Induced Structural Defects in T-Even Bacteriophage

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JOURNAL OFVIROLOGY, Feb., 1967, p. 193-204 Copyright ©1967 American Societyfor Microbiology

Vol. 1, No.1 PrinztedinU.S.A.

Induced Structural

Defects

in

T-Even

Bacteriophage

DONALD J. CUMMINGS, V. A. CHAPMAN, S. S. DE LONG, AND L. MONDALE

Departmentof Microbiology, UnziversityofColoradoMedical Center, Denver, Colorado

Received forpublication 14 November 1966

Multiple aberrant substructures of T-even bacteriophage particles occurred when

amino acid analogues orantimetaboliteswerepresentduring phage growth. Certain aberrant substructures were induced by specific analogues or antimetabolites. In

particular, it was observed by electron microscopy that L-canavanine, an arginine

analogue, gaveriseto polyheads; L-azetidine-2-carboxylic acid, a proline analogue, gave rise to polytail tubes; and 1,2,4-trizaole-3-alanine, a histidine analogue,

proflavine, and actinomycin D all gave rise to small heads. These aberrant sub-structures were similar to those reported earlier with conditional lethal mutants

(amber) of T4D ina restrictive host.

The morphology

of

T-even bacteriophages has been well described (27). These particles consist

of a polyhedral head, contractile tail sheath, tail

tube,

tail

plate,

and tail fibers.

Recently,

the

ele-gant genetic and morphological studies of Edgar and his

colleagues

on conditional lethal

phage

mutants (8-10,15) have shed some light on the

assembly

processof

phage

substructures into ma-ture virions. Under restrictive conditions, some

of

these T4Dambermutantsgaverisetoaberrant substructures such as

polyheads,

small

heads,

polysheaths,

and

polytail

tubes

(Fig. 1).

Ina

per-missive

host,

these mutants

produce

a normal phage

population. These investigations

on

sub-structure

aberrations

have

led

to theconcept (9,

15)

that

self-assembly

(or

maturation)

of

phage

protein

componentsis

regulated by specific phage

gene

products. Kellenberger (15)

envisions the

assembly

process of individual

protein

subunits intoaphagecomponent as a

sequential triggering

of

subprocesses

induced

by

different

morphopoi-etic

factors. Fromthis

point

of

view,

it should be

possible

to interfere with these

subprocesses

in

cells

infected

with

normal

wild-type

bacterio-phage.

The

approach

chosen hereto

study

the

matura-tion process was the

addition of

various amino acid analogues and antimetabolites (7, 13,

16,

21-23,

26) during

the

infective

cycle of wild-type

T-even

bacteriophage.

Substructure aberrations

were obtained with manydissimilar compounds, and some

aberrations,

such as

polyheads,

small headsor

polytail tubes,

were

produced

specifically

by

some

of

the

compounds

utilized.

MATERIALS AND METHODS

Bacteriophage growth conditions and purification.

Bacteriophages T2L, T4BO1 (an osmotic

shock-resistant mutant of T4B obtained from Bruce Ames), and T6 (obtained from M. Jesaitis), each of which have latentperiods of about 22 to 26 min and eclipse periods of about 14 to 16 min (27), were used through-out,and, unless otherwise stated, the results presented wereobtained with each of these viruses. Infection of Escherichia coli B and its derivatives was performed at amultiplicityofabout 5:1 to insure simultaneous infection of all bacteria. The bacteria were grown in a minimal medium (Na2HPO4 8.0 g; KH2PO4, 2.0 g; NaCl, 3.0 g; NH4Cl, 3.0 g; gelatin, 0.2 g; 3 X 10-4 M

MgSO4; 8 X 10-5 M CaC12; glucose, 10 g; 1 liter of demineralized water; pH 7.2) and supplemented with 100 mg ofL-tryptophan per liter when T4BO1 was employed.Routinely, 500 ml of this medium was used to obtainsufficient material for electron microscopy. When the bacteria had reached a titer of 3 X 108 to 4 X 108per

milliliter, bacteriophage

was added, and,

at the times indicated, the respective analogues or antimetabolites (proflavine or actinomycin D) were added and growth was allowed toproceedfor 2.5 hr withvigorousaeration at 37 C. Mutants of E. coli B requiringL-prolineorL-argininewere isolatedby use of the nitrosoguanidine technique of Mandell and Greenberg (19). With these mutant bacteria, the minimalmedium was supplemented with 40,ug/ml of therequired amino acid. Infection withbacteriophage

