UNCLASSIFIED

AD 400 308

ARMED SERVICES TECHNICAL INFORMATION AGENCYARLINGTON HALL STATIONARLINGTON 12, VIRGINIA

UNCLASSIFIED

FINAL No.1 REPORT ON

* CONTRACT NO DA-92L57iFEC-35775

INCLUSIVE DATES 20 January 1962 TO 19 January 1963

SUBJECT OF INVESTIGATION

r7:)

'• u. Genetic, serologic,I and-, •biochemical s tudies

•y. •onviral infection and lysogenization

RESPONSIBLE INVESTIGATOR

Dr. Hisao UETAKE

Professor of MicrobiologyDepartment of Microbiology

Sapporo Medical CollegeSapporo, Japan

[" /PR 9 ' ,iL~ hY T• I•

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U.S. Army Research & Development Group (9&52) (Far East)Office of the Chief of Research and Development

United States ArmyAPO 343

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The distribution of this report as made by USA R&D Gp

(FE) is as folloivrs:

Army Research Office, OCRD, Tashington 25, D. C. (•

Army Attache, American Embassy, Tokyo, Japan (1)

U.S. Army Medical R & D Command (4)

ASTTA

Office of Primary Scientific Liaison (1)

O

Offices of' Scientific Cognizance ( /)

II

EvýIdences are pre-aented that Iyno~enization is af fected- by vir-11gti~e+ic oro-perties ý.j by th- 'eec of the -enome o" a o&Cono. nLz--related 'phage -Ctlihe of lysogeny of phage i3 s attri-buted '-o viral g:eret~c propezrties. ly-uoigenization wi~tj c-5 *ic inter-fered by i~rey~rq.bafe J5btt not b.-ýr ohage F-15. Prop)%a. e eptablim',meint of ph~age g73 41 1 -'c-j e~~riprse in cells A(6-9 ) but not _'n

e~l -t L'w ri.- phge A] .is iriterfc-csi wi9 pr op'iageestablis~hnent ir cells 4 c~ .hj.1E, t.ot at hi~rh m -o.i.. 2ven at lowM-O.i- C15 EA(u3 4 )J estatriishe--s lyso~eny in A(C) 4 ) without irterferen::e.

Thr L no nerfernence .'n idifeCt4 'n of strainis A anndC EAJ and/or cl 5 EA(rLj.1 ): and in tniection of surains A and A(Owith 9341 [A] . Most probable explanaý-ion to these findings world bethat 1.1rsogeni~zation riay to affected~ b-~ host -controlled varLation (FCV).

'Phage c 1 5 gosudo C.CO~I- which do not allowmutlci.

L'f o: hare cl 5 [A3 by s~inglc_ J.ixecl. ion may allow phago mrowtho ,-h,ý,.nutlri-nfected.(Tfli~~~ activation"l = i A). ha ee c 1 5 sCL

1.s capable of contolbu~u-!ng to ~iA jn inafection of I-1 with F115 [AJ whilec~~~tsLI-l]~~ or5rns~vtd L

9 t-tkes little ol no MA effbct fo-0z 1"', P- ind(icatiing Lh~t t~he helper effect is influenced by minor dififerpnce(s) in DNIA st;-uc~uro!. Factors) responsible for HCV proved cchoe thermolibile, hisi<ly opecifi'c, aný( capable of i~nteracting' with ini-taJ"t >-W Th

'The Lt:~lizati on ol che~rical. mtutangens resulta-d in isolation of',o'jr.m)-at'ure sronsitivc. inutants rl'ltý *md others.

inoleiso ' lipo. iivsacchn.-rido&ý wi,.th specificity of' nix~pse par ±d ýrom Salwonell.a setnfto.;:b,ýr_ 37Aal ývhowed Lhat g<,ýIaIctose, rnjir'nose) -n' 1 rlaninoce I-.L x, -11- in colusti 'tui~ nts.

FINAL REM.RT NOQ. 1 ON

CONTRACT NO PA 92-55?-FEC.357?5

INCLUSIVE DATES 20 January 1962 TO 19 January,.196_

SUBJECT OF INVESTIGATION

Genetic, serologic,and

biochemical studieson

viral infection and lysogenization

RESPONSIBLE INVESTIGATOR

Dr. Hisao UETAKE

Professor of Microbiology

Theprtment of Microbiology

Sapporo Medical CollegeSapporo, Japan

CONTENTS

T. Text1. Purpose of researches 12. Analysis of factors affectingr cn the process of lysogeni-

zation 1a. Materials and methods 34 2b. Lysogenization with phage c of cells without carry-

ing prophage c15 2c. Tnte--ference with establishment of lysogeny of phage

cl1 in cells carrying prophage c 3 4 3,d. Test for the hypothesis (1) 3',e. Tests for the hypotheses (2) and (3) 4f. Effect of Multiple infection upon establishment of

lysogeny of phage cl 5 EA] in cells A(c3 4) 4g. Lysogeniption of strains A and A(g3 41 ) with c [A]

and/orc ')[A(g 3 4 1 )] 4h. lysogenization of strains A and A(cy) with g3 4 1 [A] 4i. Infection of A(c3 4 ) with phage g 3 4 1 5i. Discussion 5k. Conclusions 6

3. Studies on the mechanism of host-controlled variation (HCV) 7a. Materials and methodsb. Dependence on propagating strains of BOPc. Phage adsorption and injection of phage genetic materi-

als into the host cell 8d. Cell killing and lysogenization 8e. Serolo-ical properties of the restricted phage 8f. Mixed infection of cells I-1 with cl5 [A] and c 1 5 vir[A] 9g. Physiological states of cells 9

.h. Effect on EOP of multiple infection 9i. Cell killing and lysogenization after multiple infection 9j. Effect of UV-inactivated phage 9k. Contribution of c ts[A] to MA 10

1I Infection of A(c3 4 ) with c 1 5 [A] l1Pm, Rescue of Ol5 vir[A] by superinfecting Cl 5 ts[AJ 10

n, Effects on BOP of heating calls in HCV 10o. Effects on EOP of multiple infection of A with cl5 Il-] 11

p. Summary and QQnclusigns 15 124. tolaftion of mutants of the converting phage c 12

a. Mat'ials and methods ý12b. Search fo r plaque., typa.mutants 12

"c- Isolation of temperature sensitive mutants with ethylmeti'ane sulfonate 15 12

d. Properties of mutant phage c ts 15 13e. Induction of mutations in phage C with nitrous acid,

5-bromouraci]., and 5-bromodeoxyuridine 14f. Summary 14

5. Chemical Structure of somatic antigens and phage recep-tors in bacterial strains 14a. Materials and methods 15b. Extraction and purification of antigenic polysaccharide 15

iii

c. Separation cf constituent o&igosaccharides from partialacid hydro].ysate of nucleic acid-free LPs 15

6. List of papers submitted to publication under the supportfrom the Contract NO DA-92-557-FEC-35775 16

II. List of references 17

III. Appendixes1. Appendix "Al : Tables

a. Table 1 see Text 2-e,f,j 21'b. Table 2 j 21c. Table 3 see Text 3--b 22d. Table 4 b 22e. Table 5 c 23f. Table 6 d 23g. Table 7 f 24

2. APpendix 1311 : Illustrationsa. 7ig. 1 see Text 2-c,_ 25b. 7i," 2 f 26c Fit. 3 g 27d. F:,. 4 h 28e. Fig. 5 h 29f Wig. 6 see Text 3-h 30g, Fig. 7 h 31h. rig. 8 i 32i. Fig. 9 i 33

1ig. 10 M 34k. Vii. 11 a 351. Fig. 12 see Text 5--c 36

iv

j. Purpose ef researches

In the process of viral infection and lysogenization, there aremany phenomena, of which mechanisms remain to be elucidated. Thesemay be analysed genetically, serologically, and biochemically.

We have been working on antigenic conversion, viral infection, andlysogenic conversion since 1949, and main important findings are assummarized in application paper. Our system of research consists ofSalmonella strains of group E and temperate convertinm phages carryinggenes responsible for the synthesis of somatic antigens 15 and/or 34of host bacterial cells.

Basing upon the findings obtained, and by making use of the lysoge-nic conversion system mentioned above, researches are being carriedout in order to elucidate genetic and chemical factors involved in theprocess of viral infection and in the process of lysogenization. Andin practice the following research works have been designed to be car-ried out.

a. Analysis of factors affecting on the process of 1 soenization

b. Studies on enzymic activities in cells destined to lysogeny

a fter•hage infection

c. Studies on the mechanism of host-controlled variation (HCV)

And during past one year, the following results were obtained.

