis rapidly lost upon freezing and thawing, al-though normal refrigeration and even roomtemperature mnaintain the antibacterial activity.Despite the striking loss in activity, electronmicrographs did not demonstrate discerniblemorphological changes of the particles afterfreezing and thalwing. Neverthieless, these resultsappear to be consistenit witlh those of Kageyama(19), who noted a loss in activity upon lyophili-zation of pyocin. One can assume that the sta-bility of crude lysates to freezing is due to thepresence of protective impurities whichi aire sub-sequently removed during the purificationprocedure. OII the other hand, the compcparativelyslight enhaLncement of activity by laged humanand rabbit sera, but not by human hiemoglobinor serum albumin, suggests that pyocin ditfersin its mode of action from the colicine K system(1, 28, 29) which was stimulated several hundred-fold by these serum supplements.

A(K NOWLLD(;M[NTSWe are inldebtedt to Altoni Taylor ot'1Parke, I)avis &

C'o.. Detroit, M\ich.. for his technilcal adivice anid assist-aince.

This investigationi was suLpprl1tedC l[N tle PulblicHealth Service granit Al-0)5214-0)3 from the National11IstitUte of Allergy and InfcCtioLus Disea.ses.

Ll1LIZ.ATURE CITED

1. Al\ANO, T., W. F. GoI-131- AND E. M. Smixi-in.1958. Colicinc K. 111. The immunol_1ogiCalpropcrties of a SUbstancc h1avin1g colicine K ac-tivity. 1. ExpItl. Mcd. 108:731-752.

2. ARBER. W., AND) G. K1 LLLNnEIBIRIR. 1958. StUdyof the properties of' scveii defective-lysogenic

straiins derived from Esclhric'hiu (coli K12 (X)Virology 5:458-475.

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10. GOEBE., W. F., G. T. BARRY, M. A. JL.SAI'FIS,AND E. M. MIILIR. 1955. Colicine K. NatUre176:70)0 701.

1 1. HALL, C. E. 1955. Electroin densitoImetr) of'stainied virus particles. J. Biophy s. Biochicim.Cytol. 1:1-12.

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13. HONMA, J Y., ANI) N. SuzuKi. 1961. A simliiieprotein with pyocine activity isolated fromil thecell wall ol' P)seudlomoncs erlugino.s and itsclose relationi to enidotoxiin. Japan. J. Exptl.Med. 31:209 213.

14. HoNINIA, J. Y., AND N. SUZUKI. 1964. Cell-wallprotein A" ol Pseud(omonas uerlginlosa andits relationship to "original endotoxini protein."J. Bacteriol. 87:630-640.

15. IKEDA, K., M. KAGLYANIA AND F. E(oAM. 1964.StudiCes of a pocicn. 11. Mode of production ofthe pyocin. J. Biochemil. 55:54 58.

16. ISHII, S., Y. NisiII AND F. E(A\II. 1965. Thlc finiestrLuctLire of a pyocin. J. Mol. Biol. 13:428 431.

17. JACOB, F. 1954. Biosynthise indiuit et moded'actioni Cd'uLeC p\ ocinie aitibliotiCque de Pseulo-m101lO.n P tIWvo(Ym. Ainn. Inst. Pi'asteUr 86:149-160.

18. KAGLYANIA, M. 1964. Studies ol a pyocin. 1.Physical andt clemlical properties. J. Bioclhcmi.55:49 53.

19. KAGLYAMA, M., AND F. EC;ANII. 1962. Onl theturificationi andcl soimie properties of a pyocin, a

bacteriociin prodluced 1y Pseiidomonaois acn/i-guiosa. Life Sci. 9:471 476.

20. KAGLY) ANIA, M., K. IKDA, aiidI F. EGAMI. 1964.Studlies of' a piyocin. 111. Biological propertiesof the pyocin. J. BiochIemii. 55:59- 64.

21. KELLENI3I:RCiLR, E., AND) E. BOY DI LA To(lR. 1964.

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On the fine structure of normal and "poly-merized" tail sheath of phage T4. J. Ultrastruct.Res. 11:545-563.

22. MENNIGMANN, H. D. 1965. Electronmicroscopyof the antibacterial agent produced by Esc/h-eric/hido coli 15. J. Gen. Microbiol. 41:151-154.

23. REE\ES, P. 1965. The bacteriocinis. Bacteriol. Rev.29:24-45.

24. SEAMAN, E., E. TARMY, AND J. MARNlUR. 1964.Inducible phages of Bccillius sabtilis. Biochem-istry 3:607-613.

25. SHARP, D. G. 1960. Sedimentation counting ofparticles via electron microscopy. Intern.Kongr. Elektronenmikroskopie Berlin, 1958.2:542-548.

26. TAKEYA, K., AND K. AMAKO. 1966. A rod-shaped Pseudomonas phage. Virology 28:163-165.

27. To, C. M., A. EISENSTARK, AND H. TORECI. 1966.

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Structure ol mutator phage Mul of Evcherichiacoli. J. Ultrastruct. Res. 14:441-448.

28. VAN VUNAKiS, H., A. RUFFILLI, AND L. LEVINE.1964. Colicine K and endotoxin: effect ofhemloglobini anld its subunits on their antibioticandt serological properties. Biochem. Biophys.Res. Commun. 16:293-299.

29. VAN VUNAKIS, H., A. RUFFILLI, AND L. LEVINL.1965. Enhanicemiienit of antibiotic activity ofcolicinie K and alteration of serological proper-ties of colicine K and endotoxins. J. Exptl.Med. 121:261-278.

30. WILLIAMIs\ R. C., AND D. FRASER. 1956. Struc-tural anid functional differentiation in T2Bacteriophage. Virology 2:289-307.

31. ZAK, B., AND J. Co(iEN. 1961. Automatic analysisof tissue culture proteins with stable Folinreagents. Clin. Chim. Acta 6:665-670.

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