waspreceded by filtration of the bacteria on a 0.45-u membrane filter (Millipore Filter Corp., Bedford, Mass.) and resuspension in fresh minimal medium

containing 1.5 ,ug of therequired amino acidperml. This procedure kept the concentration ofthe amino acid at aminimum,but still allowed successful phage infection. In the case ofactinomycin D, infection was

precededby a similar filtration of E. coli B, and the bacteria were resuspended in

ethylenediaminetetra-acetate (EDTA) as described by Korn, Protass, and Leive (16), in order to render the bacteriapermeable

to the actinomycin D. After this pretreatment with EDTA, the bacteria wereresuspendedinfresh minimal medium and infectedimmediately withbacteriophage;

10to 15 minlater, actinomycin Dwasadded. Growth 193

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CUMMINGS ET AL.

NORMAL T-EVEN PHAGE

HEAD

SHEATH

TU3 E

DEF ECTS POLY HEAD L._ J P0LY SHEATH POLY

T UI E

FIG. 1. Schematic diagram of the morphology of T-evenbacteriophageandsomeaberrantstructures.

ofphagewasstopped byaddition ofchloroform,and thelysatewasstoredovernightinthe coldtofacilitate clarification, andwastreated with deoxyribonuclease andribonuclease. Thelysateswerepurifiedthreetimes

bydifferentialcentrifugationat7,000Xgfor10minto remove bacterial debris. It was determined that centrifugationat 28,000X gfor4.5 hrwassufficient

to sediment quantitatively each of the substructure phagecomponents. This determination wasbasedon

the factthatsubsequent centrifugationof thevarious

supernatantsolutionsat100,000Xgfor3hrproduced nonew,and littleadditional,material.This described centrifugationprocedure wouldnotsediment compo-nents havingasedimentationcoefficient ofmuchless

than50S, andthehigher forcefield utilizedtomonitor

supernatantsolutionswouldnotsedimentcomponents

with sedimentation coefficients much less than 15S. The final bacteriophage pellets were resuspended in

0.10 MNaCl, 10-3 M MgSO4, and 10-3M phosphate

buffer(pH 7.2),andwereassayed forviablephage by standardtechniques (1).

Electron microscopy. Electron micrographs of all the phage preparationsweremade byuseofthe

phos-photungstic acid negative staining method (2) andan

RCA-EMU 3G electron microscope fitted with a

modified gridcapto enhancecontrast (12). Final

re-sults were recorded on Dupont Cronar Ortho 708

Litho Afilm atamagnification of about 15,000and werephotographically enlarged fourtofivetimes.To make the observations less subjective, the actual

num-bersof eachphagecomponentinthe fieldwere tabu-lated and then were corrected to a standard basis. The standard basis selected was a specified number

(2,000) of whole phage and ghosts, withor without

tails, inthe fieldsexamined. Although this basiswas no doubt subject to some variation from lysate to

lysate, the method described was considered to be subjecttothe leasterror.

Amino acid analogues anid antimetabolites. The following compounds were added to infected-cell cultures in the amounts indicated: L-canavanine sulfate (22, 23) obtained from Mann Research Laboratories, Inc., New York, N.Y., 150 j,g/ml;

L-azetidine-2-carboxylic acid (13) obtained from Calbiochem,LosAngeles, Calif.,150,g/ml; 5-methyl-DL-tryptophan p-fluorophenylalanine, and DL-7-aza-tryptophan (4, 11), obtained from Calbiochem, 150 ,ug/ml;1,2,4-triazole-3-alanine(18) obtainedthrough the courtesy of R. G. Jones of Eli Lilly & Co., In-dianapolis, Ind., 120 ,ug/ml; proflavine (7) obtained from the Aldrich ChemicalCo.,Inc.,Milwaukee, Wis., 3 ,ug/ml; and actinomycin D (16) obtained through the courtesy of Merck and Co., Rahway, N.J., 20

jug/ml.