2. Analysis of factors affecting on the process of lysogenization

The process of lysogenization is different between phages c15 andc34; it takes approximately 3 h urs for c 1 5 to establish lyso'ýeny(Uetake et al., 1958), while c3& usually becomes a prophage very rapid-ly after infection (Ha-iwara, 1959a, 1959b; Uetake and Hagiwara, 1959).Some phages resemble to phage c 1 5 and. others to phage C3 4 in establish-ing lysogeny (see Bertani, 1958).

However, it seems premature to conclude that the difference is dueto qenetic differ-:nce(s) between the two phages, since the host cellof c15, A, is different from that of c34, A(cl5), in that the latter

carries trophage c1 5 . Hence, the question remains to be determinedwhether or not the presence of phage genome(s) affects the process oflysogenization of a second unrelated phage. From such a viewpoint,effects of the presence of phage genome(s) upon the process of 1Yrsoge-nization of a second unrelated phage were studied experimentally. AndL1,:i-s has eventually elucidated a close relationship between lysogeni-zation and host-controlled variation as described below.

a. Materials and Methods

(1) Bacterial strains: Salmonella anatum 293 (=A) and its lyso-genic derivatives, A(cl 5 ), A(c3 4 ), A(g 3 4 ), and A(cy) havebeen described elsewhere (Uchida et al., 1956; Uetake et al.,1958; Uetake and Uchida, 1959; Uetake and Hariwara, 1960a,1960b, 1961).

(2) Phase strains: Temperate phages J5, c34, and g,41 have beend,-1scribedTfUe'take et al., 1955; Uetake, 1956; Na alawa,1957a, 1957b, 1959; Uchida et al., 1956). Phage c 5 which ispropagated on strain A or strain A(g...) is described as•15[Al and or e1 5 [A(g 3 4 1 )], resnective y.

(3) Antiphage sera have also been described (Uetake et al., 1958).The neutralization velocity constants of anti-c1 5 , anti-'•41,and anti-c34 sera are 120 min-m , 180 minm , and 130 min

respective3y.

(4) Follow-up of the process of lysogenization: Growing cellswere infected with phage, and thereafter surviving cells wereplated out on agar plates at appropriate time intervals. Re-sulting colonies were tested for phage production by replica-ting onto plates seeded with indicator cells (Ledeberg andLederberg, 1952; Uetake et al., 1958; Hagiwara, 1959a). Thistype of experiments were carried out in various combinations

of phage and bacterial strains and at various multiplicitiesof infection.

b. Lys. enization with phage c34 of cells without carryi

phae--C15 .- -o tesl: the effects of prophage--5 c on. ysogenization pro-cess with c3 , cells A without carrying prophage cl were infedted

with C3 To carrY out these experiments special design of experimentwas inade because colls A do not adsorb phage c3 4 at all.

When cells A aro infected with c15 at a mul.tiplicity of infection(m.o.i.) of about *, cells which survive infection segregate c 1 5 -car-rier and non-carri,:r cells in their progeny. At about 3 hours afterinfection, about 80% of cells in the culture possess receptors for c34without carryinR J1 5 (Uetake et al., 1958). Therefore by infectingthe non-carrier progenies with receptors for C34 by c34, cells A might

probably be lysocTenized with 034 without carrying c 1 5 (Uetake and

Hagiwara, 1960a, 1960b, 1961). In practice, this was carried out asfollows.

Growing cells of A were infected with c15 at m.o.i. of 7.9. After10 minute adsorption, the mixture received anti-.phngo serum to removeunadsorbed phages. After another 10 minutes, a dilution was made intobroth containing anti-c 1 5 serum, and kept at 37°C without aeration.After 3 hours, cells were chilled, collecte• by centrifugation, re-suspended in broth at a density of about 100 cells per ml, and infected

2

with c34 at m.o.i of 1.8. After 10 .-inute adsorption and another 10minute treatment with anti.-c34 seruas, a dilution was made into brothcontaining anti-c34 serum, and keot at 370 C for 6 hours, during whichtime cells werýe plated out on aga.- plates for viable count and result-ing colonies were rep]ictted on both plates seeded with cells A andthose with cells A(c15) to test for )roduction of phage c15 and c34.respectively.

34Approximately 50% of cells survived infection with c . Among

survivors about 80% were c3 4 -producers at first, 70% after ,60 minutes,60% after 90 minutes and thereafter, indicating that c3 4 establisheslysogeny within 60-90 minutes, This result is consistent with thosein experiments in which strain A(clS) was infected with c34 (Hagiwara,1959a, 1959b, 1959c; Uetake and Hagiwara, 1959). Therefore, it canbe concluded that phage c establishes lysogeny soon after infection,irrespective of the presence of prophage c15. In other words, itindicates that rapid establishment of lysogeny in c34-infected cellsis mainly attributed to genetic properties of phage 034

c. Interference with establishmer.b of_ geny of 1ha5e c.. incells carrying proph5ge c5t To test effects of prophage c-lkon lyso-genization with phage c---'cells A(34),; which were obtained as des.-cribed above (Uetake and Hagiwara, 19 6 0a, 1960b, 1961), were infectedwith clS[A] at m.o.f, of 6-9 and the process of lysogenization wasfollowed up as described. The results of one of these experiments isshown in Fig. 1.

In strain A as control, lysoerenization was established in about 3hours after infection, cons:i.stent with the experiments reported byUetake et al. (1958), but in cells AMc 3 4), lysogenization with c L[A]

was so difficult that survivinL.: cells continued to segregate c 1 5..car_rier and non-carrier cells in their i-orogeny even until 6 hours afterinfection. This seems to indicate that the existence of pro-phage c3in cells A interfers with phage c-15 to establish lysogeny, and forexplanation following possibilities may be considered: (1) Somemetabolic changes in cells due to the presence of ,34 may be responsi-ble; (2) efficiency of piating (M)P) of C1 5 A] on A(034) is about 1/3of that on A, suggesting bost-controlled variation (HCV) (Uctake etal. in press), and the HCV may possibly be correlated with the diffi-culty of lysogenization; (3) steric hindrance due to the attachmentof •4•genome to bacterial chromosome may be responsible.

d. Test for the hypothesis (1). To test the hypothosis (1), cellsof A were infected witý'h" 1 and 30 minutes later superinfected withc3 4 . In this case, phage lS1 estabJlisped itself as a prophage in 180minutes as seen in the case without c3 -superinfection (UWetake et kl.,1958) ±while phage c34 had difficulty in establishing lysoreny andnon-c~+ carrying progeny cells were segregated out from c3,infectedcells for longer than 3 hours.

3

It can be said, from the aboves that lysogenization with J15 isremarkably affected by prophage c34 but not by c34 when it is in pre-lysogenic state, and, in addition, that phage C34 becomes a prophagevery easily and rapidly in cells carrying prophage c15 and/or no c 15,while lysogenization with 04 is interfered in freshly c 1 5 -infectedcells in which c15 is still in prelysogenic state. And it seems un-likely that the metabolic changes due to genetic functions of phagec34 greatly arfect the lysogenization process with c15.

e. Tests for the hyp theses (2) and (3). To test the hypotheses(2)-and(3), cells A( c;) were infected with c15, which were pro a-gated on cells of A(c3 4 ), at m.o.i. of about 5-1.0, instead of Tpropagated on mlls A. As shown in Table 1, c15 established lysogenyin about 3 hours as phages c1 5 [A] do in infection of cells A, indicat-ing that lysogenization is affected by phage propagating strains, i.e.probably host-controlled variation. This also indicates, on the otherhand, that the interference with prophage establishment of Ol5 [A] inA(c34) is not attributed to the steric hindrance due to the presenceof prophage c34, excluding the possibility of th'e hypothesis (3).

f. Effect of multiple infection upon establishment of lysogeny ofphage £U[A] in cells of A(c•4). As described in Section 3, experi-ments of HCV which were carried out in parallel with these experimentsshowed that multiple infection with restricted phages may lead to,,age multiplication even in cells which do not allow phage growthwhen singly infected.

Basing upon these findings, effect of multiple infection on estab-lishment of lysogeny of c1 5 [A] in cells of A(c3 4 ) was tested. Asshown in Table 1 and Fig. 2, when cells of A(c3 4 ) were infected with

at high m.o.i. (20.0; 24.2), establishment of prophage stateof c15 was completed in about 3 hours after infection without inter-ference. This also favors the hypothesis (2).

•. Lysogenization of strains A and A(g9 4 1 ) with J1 5 [A] and/or1C 5 [A(g__)J There is no HCV when phage C15 is propagated on A and

on A(g3 4 1 ). If HCV were responsible for the interference with pro-phage establishment, no interference would be expected in this system.And this proved to be true in the following experiments.

Each of bacterial strains was infected with either 15 [A] or J15[A(g 3 41)] at m.o.i. of about 5, and the process of lysogenization withc15 was followed up with results shown in Figs. 1 and 3.