RESULTS

Some analogues suchasp-fluorophenylalanine

(11), 5-methyl tryptophan (4), and DL-7-azatryp-tophan (11) can be regarded as rather gross

inhibitors ofcell growth, and these were chosen to determine what structural defects could arise inphagegrownin theirpresence. As canbe seen

inFig. 2,manydefects, suchas polyheads, small

heads, polysheaths, and polytail tubes, were

ob-tained with no apparent specificity due to the

analogue used. These substructures were identi-fied by virtue of their general appearance and their occasional association with other phage

parts (see Fig.3 and7).Most of the aberrant sub-structures were readily identified. However, the

polytail tubes were not often associated with heads or plates (about 0.5% of those polytail

tubes observedwere attached toeither ahead or a tail plate). The possibility that these substruc-tures represented bacterial pili (3) was excluded since results with uninfected cultures and with

some analogues revealed few if any such sub-structures. The actual frequencies with which different substructures occurred are presented in Table1,where itcanalso beseenthat,asexpected

(4, 7, 18, 21), thenumber of virions obtainedwas

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STRUCTURAL DEFECTS IN T-EVEN BACTERIOPHAGES

(r

w4.t

X

.i

-IOOOA

FIG. 2. Electronimicrograph oftlhepurifiedlysate obtainedafteradditionl of

p-fluorophentylalaninze. Int

thiscase, the analoguewas added 10miii after

infectioni

ofEscherichia coli B withl T2L. The arrows

inzdicate

the various

aberranlt substrluctuires obtainled, suchaspolyheads,polysheaths,polytailtutbes,

anid smyiall

heads,

anid

these

corre-sponid

tothose depictedinFigure 1.

quite low. Once it had been observed that

aber-rantsubstructures dooccurin anormal bacterio-phage system, the question arose as to whether

particular compounds

wouldgive rise to

preferen-tial defects. Certain choices could be made from what was known about the physical and chemical

properties

ofmature phage. Regarding the head structure, Kozloff and Lute (17) showed that

VOL. I, 1967

195

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CUMMINGS ET AL.

TABLE 1. Distributioni of iniduced defects in T-evenbacteriophagea

Compound Aminio acid L-Tyrosine

I

L-Tryptophan

(

L-Phenylalanine

(

L-Arginine

J

L-Histidine L-Proline Anitimetabolites Actinomycin D Proflavine Alnalogue

(p-Fluorophenyl-

d alaninine 5-Methly-DL-tryp-tophan 1DL-7-Azatryptophan L-Canavanine 6min

10and 14 min 18 min Triazole-3-alaninee T2L T6 L-Azetidine-2-carbox-ylic Phage

(

T2L T4 T6 T2Lf

I T4 orT6

Viability c

1 X 10-a

I X 10-2 2 X 10-3 4 X 10-3 3 X 10-1 0.5 6 X 10-4

L.5 X 10-2 7 X 10-2 0.4 5 X 10-1 2X 10-5

Bacteriophageb XW'hole Ghost 830 1,170 260 280 1,170 1,870 1,470 920 1,390 1,020 1,110 30 1,740 1,720 930 130 530 1,080 610 980 890 1,970 Poly- Poly-head sheath 6 56 51 2 0 2 2 0 1 0 2 Poly- Small tail head tube

I_

33 32 9 170 35 9 8 3 1 1 7 2 5 180 10 1 10 4 3 0 5 2 30 3 5 8 110 580 2 40 370 150 750

aWith the centrifugation procedure described in Materials and Methods, theonly aberrations re-covered werepolyheads, polysheaths,polytail tubes, and small heads. Itis possiblethatother defects occurred and were not detected, but attemptsto isolate such unknown structures were notsuccessful. Itis also possible that the structures isolated represent only themoststableproduced under these condi-tions.

IInall cases, from 2,000 to13,000actualparticleswerecounted,and allfigureswerethen normalized

to2,000 total particles. Allof thesetabulations represent the averages obtained fromat least two se-paratelysates; the figures from individual lysates of thesamephageorofdifferent phage agreedto +t 10%.

cViabilitybased oncomparison with growth under thesame conditions,but in the absence of

ana-logue.

dTheyieldof T4in the presence of tryptophan analogues wasalmostnormal, probably owingtothe presenceof the L-tryptophan necessary for adsorption; hence, these data wereobtained withT2Land T6.

eIn the case ofT2L the L-histidine analogue was administered from 3 to 14 min after

infection,

with no significant differences observed in the number ofsmall heads observed. For T6, the analogue was administered at 3 min only.