Phage l5E[AJ lysogenizes strain A in 3 hours and strain A(g 3 4 1 ) in4 hours, whereas cl 5 [A(g 3 4 1 )] lysogenizes strain A in 4 hours andstrain A(g 3 41 ) in 3 hours. However, the difference between 3 and 4hours seems too small to be regarded as significant.

h. Lysogenizatio of strains A and A(cy) with g 3 4 1[A]. There is

4

no HCV either in phage g3 4]. when plated on A and on A(cY). and simi-lar result to the above E was obtained in this system, too.

In either bacterial strain, phage infection was carried out at,n.o.i. of 0.8-4.8. As shown in Figs. 4 and 5, in either strain g3 4 1[A]established lysogeny in 60-90 minutes after infection. There was nodifference between the two strair- -'niatinq that prophage CY has noeffect on lysogenization with g3 4 1 -

i. Infection of A(c34) with hae 934 1 . The strain A(c34 ) is resis-ta nt to infection with phage C 41, a vI.rulent mutant of g3 4 1 , althoughit adsorbs C341 to a considerable extent (Uetake and Ha.aiwara, 1960a,1960b, 1961). The same was true in infection with g 3 4 1 . Adsorption

f g34 1 to A(c3 4 ) is irreversible but there is neither killing of cellsto a demonstrable extent nor lysogenization.

,. Discussion. From the above experiments it sbould be pointed outthat the presence of phage genome(s) may affect on the process of lyso-genization of a second unrelated phage. An extreme case is a completesuppression of multiplication and lysogenization of phage g 3 4 1 inA (c4), but more interesting is a relationship between 415 and c3 4 .The experiments described in b and c indicate that the rapid establish-ment of the prophage state of-phage-c3 4 is attributed to genetic pro-perties of the phage, being not correlated to the presence of the pro-phage c15 in cells A. On the other hand these experiments also indi-cate that the effect of c15 genome on c34 varies with the state of cl5

genome. When it is in prelysogenic state, c34 is excluded.

Phage J15 is interfered with establishment of lysogeny in cellscarryin.g 0po•!• •3 4 as shown in experiments c and the mechanism is.crded to be due to HCV ý ýhown in d, e, f, S and h. Efficienciesof Plating of phages 615 and g341 propagated on various strains, aresummarized in Table 2. Comparing Table 2 with the above experimentalresults, one can notice a remarkable correlation between HCV and inter-ference with lysogenization. The interference is observed only in com-bination of phage and bacterial strains in which HCV is also observvd.And even in such a combination, infection of cells with restrictedphages at high m.o.i. may lead to prophage establishment without inter-ference (, This is consistent with a phenomenon "Multiplicity activa-tion (MAT" observed in experiments on the mechanism of HCV. (see Sec-tion 3), favoring the explanation proposed.

On the one hand. phages slS[A(c34 )] show hi'-her EOP on cells Athan on cells A(c34 ), and on the other hand, lysogenization of cellsA with cl [A(c3 4 )] is completed in 3 hours even at low m.o.i. (e), asseen in infection of cells A with cl 5 [A]. This also favors our hypo-

thesis.

However, there still remains a following possibility to be exclur1irls.The higher the m.o.i., the more fiequont would be th. co]lii:.s be-

5

tween phage DNA and bacterial chromosome, leading to more rapid lyso-genization. But this should not be the case, because in infection ofcells A with cl5EAL 1vsogenization is completed in 3 hours even at aslow m.o.i, as 1 (Tabl, 2,.

The continuation for a lonýu time of seqre ation of sensitive pro-geny cells from cl 5 H] -.infected cells of A7(4, suggests that injectedphage genome persists in the cytoplasm without replication. And both"i in'ection of A with r15L1A] and in infection of A(c34 ) with c1 5

EA(c_34], phao:e P'enome seems to persist in preprophage state for sometime before establishing prophage state. Nevertheless the preprophagestate persists for a longer time in the former case than in the latter.What is the explanationr:

If the structure of DNA of c15A] is identical with that of J5[A(c34)], the aboves should not be expected. These may rather suggestthat DNA of c-lS[AJ and that of ci 5 [A(c3 4 )] have different structures,each of which is fit fox, attaching to bacterial chromosome of A orA(634), respectively ITf so, such structures must be produced aftermultiplication of rl15 on A and or A(e34), respectively. Then, if DNAof E1 5[A] is reulicated following infection of A(c34)', de novo repli-cate• DNA must have a structure fit for attaching to chromosome ofA(M3V). ) igh multiplicity infection (f)may result in replication ofinjected phage DNA and eventually lead to unimpaired establishment oflysogeny. The fact that c 5 [A(034)1 has a higher EOP on A than A(c3 4 )may indicate that Cl5[A(C34)] replicates and multiplies more easily inA thar in A(34)., and in this case lysogenization of A with cl5'A]proceeds without retardation.

In short, by assuming that the structure of phage DNA is modifiedu. ._-e extent bh the host cell on which it is propagated and thismodification reflects on the ability to replicate-in bacterial strainsto he infected, and that when not easily replicated, infecting phageDNA maintains its structure as it is and has difficulty to establisha prophage state, while when easily replicated, do novo replicated DNAtakes a structure fit t6 a new host cell, having no difficulty inestablishing lysogeny, all of the above data can be explained clearlyand in addition these assumptions are compatible with our findings inexperiments on the mechanism of HCV (see Section 3) and recent reportmon phage X (Arber and Dussoix, 1962; Dussoix and Arber, 1962). Andune replication of phage DNA before establishment of prophage statehas been suggested in phages ýk (Stent and Fuerst, 1956), P22 (Luria etal., 1958), and P2 (Bertani, 1962). In phage P2 lysogenization is notaffected by moo.i. and may be multiple even after single infection,and this may be easily explained by assuming that P2 DNA replicatesvery easily and immediately after injection (Bertani, 1962).

k. Conclilsions. Phge C1 5[AJ est&-'11hes lysogeny in about 3 hours

in cells A, wherea. ic uic , •• ..... than 6 rniirs in cells A(c34 ) wheninfected at low m.o~i,. On the other hand, lysc -iJi' ;b 415

6

can be completed in 3 hours even in Sels A(c)34 when infected withcl 5 [A] at high m.o.i. and/or with ClS[A(c, )] at low m.o.i.,

In contrast, there is no interference with lysogenization in in-fection of strains A and A(-j34) with c15 [Aj and/or c1 5 [A(g 3 41 )] andin infection of strains A an. A(c ) with g3 4 1 [A].

Most probable explanation to these findings would be that the pro-cess of lysogenization may be affected by host-controlled variationof the infecting phage of which the mechanism was discussed in detail.

In addition, it •as also been shown that the rapid establishmentof lysogeny with c3 4 is attributed to genetic properties of phage c3 4 .

3- Studies on the mechanism of host-controlled variation (HCV)Phages c1 5 and c34 go under HCV. HCV is a well-known phenomenon

but its exact mechanism is still obscure (Felix and Anderson, 1951;Anderson and Felix, 1952, 1953; Luria and Human, 1952; Ralston andKrueger, 1952; Bertani and Weigle, 1953; Luria, 1953; Garen and Zinder,1955; Maio and Zahler, 1958; Arber and Lataste-Dorolle, 1961; Chris-tensen, 1961, 1962; Drexler and Christensen, 1961; Arber and Dussoix,1962; Dussoix and Arber, 1962). Basing upon suggestions which we gotrecently, the analysis of the mechanism has been carried out.

a. Materials and Methods

(1) Bacterial strains: Salmonella anatum 293 (=A), and its deri-vative A(c-j5 are described, in Section 2. Salmonella butantan(=I-1) is an international standard strain of group E1 Salmo-nella.

(Ž Phage strains: Temperate phage c5, its virulent mutant•7r (Uptake et al., 1958) and its temperature sensitive

mutant Clts (see Section 4) were employed.

(3) Anti-c5 serum is described in Section 2.

(4) Serol.ogical tests: Antibacterial antisera, anti-lO and anti-15 sera were prepared by routine procedures, and slide andtube agglutination tests were done as usual (Kauffmanni 1954)..

(5) Tests for 1oyso-enicity was done by replica plating (Lederbergand Lederberg, 1952; Uetake et al., 1958; Hagiwara, 1959a).

(6) Ultraviolet light irradiation: Growing cells from aerated;oth culture were restspcendcf in uhosphate buffer (pH 7.0;

M/150). Samples were irradiated in Petri dishes at 80 cmdistance from a Termicidal lamp (Matsuda GR 1510, 15W).