fUnder theminimal medium growth condition used here, the development ofT2Lintomaturephage particleswaslargely unaffected by proflavine, in sharp contrast toT4 or T6.Whereasonly

5C%

viability of theT2Lparticles was measured, much of this inactivation wasprobablyduetophotodynamic effects, sincepurification in the light yielded 103-foldfewer active particles.

argininehasadisruptive effectonthe phage head

protein, andCummings (5, 6) obtained evidence indicatingthat histidine is involved in head transi-tions of bacteriophage T2L. Consequently,

L-canavanine, an arginine analogue (22, 23), and

1,2,4-triazole-3-alanine (18), a histidine

ana-logue, weretried,and itwasfound that both

ana-logues exerted their effects largely on the phage

head. L-Canavanine was the only analogue

ex-amined which "induced" the formation oflarge numbers of polyheads (Fig. 3 and Table 1). As

isolated, these

polyheads

wereessentially devoid of

deoxyribonucleic

acid (DNA), but thismayhave

been due to the treatment of the lysate with

deoxyribonuclease. There was no significant dif-ference in the number ofpolyheads obtained when L-canavanine was added from 6 to 14 min after infection, but after 14 min the relative number decreasedsharply. Moreover, attheearliest time (6

min)

of addition, thenumberofpolytail tubes

was much higher than atany other time. It may also be noted that, in contrast to the

polyheads

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FIG.3. Electron micrographs demonstrating the effect of L-canavanine. In A, L-canavaninewasadded14min after infection of Escherichia coliBwith T2L.Notice that the polyheadsobtained are of varying lengths, and that the insert has apolyhead withatail attached. Such polyheads with attached tails were observed inI to 2% of the polyheads and primarily on the shortestpolyheads. In B, these particles resuiltedfrom a T4BOI lysate where L-canavanine wasadded10minafter infection. This micrograph illustratesacluster of polyheads on the electron microscope grid, and the apparent rigidity of theseheads; the fact that most are closed and have indentations (arrows) at one end isalso evident. Similar results were obtainedwhen L-canavaninewas added at 6min after

infectioni.

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CUMMINGS ET AL.

FIG.4. Electron

micrographls

showinig

the effect of 1, 2,4-triazole-3-alanineonthedevelopment ofbacteriophage (A) T6 and(B)T2L.InA,1,2,4-triazole-3-alaninewasaddedat3min,andinB, at1Iminafterinfection.Similar numbersof small heads (arrows) wereobtained with1, 2, 4-triazole-3-alanine at3, 8, 11,and 14 min.

observed

with the amber mutants (10, 15), the

of

the polyhead, an

indentation

was

frequently

ends of the

polyheads

obtained with L-canavanine observed, and it is

likely

that this may be the

are

mostly

closed and appear

normal,

evento the juncture between the head and

tail,

since tails

al-extent

of

being

attached to the tails. At one end ways appear to attachatthissite.

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STRUCTURAL DEFECTS IN T-EVEN BACTERIOPHAGES

FIG. 5. Electron micrograph depicting the effectsofadding proflavine to agrowing phage culture. (A) T2L, proflavine addedat8 minafterinfection. Theonlyaberration notedwassmallheads(arrows),andprimarilywhole phagewererecovered whether theproflavine wasaddedat 8, 11,or 14 min. (B) T4BOI,proflavineaddedat 11

min.Again, small heads were obtained,but with veryfew tailsorwholephage.

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FIG.6

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STRUCTURAL DEFECTS IN T-EVEN BACTERIOPHAGES

Although

TRA also exerted its effect on the head, it was not to give polyheads but rather to give small heads, both with and without DNA, having a length

of

650 A

compared

with the typical headlength

of

1,000 A. No change in width was noted, and no otheraberrant structures appeared to asignificant degree (Fig. 4 and Table 1). These

small-headed

particles may be similar to the in-complete phage

particles reported

by Mosig (20). Antimetabolites,

acridine-like

compounds in particular, have

been used

in many investigations of

phage growth (7, 14, 16,

21). The effect of

pro-flavine

and

actinomycin

D onthe

development

of

T-even bacteriophages was examined, and, in both cases, itwas

found

that the

only

substructure

defect observed

was small

heads, either

attached

totails ornot.