7

b. Dependence on propagating strains of EOP. Phage c propagatedon -cells of A sh;oow-sTEOP oof *P- 2 2 on cells of I-1, while C 5 pro-a-gated on cells I-1 shows TOP of lO-4 on cells A (Table 3). Phagescontained in a single plaque on one strain shows similarly lower MOPon the other strain. Single cycle growth of the phage on one strainresults in production of phages with lower EOP on the other strain(Table 4).

This reciprocal shifting of EOP proved experimentally to be '1, -HCV and not to host range mutation by the following experim•._ts.

c. Phage adsorption and injection of p_ hae genetic materials intothe-host cell. Regardless of the difference in EOP, phages can beadsorbed well even onto cells with lower EOP as shown in Table 3.

• a 15Since it has been shown that when cells A are infected with c ,

de novo synthesis of somatic antigen 15 on phage-infected cells canbe demonstrated several minutes after infection (Uetake et al., 1958),injection of phage genetic material into cells can be tested by denovo synthesis of phage-cortro1 1 -1 ens.

Growing cells of A and of I-1 were infected with either cl 5 [A] orc 1 5 [I-1] at m.o.i. of 5-10, and 25 and 50 minutes thereafter, aliquotswere removed and heated at 100 0 C for 4 minutes. After centrifugafi-ncells were resuspended in 0.2% saline and tested for antigen 15 bytube agglutination. As shown in Table 5, in any of combinations ofbacterial and phage strains de novo synthesis of somatic antigen 15was demonstrated, indicating the injection of phage DNA into bacterialcells.

d. Cell killing and lysoRenization. Cells A and I-1 were infectedwith OI1A] at m.o.i. of about 9. At. i'ý rhinute adsorption and ano-ther 10 minute treatment with anti-c1 5 serum, cells were plated outonto agar plates for viable count, and resulting colonies were tested

"". _.,nicity by replica plating.

As shown in Table 6, more cells were killed in 1-1 (92.6%) than inA (66.4%), whereas lysogenics among survivors were 99.8% in A and30% in I-1.

Similar experiments were made in infection of cells A and I-1 withc 1 5 [I-l1'. Surviving cells were 100% in A and 15% in I-1. Lysogenicsamong survivors were 60.5% in I-1 but not to a demonstrable ixtent inA (Table 6).

These indicate that in infection with restricted phages infectionleading to abortion and curing is very frequent.

e. Serological properties of the restricted Dhage. Phage 15[I-1]

is neutralized to the same extent as C-'LAJ by anti ,-,m ---- nP-edagainst cI 5 [A].

8

f. Mixed infection of cells I-I with clA) and clbvirEA]. Whencells I-1 were mixeýdly 'infected with &LAJi and 5virt-, umber of

mixed yielders was far mori than that calculated under the assumptionthat each of cl 5 [A] and cl vir[A] contains a host range mutant capableof attacking cells I-1 at a proportion of about 10-2 and 2xlO 2 , res-pectively, favoring our hypothesis that the difference in EOP shouldnot be attributed to h mutant but to HCV (Table 7).

. hysiological states of cells. UV irradiation of cells did notaffect EOP at all. However, when cells of I-1, which were grown underaerauion for about 5 hoors after entering stationary phase of growth,were infected with restricted phage J 5 [A] at m.o.i. of 0.02, EOP in-creased to 10-1, showing the dependence of EOP on the physiPlogicalstates of bacterial cells (see also MA in synthetic medium),

h. Effect on BOP of multiple infection. DurinT the course of theabove experiments, the data were found which suggest that multipleinfection may lead to phage multiplication even in cells which do notallow phage growth when singly infec-.:ed. This was studied in detail.

Cells of I-1 were infected with J 5I[A] at various m.o.i., rangingfrom 0.03 to 16.5, and numbers of infective centers and killed cellswere examined.

In experiments in nutrient broth, as shown in Fig. 6, proportion ofplaque formers to phage-infected cells increases with increasingm.o.i., reaching 1.0 at m.o.i. of about 10. The data in Fig. 6 .alsoindicate that about 5 particles per cell are required for phage mul-tiplication. Essentially the same results were also obtained in ex-periments carried out in synthetic medium, and the only difference wasin that fewer particles were sufficient to induce phage multiplica-tion (Fig. 7).

i. Cell killing and lysogenization after multiple infection. Incomplete paralell to the above "Multiplicity activation", it was alsoshown that number of cells I-! killed by cl [A] increases with in-

creasing m.o.i., reaching to more than 99% at m.o.i. of 10 or more(Fig. 8).

The proportion of lysogenics among surviving cells also increaseswith increasing m.o.i. as shown in Fig. 9. But the proportion oflysogenic cells to input cells were not variable, ranging from 1.6%to 2.4%.

These findings indicate that it is the number of killed or lysingcells that increases with increasing m.o.i.. In other words, multipleinfection may lead to phage multiplicotion and lysis in cells which donot allow phage growth when singly infected.

1. Effect of UV-inactivatedytagýe. Multiplicity reactivation is awell-known phenomenon in UV inactivated phages (Luria, 1947; Luriaand Dulbecco, 1949; Harm, 3.956; Tesuiran and Ozaki, 1957; Epstein, 1958;

9

Baricelli, 1960). The question wa. to be determined if a similarmechanism is involved in MA. Phags which were inactivated to 0.06%survival by UV irradiation were tested for their ability to contributeto MA, but positive results were obtained in neither cHEAJ norClB[I-11. So it seemed likely that MA is different from multiplicityreactivation in its mechanism.

On the other hand, this experiment led us to the suspision thatthe spacial configuration of UV-inactivated phage DNA may be differentfrom..that of the native one and it is responsible for the inability tocontribute to MA. To test this, c' 5ts was employed. Phage cl 5 ts isable to multiply at 250C but not at 37?C.

k. Contribution of c15ts[A3 to MA. When growing cells of 1-1 weremixedly infected at 37 with c--Aj and ..15ts[AJ at m.o.i. of about0.015 and 9 respectively, 99% of c 1B[A]-infected cells gave rise toplaques, while without adding cl 5 ts[AJ only 1% of cl 5 [A]-i ectedcells were plaque formers. And in addition, when phages c ts[I-1]were employed in place of c1 5 [A] in the above experiment, about 35%of c15[A]-infected cells were plaque formers, probably being due togenetic recombination.

These results suggest that to give rise to MA, phage DNA should beintact and propagating strain-specific.

'34 15 11. Infection of A(c ) with c 5[A]. EOP of clB[A] on cells of

A(C 34 )js about 1/3 of that on A. Cells A(c3 4 ) were infected withC1 5[AJ at m.o.i. of 5-10. Surviving cells were plated out 20-30minutes after infection and resulting colonies were tested for phageproduction by replica plating. As shown in Table 1, the proportion oflysogenic colonies among survivors was smaller than that calculatedfrom the number of cells infected Lheoretically. This suggests thatinjected DNA of phage c1 5 [A] may be destroyed some time after infec-tion. Similar suggestion comes from the fact that 100% of cells Arecover from infection with cl3[I-1] (Table 6).

m. Rescue of cl 5 vir[A] bJysuperinfectinp-c15 tsEA]. If injectedphage DNA were destroyed, it would be expected that the longer thetime elapses before adding a second rescuing phage, the lower theefficiency of rescuing a primary restricted phage would be.

Growing cells of I-1 were infected with c5 virEA3 at low m.o.i.and at appropriate time intervals tbereafter superinfected withcl 5 ts[A] at high m.o.i. to see the efficiency of rescuing c vir. Asseen in Fig. 10, the results showed that the efficiency of rescuingCl 5vir decreases with prolongation of time intervals between primaryand secondary infections, suggesting the destruction of injected DNAof primary phage cl 5 vir[A].

n. Effects on EOP of heating ce2ll in HCV. What factor is res-

ponsible for the destruction of DNA of the restricted phage? To look

10

into the nature of the responsib]ý factor, heat effect was tested.

Growing cells of I-1 were heated in water bath 0 for 30 seconds --5 minutes at temperatures ranging from 44 C to 55 Cl transfered backto 37 C, kept for 1-2 minutes, and infected with c [A] at low m.g.i..As shown in Fig. 11, EOP increased remarkably by heating at 49-50 Cfor 2 minutes. This suggests that the responsible factor is thermo-labile.

o. Effects on E0P of multiple infection of A with c1 5 :I-1. Ininfection of cells A with c-lJ[I-1, MA effect was not so remarkable.At m.o.i. of about 50, plaque formers increased by 10 times. But evenunder this condition, the proportion of plaque formers to infectedcells is only 10-3.

1. .Summary and Conclusions. Under the conditions employed, phageC "propagated on one strain shows lower BOP on the other, and thisreciprocal shifting of EOP proved to be due to HCV by experiments des-cribed in c through o.

cl [A) and cl5[I-1] are serologically indistinguishable, By infec-tion phage genetic material is injected: into the host cell regardlessof BOP'. Only the fate of injected DNA seems to be different and ab-normal when injected into cells on which phage shows lower EOP. Amongthe data presented, most remarkable is MA. The results showed clearlythat cells of I-1 which do not allow multiplication of c 5lA] by sin-gle infection may allow phage multiplication when multiply infected.As for the mechanism of MA, following possibilities may be considered:(1) Genetic cooperation or complementation; (2) genetic recombination;(3) removal of inhibitors of phage multiplication.

cl5ts is characteristic in that it is unable to multiply at 37 C."veruheless cl ts[A] is capable of contributing to MA in infection

of I-1 with Cl5 [A] while el 5 ts[I-l] is incapable (k). This favors the

hypothesis (3) but not (1) or (2).

On the other hand, the fact that UV-inactivated JlS[A] is inca ab].of contributing to MA (1) suggests that the intact structure of c15[A]DNA is necessary for MA. And the fact thafv1cl5[I-l] or cl 5 tsEI-l]takes little or no MA effect for cl 5 [AJ, suggests that the helpereffect is controlled by only a minor difference in DNA structure.

In addition, the specific destruction of DNA of restricted phageis suggested (1), and the factor(s) responsible for this destructioncan be overcome by multiple infection (Section 2-f) and is thermolabile(n)._

Summing up the above findings, the factor(s) responsible for HCV-_, 'n be thermolabile, highly specific, capable in interactin7 with

intact phage DNA, and present in a small amount in a cell. If thefactor(s) with such zogper 4-• c .. e ssumed to be a DNase, all the

11

I

data could be explained quite easily. The experiments along theselines are under way, and some of preliminary experiments have shownlthe results favoring the hypothesis but the details will be describedin future reports. Recent papers on phage ; (Arber and Dussoix, 1962;Dussoix and Arber, 1962) seem to be along similar line.

4. Isolation of mutants of the converting phage c15

For carrying out experiments on HCV and others, it became desirable4_0 •'olate mutants of the phage c15, since a few mutants with abnormalconverting properties were on hand but they were found not to be ade-quate for these purposes.

a, Materials and Methods

(1) Bacterial and phage strains, anti-c15 serum, antibacterialantiserum, anti-10 and anti-15 sera are described in Sections2 and 3.

(2) Physiological saline, phosphate buffer, and M-9 salt solu-tions were sterilized before use.

(3) Procedures for slide and tube agglutination tests, and testfor lysogenicity are also described in Sections 2 and 3.

(4) Phage adsor•tion, neutralization, single-step growth experi-ment were done by routine procedures (Adams, .1959).

(5) Bresch's medium (Bresch, 1952; Bresch und Trautner, 1956) wasemployed for selecting plaque type mutants.

(6) Mutagenic substances. Ethyl methane sulfonate (Bautz andFreese, 1960; Strauss and Okubo, 1960; Green and Krieg, 1961;Strauss, 1962), nitrous acid (Gierer and Mundry, 1958; Boeye,1959; Tessman, 1959; Vielmetter und Wieder, 1959; Sinsheir- ,'q60; Vielmetter and Schuster, 1960; Bautz-Freese and Freese,WU1l; Granoff, 1961; Baylor and Mahler, 1962), 5-bromouracil

(Brenner et al., 1959; Litman and Pardee, 1956, 1959, 1960a,1960b), and 5-bromodeoxyuridine (Freese, 1959a, 1959b; Lawleyand Brookes, 1962) were employed as chemical mutagens.

b. Search for plaque type mutants. Attempts were made tq isolateplaque type mutants of the phage C'I by using Bresch's medium, addingas one of inFradients one of carbohydrates such as dulcitol, i-inosi-+,I, inulin, maltose, d-mannose, raffinose, rhamnose, trehalose, d-xylose, and 1-arabinose. A few plaque type mutants were isol'ited, butthe difference from each other and from wild type of plaque was notremarkable that they were not useful in practice.

c. Isolation of temperature sensitive mutants with ethy. methane

12

sulfonate. Phages c 15[A] were exposed to ethyl methane sulfonate(0.4m; O6m; IM) in broth and or in M-9 salt solution at 250 C for 4hours. At a concentration of O,4M for 4 hours, about 95% of phageswese inactivated. Surviving phages were propagated on cells A at25 C for 3 hours for single-step growth. Released phages were platedat 250C and each of resulting isolated plaques was transfered in du-plicate onto plates seeded with cells A, of which one was incubatedat 370C and the other at 25°g, and phage strains which gave rise toplaque at 2ý C but not at 37 C were selected out. Only two suchstrains (Cl ts) were isolated among 9080 plaques examined.

.d. Properties of mutant phage F ts. J ts is able to multiply at

25°- but not at 37-C. At 37C c--ts-infected cells are either killedwithout lysis and phage release, or lysogenized. The proportion ofsurviving cells among el 5 ts-infected cells is largsr at 250C than at37 C when compared at the same m.o.i.. Even at 37 C cells survivinginfection segregate c 1 5 ts-carrier and non-carrier cells in their pro-geny for about 3 hours after infection, by which time the establish-ment of lysogeny is completed, as observed in the case of infectionof cells A with wild type of phage c]-5 ts+ (see Section 2).

When cl5ts-infect 8 d cells, which are to be killed and kept at 370C,are transfered to 25 C before the end of rise period, phage multipli-cation may be restored. This restoration phenomenon is now underfurther investigation and the details will be described in future re-ports.

Whnsord*15When stored a n broth and/or physiological saline, phage c ts is

as stable as _ ýts+. In broth inactipatisn was not observed to ademonstrable extent for 6 months at 0 C-5 C. It is resistant to chlo-roform as the wild type is. Plaques produced by c 1 5 ts are indistin-guishable from those by c 1lts+. The adsorption rate constant of c1 5 tponto cells A is 2.5xlO- 9 ml min- 1 at 370C and ,.3x10- 9ml min-' at 250C.Cl5ts is neutralized to the same extent as Jl ts+ by antiphage serumagainst cl5ts+. Single-step growth experiments at 25 C showed thatthe latent period is about 80 minutes, the rise period 100-110 minutes,and the average burst size about 600.

The spontaneous back mutant was found 4/105 in one strain and 3/104in the other.

The antigen converting property of c 5 ts is the same as that ofC1 5 ts+. Strain A(cl 5 ts) was obtained by lysogenizing strain A withphage c 1 5 ts. By both slide an• tube agglutination tests, and by ag-glutinin absorption test, A (c1~ts) was confirmed to possess 4ntigbns3.15 as somatic antigens, the same structure as that of A(c _1ts ) Noantigenic difference was found between cells A(c1 5 ts) grown at 250Cand those grown at 37 C.

cl 5 ts has also Droved to be ver T useful for the researches on HCVas al.read-, described in Section 3.

e. Induction of mutations in phage c 1 5 with nitrous acid, 5-bromo.uracil, and 5-bromodeoxyuridine. By zreating phage C' with nitrousacid (5x1_0_M), 5-bromouracil (50 -g/ml) and or 5-bromodeoxyuridine(250 1,.g/ml), mutants, especially of plaque type, have been searchedfor. Several strains which give rise to larger plaques than that bywild type of c15 have been isolated and their properties are now unde.investigation in detail.

f. . Search for mutants of J5, which are useful for carry-ing out experiments on HCV and others, has been attempted by usingBresh's color medium with various carbohydrates and or by chemicalmutagens.

Temperature sensitive mutant strains c 5ts were isolated aftertreating c 1 5 with ethyl methane sulfonate. Their properties are asdescribed in 4-d, among which most characteristic is their inabilityto multiply at 37 C. And on account of this special character, cl tshas provided a very useful tool for the researches into the mechanismof HCV as described in Section 3.

Several other mutants have also been isolated with the use of otherchemical mutagens.

5. Chemical structure of somatic antigens and phage receptors in bac-terial strains.

Our analysis of monosaccharide composition in specific somaticantigens of 7roup E1 (3.10), E2 (3.15), Eý ((3)(15)34), and E4 (1.3.19)revealed that they are qualitatively indistinguishable from each o-ther, showing glucose, galactose, mannose, rhamnose, xylose and gluco-sanine (Ise, 1954; Nakagawa, 1954; Sasaki, 1955, 1956a, 1956b). These,lso seemed to suggest that different specificities of somatic anti-gens might be determined by different configurations of polysaccharidechains. And further studies have been being carried out to clarifythe structures of determinant groups, in collaboration with the Massa-chusetts Institute of Technology, Cambridge, U. S. A., where we sentDr. T. Uchida, one of our collaborators. Receptors for phages J1 5 ,C, 4 1, and or c34 are associated with somatic antigens 3.10 or 3.15 buttheir exact properties remain to be determined.