Proflavine,

especially (Fig. 5),

gave rise to more than

30%/o

small-headed particles in

the T4 or T6

preparations

examined.

Although

little difference in the response

of T2L,

T4, orT6

to

proflavine

was

expected,

with the use

of

mini-mal

medium

rather than

complete

medium

(1,

7),

T2L

responded quite differently.

Whereas about 97

%l

of

the T4orT6

particles

observedhad

empty

heads and

in fact veryfew

tails,

the T2L

particles

appeared

tobe

mostly

normal

phage (as

also

evi-denced

by

the difference in

viability)

and the

population

contained

fewer

small-headed

phage.

This

decreased

frequency

of small heads in T2L maybea

reflection

of

the

fact

that inour labora-tory we

frequently

observed some

(2 %)

small heads in

normal

stocks

of

T4orT6but have yet toobserve any small heads in normal T2L stocks.

With

actinomycin

D

(Fig.

6 and Table

1),

fewer

small

heads were

obtained,

and in all cases the phage

populations

containedagreaternumber of whole

phage.

This lesser effect of

actinomycin

D

relative

to

proflavine

may be a consequence of

permeability

differences,

or it may be that

pro-flavine

is a more potent maturation inhibitor. It is

pertinent

to

point

out that

small-headed

phage have also been

observed

in

proflavine

lysates

of

bacteriophage

x

(14),

a

phage quite

different

fromthose examined here.

As

indicated

earlier,

an additional aberrant

substructure,

polytail tubes,

was also

observed.

An

usual chemical

feature

of

the normal

sub-structure is its

high

(6.5%/,)

proline

content

(24).

For this reason

L-azetidine-2-carboxylic

acid.

an

L-proline

analogue,

wasexamined for its effecton

phage maturation. In

Fig.

7 and Table

1,

it can

be seen that the

only

aberration obtained was

polytail tubes.

Although the quantitative effect of this particular analogue was convincing, polytail tubes were also observed in the

p-fluorophenyl-alanine,

5-methyl-DL-tryptophan, and DL-7-aza-tryptophan, and in one of the L-canavanine ly-sates. If polytail tubes were specifically induced by L-proline analogues, then the effect should be enhanced by using L-proline-requiring mutants. Such wasfound to be the case (Fig. 7 and Table 2). The number of polytail tubes was increased about eightfold when proline-requiring E. coli B was infected in the presence of low concentra-tions

of

proline and L-azetidine-2-carboxylic acid was added at various times after infection. The number of polytail tubes isolated remained essen-tially constant until at least 14min after infection and then decreased precipitously. Readdition of L-proline rather than the analogue gave rise to

an essentially normal phage population. This success with L-proline mutants led us to re-ex-amine the effect

of

L-canavanine with an arginine mutant. However, under a variety of conditions, few if any aberrations and few phage were ob-served, probably owing to the lethal action

of

L-canavanine onE.coli (25).

DISCUSSION

These

observations

by means of electron mi-croscopy have indicated that it is possible to

in-terfere

with the subassembly processes of phage protein components. When particular amino acid analogues or antimetabolites were added to a growing bacteriophage culture at some time dur-ing the

eclipse period,

the

formation of

either

polyheads,

small

heads,

or

polytail

tubes was

"induced," depending

on the compound. The aberrant substructures obtained were similar to those

reported for conditional

lethalmutants(10), but whether the type

of

control mechanism(s) in the two instances is the same is not known. The mechanism

of

action

of

thesecompounds has not been

established

(11, 26); some analogues are known to be

incorporated

(11, 13, 22, 23), but it is apparent that

incorporation

is not the only meansbv which bacterial cell growth is

inhibited.

Regulation by

means of phage gene

products

is also notwell

understood,

and itwould be

prema-ture to

postulate

a

mechanism(s)

linking

the

sub-structure aberrations obtained here with those obtained

by

means of

genetic

defects.

Although

the

approach

presented

here

strongly

suggests

FIG. 6. Electronimicrographs illustratinig that actinomycin D alsogives rise to small heads (arrows). (A) T6, actinomycin Daddedat 15miti; (B) T2L,actiniomycin D addedat 13 mitt. Thesameresultswere obtainedalso at Jami,i after infectioni. Otiearrowpointsout apolytail tube in thispreparationl aiidillustrates theoccasional appearanceofsomesubstructures other thaii thatobtaiiiedspecifically.