Since Uchida, with collaboration of Dr. P.W. Robbins, has clarifiedthe structure of determinant groups of antigens 10, 15 and 34, ourmain concern at present has been directed toward the structure of an-tigen 7. And for this purpose, strain 87Aa', a mutant of SalmonellasenftenberT (Ise, 1954), was employed, since it possesses factor 3 a-lone, which is common to group E Salmonellas, as somatic antigen. Al-though researches are on the way and the results obtained are prelimi-Pary, the followings have been observed so far.

14

a. Materials and Methods

(1) Bacterial strains: Strains A, A(c 1 5), A(c34), and A(cl5,C34

are described in Ssction 2. ttoains 87A and 87Aa' of Salmone.-lla senftenberg were reported by Ise (1954).

(2) Bacterial. clls: Strain 8'Aa' was grown in nutrient brothenriched wit!, yeast extrac:, under aeration, and bacterialcells were harvested by Sha~rles centrifuge.

(3) Antibacterial- antiserun, anti-10 and anti-.15 sera are des-cribed in Section 3. Anti.-7Aa' 0 serum was also employed.

b. Extraction and purification of antigenic polysaccharide. Acrude lipopolysaccharide (LPs) was -1'p repa-r-'ed--fro-m-- cell--ýof-7-Aa' byr~ot phenol extraction (Westphal et al,, 1952), dialysis of the aque-ous phase, and repetitive acetone precipitation, followed by lyophili-zation. The crude LPs preparation was separated by ultracentrifugeinto sediment and supernatant, both of which contained LPs and a smallamount of protein. This protein was removed by passing through anionexchange resin column. The supernatant fraction showed a strong ab-sorption at 260 mu, indicating the presence of nucleic acid, while thesediment fraction showed no absorption at 260 mu, being free from nuc-leic acid. The nucleic acid moiety in LPs-NA was confirmed to be ofribonccleic acid type by parer chromatography and by fractionationmethod of Schmidt-Thanbauser. Among several attemptA to seParate RNAfrom LPs in LPs-RNA preparation, only alkali hydrolysis was found tobe effective. These findings are suggestive concerning the type oflinkage between LPs- and RNA-moieties in the LPs-RNA fraction. Bothfractions are reactife with anti-87Aa' 0 serum to a similar extent,containing glucose, ýalactose, mannose, rhamnose and a trace of xyloseSmonosaccharide components. An add-.tional monosaccharide component,

ribose, was found in LPs-RNA fractions.

c., Separation of constitiuent oligosaccharides from partial acidh~y~rolsate of nucleic acid-free LPs. LPs was hydrolysed partiallywith N-stlfuric acid at 100 C for 20 minutes. Hydrolysate was neu--tralized with Ba(OH) 2 1 centrifuged and concentrated for charcoal-celite chromatography., Fractionation was carried out by H2

0 -ethanol7--adient elution, and ;he sugar content in each of separated fractionswas measured by phenol-sulfuric acid (Fig. 12). Paper chromatography-evealed that fractions 5, 6, 7, and 8 contain separable oligosaccha-rides, although overlapping each other. Fractions 12 through 18 alsocontain oligosaccharides indistinguishable by paper chromatography.It is noteworthy that galactose, mannose, and rhamnose are main con-stituent monosaccharides in partial hytdrolysate (peaks 3 and 4) and e-ven in complete hjdrolysate. These firdin!s are suggestive in thatmain structure of polysecchgride Wodld be composed of these 4-bree monn-saccharides.

15

6. List of papers submitted -to publication under the--support from theContract No DA-92-557-FEC-35775

Uetake, H. and Kariwara. S.: Effects o'f the unrelated phage uponlysogenization, Virus (in Japanese)

Hagiwava, S., Uetake, !T. and Toyamna, S.: Lysogenization and host-controlled variation. Virus (in Japanese)

Uetake, H., Toyamia, S.i ar0 S'niwara, H.: Host-controlled variationin Salmonella phase c 1 5 . Virus (in Japanese)

Uetake, H.', Toyama, S. and Hagiwara, S.: Host-controlled variationand multipPl-ity activation. Virus (in Japanese)

16

REFERENCES

½sM.H.: Ia.t gs(1959) Interseience Pubi. New York.Andei ;ci,7 -2' & Felix, A.: v:,ation in Vi-phage II of Salmonella

typhi. Nature 170, 4ý92-494 (19;:2)Anderson, E.S. & Felix, A.: The Vi-tjype determining phages carried

by Salmonella typhi. J.Gen.Microbiol. 9, 65-88 (1953)Arber, W. & Dussoix, D.: Host specificity of DNA produced by Escheri-

chia. coli. I. Host controlled modification of bacteriophage ~J.Mol.Biol. 5, 18-36 (1962)

Arber, W. & Lataste-Dorolle, C.: Erweiterung des Wirtbereiches~desBakteriophagen auf Escherichia coli B. Pathol.Mikrobiol. 24,1012-1018 (1961)

Baricelli, N0.A.: An analytical. approach to the problems of phage re-combination and reprodu ction. I. 'Multiplicity reactivation and thenature of radiation damages. Virology 11, 99-1-35 (1960)

Bautz, E. & Freese, E.: On the mutagenic effect of alkylating agent.Proc.Natl.Acad.Sci., 46, 1585-1594 (1960)

Bautz-Freese, E. & Freese, E.: Inducl-ion of reverse mutations andcross reactivation of nitrous acid-treated phage T4. Virology ~19-30 (1961)

Baylor, M.B. & Mahler, H.R~.: Effects of nitrous acid on intracellularT2 bacteriophage. Virol.ogy 16, 444-451 (1962)

iBertani, G.: Ltysogeny. Advances in Virus Research 5, 151-193 (1958)Bertani, G.: Multiple lysogeny from sin.g;le infection. Virology 18,

131-139 (1962)Bertani, G. & Weigle, J.J.: Host-controlled variation in bacterial

viruses. J.Bacteriol-. 65, 113-121 (1953)SA.: Induction at ý-mutation in poliovirus by nitrous acid.

Virology 9, 691-,,oo (1959)Brenner, S. & Smith, J.D.: Induction of mutations in the deoxyribo-

nucleic acid of phage T2 synthesized in the presence of ohloram-phenicol. Virology 8, 124-125 (1959)

Bresch, C.: Unterscheidung versci~iedener Bakteriophagentypen durchFarbhindi1tnrn'!hrboden. Zbl.Bakt. I Orig. 159, 47 (1952)

Bresch, C. u. Trautner, T.: Die Bedeutung des Zw-eifarb-Nithrbodensfffr qenetisohe Untersuc1-unren am Bakteriophagen Tl. Zsch.Indukt.Abstam~ungs- und Vererbungslehre. 87, 590-594 (1956)

Christensen, J..R.: The fate of genes from restricted Tl in lysogenicShigella dysenteriae, Sb(Pl). Virology 16, 133-139 (1962)

Christensen, J.R.: On the process of host-controlled modification ofbacteriopohage. Virology 13, 40-43 (1961)

T-axler, H.'.& Christensen, J.TR.: Genetic crosses between restrictedand unrestricted phage Ti in lysogenic and nonlysogenic hosts.Virolo'-y 1.3, 31-39 (1961)

DussoiLx, D. & Arber, W.: Host specificity, of DNA produced by Escheri-chia coli. II. Control over acceptance of DNA of bacteriophage ,

J.Mol.Biol. 5o 37-49 (1962)Epstein, P.H.: A study of multiplicity-reactivation in the bacterio-

phage T4. kVirology 6, ý82-404 (1958)Felix, A. & Anderson, E.S.: BacterioPhages carried] by the Vi-phage

types of %lm~yonella typhi. Nature 167, 603 (1951)

17

Freese, E.: The difference between spontaneous and base-analogueinduced mutations of phage T4. Proc.Nati.Acad.Sci. U.S. 45,622-633 (1959a)

Freese, E.: The specific mutagenic effect of base analogues on phageT4. J.Mol.Biol. 1, 87-105 (1959b)

Garen, A. & Zinder, NoD.: Padiolonical evidence for partial genetichomology between bacteriophage and host bacteria. Virology 1,347-376 (1955)

Gierer, A. & Mundry, N.W.: Production of mutants of Tobacco mosaicvirus by chemical alteration of its ribonucleic acid in vitro.Nature 182, 1457-1458 (1958)

Granoff, A.: Induction of Newcastle disease virus mutants withnitrous acid. Virology 13, 402-408 (1961)

Green, D.M. & Krieg, D.R.: The delayed origin of mutants induced byexposure of extracellular phage T4 to ethyl methane sulfonate,Proc.Natl.Acad.Sci. 47, 64-72 (1961)

Hagiwara, S.: Analysis of the process of lysogenization in phage c34

system (I). Segregation of nonlysogenic progeny. Virus 9, 468-472(1959a) (in Japanese)

Hagiwara, S.: Analysis of the process of lysogenization in phage 034

system (II). Appearance of 0 antigen 34 in freshly phage-infectedcells. Virus 9, 472-475 (1959b) (in Japanese)

'-7agiwara, S.: Analyais of the process of lysogenization in phage c34

system (III). Superinfection resistance in phage-infected cells andtheir progenies. Virus 9, 531-535 (1959c) (in Japanese)

Harm, W.: On the mechanism of multiplicity reactivation in bacterio-phage. Virology 2, 559-564 (1956)

Ise, T.: C,,anges induced in the 0 antigens of Salmonella senften-:berg and cbemical analysis of specific 0 antigens. III. Mono-.sacchatide composition of specific antiginic polysaccharides of theinduced variant and sumifiary of three reports. Sapporo Med.J. 5,1l0-106 (1954) (in Japanese)

Kauffmann, F.: Enterobacteriaceae (1954), Munksgaard, CopenhagenT awley, P.D. & Brookes, P.: Ionization of DNA bases or base analogues

as a possible explanation of mutagenesis, with special reference to5-bromodeoxyuridine. J.Mol.Biol. 4, 216-219 (1962)

Lederberg, J. & Lederberg, E.M.: R1eplica plating and indirect selec-tion of bacterial mutants. J.Bacteriol. 62, 399-408 (1952)

Litman, R.M. & Pardee, A.B.: The induction of mutants of bacterio-phage T2 by 5-bromouracil. III. Nutritional and structural evidenceregarding mutagenic action. Bioch.Biophys.Acta 42, 117-130 (1960a)

Litman, R.M. & Pardee, A.B.: The induction of mutan-s of bacterio-phage T2 by 5-bromouracil. IV. Kinetics of bromouracil-inducedmutagenesis. Bioch.Biophys.Acta 42. 131-140 (1960b)

Litman, R.M. & Pardee, A.B.: Mutations of bacteriophage T2 inducedby bromouracil in the presence of chloramphenicol. Virology 8,125-127 (1959)

Litman, R.M. 8 Pardee, A.B.: Production of bacteriophage mutants bya disturbance of deoxyribonucleic acid metabolism. Nature 178,529-531 (1956)

Luria, S.E.: Host-induced modifications of viruses. Cold SpringHarbor Symp.Quant.Biol. 18, 237-244 (1953)

18

T~r~a SE.:~eacivaionof irradiated bacteriophage by transfer of

self-reproducing un-fts. Proc,!iatl,A~cad.Sci. U.S., 33, 253-264(1947()

Luria, :ýE. F. D-ibecco, ". Tnetic recombination leading to produc-tion of active bacteriophage froiu ultraviolet inactivated bacterio-phage particles. Genetics 341 93.-125 (1949)

Luria, S.E,, Fraser, D.{., Adams, J.N.~ & Burrous, J.W.: Lysogeniza-tion, transduiction, and genetic recombination in bacteria. ColdSpring Harbor Sy-,mp~quant.BioJ0 23 71-82 (1958)

Luria, S.E. 8, Human, LI.L. : A nonliereditary, host-induced variationof bacterial viruses. J. iBacteriol. 64, 557-569 (1952)

Maio, J. J. S. Zahiler, S.-A. : Cbservations on the host-induced modifi-cation of T2 bacterionhage with special reference to the effects Of'x-keto acids. J.Bacteriol. 76, 256-263 (1958)

Nakagawa, A.: LysoG-enc conversion o:1 0 antigen 34 in Group E Salmo-nella (I). Jap.J.Bacteriol. 12, 46-52 (1957a) (in Japanese)

Nakargawa, A.: Lysogenic conversion of 0 antigen 34 in Group E Salmo-nella (II). japJ.JBacteriol. 12, 107-110 (1957b) (in Japanese)

NakaawaA.: ysoenic conversio of 0 antigen 34 in Group E Salmo-nella (III). Jap.JBacteriol. 14, 693-697 (1959)

Nakagawa, T.: Changes induced in the antigens of Salmonella (V).Chemical analysis of antigenic polysaccharide of Salmonella Egýroup. Sapporo Med.J. 5, 220-223 (1954) (in Japanese)

Dalston, D.J. & Krueger, A7.P: Phage multiplication on two hosts.i,;olation and activity of variants of Stapjylococcus phage P1.I-roc-Soc.Exptl.Biol.Med. 80, 217-220 (1952)

Sasaki, T.- Chemic.)l analysis of bacterial polysaccharides. I.Studies on the monosaccharide constituents of the specific poly-saccharide of 0 antig~ens of Salmonella anatum (Group El). Prepa-rations and purifications of the antigenic polysaccharides.JapJ.Bacteriol.. . 10, 997-1002 (19".5) (in Japanese)

Sasaki, T.: Chemical analysis of bacterial polysaccharides. II.Studies on the monosaccharide constituent8 of the specific poly-saccharide of 0 antigen3 of Salmonella anatum (Group E1 ). Mono-jaccharide constituients of the antigenic polysaccharides.Jap.J.Bacteriol. 11, 11-15 (1956a) (in Japanese)

Sasaki, T.: Chemical analysis of bacterial polysaccharides. III.Studies on the monosaccharide constituents of the specific poly-saccharide of 0 antigens of Salmonella senftenberg (Group B3).Jap.J.Bacteriol. 11,1 107-110 (1956b) (in Japanese)

Sinsheimer, R. L.: Thýe biochemistry of genetic factors. Ann-Rev.Biochem. 29 503-5211 (1960)

Stent, 0.5. & Fuerst, C.P.: Decay of incorporated radioactive phos-phorus durinq development of a tempnerate bacteriophage.Virology 2, 737-752 (19,56)

Strauss, B.S.: Response of Escherichia coli auxctrophy to heat aftertreatment with mutagenic alkyl methane siilfonates. 'J.Bacterial.83, 241.-249 (1962)

Strauss, B.S. & Okubo, S.: Protein synthesis and the induction ofmutations in Bscherichia coli by alkylating agents. J. Bacterio'.79, 464-473 (1960)

19

Tessman, I.: Mutagenesis in phages $X174 and T4 and properties of thegenetic material. Virology 2, 375-385 (1959)

Tessman, I. & Ozaki, T.: Multinlicity reactivations of bacteriophageTl. Virology 4, 315-327 (1957)

Uchida, T., Nakagawa, A. & Kai, M.: Varintion of the surface struc-ture of bacterial cell by lysogenization. Sapooro Med.J. 2,268-271 (1956) (in Japanese)

Uetake, H.: Lysogenic conversion in bacterial antigens. Proc.Intern.Genetics Symp.Tokyo & Kyoto, 645-650 (1956)

Uetake, H. & Hagiwara, S.: Genetic cooperation between unrelatedphages. Virology 13, 500-506 (1961)

Uetake, H. & Hagiwara, S.: Lysogenization and nuclear segregation.Virus 2, 574-577 (1959) (in Japanese)

Uetake, H. & Hagiwara, S.: The relationships between somatic antigens15 and 34 in Salmonella. Virus 10, 150-154 (1960b) (in Japanese)

Uetake, H. & Hagiwara, S.: Somatic antigen 15 as a precursor of anti-gen 34 in Salmonella. Nature 186, 261-262 (1960a)

Uetake, H., Luria, S.E. & Burrous, J.W.: Conversion of somatic anti-g-ens in Salmonella by phage infection leading to lysis or lysogeny.Virology 5, 68-91 (1958) 15

fetake, H. & Uchida, T.: Mutants of Salmonella phage c with abnormalconversion properties. Virology Z, 495-505 (1959)

Vielmetter, V.14. & Schuster, H.: The base specificity of mutationinduced by nitrous acid in phage T2. Biochem.Biophys.Research Comm-

uns. 2, 324-328 (1960)V ielmetter, V.W. & Wieder, C.M.: Mutagene and inaktivierende Wirkung

salpetriger Sgure auf freie Partikel des Phagen T2. Z.Naturforsch.

14b, 312-317 (1959)Westphal, 0.: Ueber die Extraktion. von Bakterium mit Phenol/Was.ser.

Z.Naturforsch. 7b, 148-155 (1952)

20

APPENDIX "Y" TABLE

Table 1 Time required for establishing lysogeny at variousmultiplicities of infection in various combinatifnsamong bacterial strains

I 15Surviving c - Prophage

Bacterial Phae moi clls* producing state wasstrain g ~ c olonies established

(%) (%) (in hours)

A 1.05 47.1 43.3 ca 35.0 35.0 99.9 ca 3

15c [A] 6.1 26.1 44.8 .167.5 25.0 51.2 >6

A(c34) 8.9 36.7 86.7 >620.0 65.5 98.7 ca 324.2 74.6 98.9 ca 3

15 34 4.8 46.2 92.7 ca 3C [A(c )j 9.4 53.3 98.9 ca 3

10.2 69,1 99.1 ca 3

3.9 23.3 33.6 > 6Heated 1 5.0 21.9 42.9 >6

A(c34 ) lLA 15.0 17.1 97.3 ca 3-415.4 20.2 98.1 ca 3-4

4:Surviving cells were plated out 20 minutes after infection and the

resulting colonies were tested for c15 lysogenicity by replicaplating.