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CUMMINGS ET AL.

FIG. 7. Electron micrographs ofT2L lysates afteradministration of L-azetidine-2-carboxylic acid. (A) The analoguewasadministeredtoEscherichiacoliBat14minafterinfection, anditcanbenotedthat theonly aberra-tionobtainedwaspolytailtubes. Theinsertcontainsaparticlewithatail tube abouttwo times itsnormallength still attachedtothe headatone end andatailplateatthe other. Similarresultswereobtained with T2L,

T4BOJI,

andT6 at 10 miii. (B) The enhancedeffect ofthisprolineanaloguewithaproline-requiringmutantofBandT2L.

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STRUCTURAL DEFECTS IN T-EVEN BACTERIOPHAGES

TABLE 2. Effect

ofjL-azetidine-2-carboxylic

acid withEscherichlia coli B

pro-Bacteriophage

Analogue Viability Poly- Poly- Polytail Small

head sheath tube head

WN'hole Ghost

L-Azetidine-2-carboxylic acid

6min, T2L 3 X 10- 350 1,650 0 0 1,620 0

10min,T2L 1.3 X 10-4 270 1,730 0 0 1,010 2

T6 3 X 10-5 190 1,810 0 9 1,330 0

14min, T2L 4 X 10]- 610 1,390 0 0 1,240 0

18 min, T2L 2 X 10- 1,470 530 0 0 63 7

L-Proline

8 min, T2L 1.0 1,940 60 0 2 0 2

that there is some specificity in the selection of particular defects in substructure, the same aber-ration was often obtained by different means. For example, polysheaths were obtained in all

casesto somedegree, and none of the compounds examined selected this aberration preferentially; polytail tubes were observed in significant

amounts in two cases other than that utilizing the proline analogue; and small heads were

"induced" by such disparate compounds as pro-flavine and triazole-3-alanine. Whereas these levels of apparent nonspecific induction of aber-rations raise some questions about the control mechanism(s), they do not argue against the designated specificities of certain analogues or antimetabolites, since the frequency of appearance ofa specificaberration was increased by atleast

anorder of

magnitude.

ACKNOWLEDGMENT

Thisinvestigationwas supported byPublic Health Serviceresearchgrant

Al-06472.

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Figure

FIG. 1.T-even Schematic diagram of the morphology of bacteriophage and some aberrant structures.
FIG. 1.T-even Schematic diagram of the morphology of bacteriophage and some aberrant structures. p.2
FIG. 2.aberranltsponidthe Electroni micrograph of tlhe purified lysate obtained after additionl ofp-fluorophentylalaninze
FIG. 2.aberranltsponidthe Electroni micrograph of tlhe purified lysate obtained after additionl ofp-fluorophentylalaninze p.3
TABLE 1. Distributioni of iniduced defects in T-even bacteriophagea

TABLE 1.

Distributioni of iniduced defects in T-even bacteriophagea p.4
FIG. 3.polyheadsaftermicroscopecanavaninethe(arrows) Electron micrographs demonstrating the effect of L-canavanine
FIG. 3.polyheadsaftermicroscopecanavaninethe(arrows) Electron micrographs demonstrating the effect of L-canavanine p.5
FIG. 4.numbers(A) Electron micrographls showinig the effect of 1, 2, 4-triazole-3-alanine on the development ofbacteriophage T6 and (B) T2L
FIG. 4.numbers(A) Electron micrographls showinig the effect of 1, 2, 4-triazole-3-alanine on the development ofbacteriophage T6 and (B) T2L p.6
FIG. 5.proflavinephagemin. Electron micrograph depicting the effects of adding proflavine to a growing phage culture
FIG. 5.proflavinephagemin. Electron micrograph depicting the effects of adding proflavine to a growing phage culture p.7
FIG. 6200
FIG. 6200 p.8
FIG. 7.analogueandstilltion Electron micrographs of T2L lysates after administration of L-azetidine-2-carboxylic acid
FIG. 7.analogueandstilltion Electron micrographs of T2L lysates after administration of L-azetidine-2-carboxylic acid p.10
TABLE 2. Effect ofjL-azetidine-2-carboxylic acid with Escherichlia coli B pro-

TABLE 2.

Effect ofjL-azetidine-2-carboxylic acid with Escherichlia coli B pro- p.11

References