Table 2 Efficiency of plating (EOP) of c 15

propagated on various strains and of g 3 4 1 [A]

EOP on

Phage strain

A A(c34) A(g 3 4 1) A(cy)

c15[A] 1 3x10 1 I15EA 34 11c [A(c)] 1 5xlO-c1 5 [A (g341)]

g3 4 1 [A] 1 0

21

APPENDIX "A" TABLE

Table 3 Efficiency of plating and adsorption rateconstant

15 Adsorption rate constantSEOP (x-10 9 ml min-1)

propagatedon A I-i A I-i

A 1 lO"2 4.9 5.1

I-1 lO-4 1 5.1 4.3

A = S. anatum 293I-1 = S. butantan

Table 4 One-cycle growth of J5 [I-1] on cells A

Indicator strain

A I-1

Initial phages of c 1 5 [I-l 3.7xlO3 /ml 3.65xlO7 /ml

Initial cells of A 1.O4x1O8 /ml

Free phages after neutralization 22.xlO2 mlof free phages

phages adsorbed in 10 min 3.64xlO7/ml

m.o.i. = 0.35

Plaque counts after burst 1.16xlO5/ml 1.OxlO3/ml

Burst size 31.4

22

APPENDIX "A" TABLE

T• e 5 Formatiap pf a ~t.ep •5 pn f•§4 ph4g@-infeqted cell@

Bac- 15 after Dilutibn of anti-15 serum Salineterial Phage Ci infec- -estrainý tion 1:80 1:160 1:320 1:640 1:1280 1:2560 control

(min)

25 + + + +50 + + + ±c15Il]•

25 + + +A I 50 + + +

1-1 25 + + + - -

I Cl5A 50 + + + + - -

A 25 + + + -

50 + + + •.

I-i - -. . . . ...

A 5 + + + + +

+ = positive agglutination-= no agrlutination

Table 6 Proportion of cells killed or lysogenized byinfection with host-controlled variants

Phage J5 Cells killed by Cells lysogenizedpragte o * phage (%) among survivals (%)

propagated on

A I-1 A I-1

A 66.4 92.6 99.8 30.0

1-1 0 85.0 0 60.5

23

APPENDIX "A" TABLE

Table 7 Mixed infection of cells I-1 with c 1[A]' andc 15ir[A]

Cell input 1.OlxlO8 /ml

c 5[A) input 7.1 x10 7 /ml

c 1 5 vir[A] input 7.2 xlO7 /ml

Infective centers 2.7 8 xlO6 /ml

Mixed yielders 1.8 x10 5 /ml ) 5.lxlO5 /ml

Mi.xed(?) yielders 3.3 xlO5 /ml..

Calculated number of mixed yielders* l.87xlO4 /ml

*Theoretical calculation was made by assuming that each phage

suspension contains h mutant at a proportion of 10-2 and2xlO" 2 , respectively.

24

APPENDIX 117<" L!' STRATION

ltAtL v~~Lo NCLLSe

/

Its

A(43 4

)

Fig. 1 Process of ]ysoerii.zation in strains A,r(, 34), aid A(C3 4 ) infecter] with clS[A]

Crowinp: cells of e',ch str-.ir were inf'cted withcl 5 [A] and proportions of phage-carrier cells werefollowed up at appropri;tce time intervals.

Since experiments were not carried out at the sametime, total viable ce1la in the experiment with cell.A were taken, for comra)arison, as standard for viablecells, and phage-carrfcr cells were plotted accordin,to the ratios of car:rier to total cells.

APPENDIX "T '.LL1•1 STRATICIN

Tidd 'hbk -iis

A

• J /

z //o, •-

.. ....

10' ,

00 120 00 Mll 240 000 06

Fig. 2 Process of lysogenization in strains Aand A(c3 4 ) infected with phage c 1 5[.J atm.o.i. of 5 and 24.2, respectively

1 Growing cells of A(c34) were infected with[AJ at in.o.i. of 24.2, while cells A as con-

trol at m.o.i. of 5.

26

APPENDIX 11" T LL- STRATION

FtALLs /aW celti

'It

tie//

///.4 ' / / /o

I0s, * e,/"

Fig. 3 Process of lysogenization in strains Aand A(g 3 4 1 ) infected with c 1 5 [A(g 3 4 1 )]

Growjng cells of A and A(g 3 41 ) were infectedwith c [A(g 3 4 1 )] and proportions of phage-carrier cells were followzd. up at appropriatetime intervals.

27

APPENDIX "B" ILLUSTRATION

toCvatmI

ceLLs

iI I

10, 0-0fde

1 0 1.

Fig. 4 Process of lysogenization in strain A

infected with g341[ .] |Growing• cells of A we.:.e infected with g J41 [A]

at various m.o.i., and y~roportions of pha e-carrier cells were f ollow•d up at appropriatetime intervals.

28

APPENDIX "B" ILLUSTRATION

I0 0

iotat viS14 muLs

ui ninfec de d CevLs

10 1

•2.o1.2

?ig, Process of .lysogenization in strain A(cy)infected with g 34D.[A]

Growing cells of A(ey) were infected with

g.h[A] at various m.o.i., arid proportions ofphaqe-carrier cells w•ere followed up at app-

ropriate tiwe intervals.

29

APPENDIX _,, -3TRAZ&Is)ON

!/!

,01j0.01

5.1, 11. 1 .1. o. l~ .t

FiT. 6 Effect of multiple infection upon efficiency of plating(plaque formers/infected cells) in cells grown in nutri-ent broth

The proportion of plaque formers to infected cells was deter-mined (9) at various m.o.i.. At each m.o.i., theoretical valueswere also calculated under the assumption that each cell may be--come plaque formner, which receives at least 4(o), 5(a), and/or6(e) phage particles, according to the formula shown in Fig. 7,where Pr = proportion of plaque formers to infected cells

m = m.o.i.r = number of phage particles adsorbed to a single celln •OP in single infection

30

APPENDIX "B" ILLUSTRATION

S..1

LLZ vi ,Ihe--

gr = - -e - /

Fir. 7 Effect of multiple infection upon efficiency ofplatinig (plaque formers/infected cells) in cells grownin synthetic medium

Notes are the same as in Fir. 6, except thn.t calculationswere mad, unde-r the assimption that each cell may become pliaunformer, which receives at least 2(e), or 3(a) phage particles.

31

APPENDIX "B"1 ILLUSTRATION

100

/7/.50

• .e

5 10 15 20

Fig. 8 Proportion of cells killed by phage infection atvarious m.o.i.

The proportions of killed to infected cells were determined(e) at various m.o.i.. At each ia.o.i., theoretical valueswere also calculated, assuming that each cell may be killedby infection, which receives at least 4(o), 5(e), and/or 6(e)phage particles.

3a

APPENDIX ILLUSTRATION

100

10

/"

SL

/

.1 10.0 15.0 20. o,,.o.1.

Fig. 9 Proportion of lysogenic cells among survivors atvarious m.o.i.

Phage-infected cells of I-1 were plated for surviving 'ells20 minutes after infection, and resulting colonies were testedfor lysogenicity by replica plating.

33

APPENDIX "B" ILLUSTRATION

UE

0 20 30 40

Minutcs After Inf3ctin.4

Fi7. 10 Rescue of c 5vir[AJ by superinfecting with -5 ts[AJ

34

APPENDIX "'' -,".,,STRATION

I.0C

.LO

45 / S

FiT, ii Effect of heating cells on efficiency of plating(plaque formers/infected cells)

Growinv cells of I-1 were heatedt for 2 minutes at varioustjp.ratiures, transferred back to 370C, infected with phageC .A3 at low (0.i) m.o.i., and rlated out for infectivecenters with cells of I-i before lysis.

APPENDIX "B" ILLUSTRATION

4O.0.

P-3

P-4

-P-1 P-7

P-6

P-21

P-Il ~g; I14 ~ P- 9

04

-2 p~jF1 60-

Fig. 12 Cbarcoal-celite column chromatography of acidllydrolysate of S7Aa' linopolysaccharide

Partial acid hydrolysis: N I12 SO4, 20 mrin.

Column: Acid-treated norib A(50 pm)/ceJlite 545(100 gin)Color development: Pbencd- 7 ,02 'C 4 reaction

56