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Copyright ©1969 American Society forMicrobiology Printed inVol.4, No.U.S.A.3

Delayed

Lysis

with

a

Mutant

of

Salmonella

Bacteriophage

P22

LARRY W. COHEN

SeaverLaboratory ofBiology, PomonaCollege, Claremoiut, Ccaliforlnia91711

Receivedforpublication21 May 1969

A mutantofbacteriophage P22

(Lys-)

wasisolated which showsa plaque

mor-phologyonmixed platescomparable to ther+ plaques of the T-even phages. When

Lys-and normal Lys+plaquesarejuxtaposed on apetridish, theLys+plaque

ex-hibitsaflat side adjacenttotheLys-plaque.ThemutantisidenticaltoP22underan

electron microscope, is inactivated atthesameratebyantiserumand heat, andhas thesamekinetics ofattachment. ItdoesnotplateonSalmonellalysogenic forphage

P22nor onstrainSt/22. In liquid culture, the lysis ofmutantinfections in M9CAA

medium isdelayed between20and40min. Cellsmixedlyinfected in M9CAAwith Lys-and Lys+ phagelyse later thanLys+-infected cells andevenlater than

Lys--in-fected cells. Inunsupplemented M9 medium, however, mixedlyinfected cellsagain lyse later thanLys+-infectedcells, butLys--infected cells requiremorethan 3 hrto

lyse. In supplemented and unsupplemented M9 media, intracellular phage de-velopment andendolysin synthesis proceed inLys-infectionsatleastasrapidlyas

inLys+-infected cells. Indiluted infections, the latent andeclipse periods of

Lys-andLys+ infectionsareindistinguishable. Thepossiblemechanismsinvolved inthe

control andtiming of lysisarediscussed.

T-even wild-type phage plated on E;scherichia coli B produce plaques characterized by their smallsize anddiffuse borders. Mutant T4phage (r-) produce larger plaques with more sharply defined halos (9, 10). The wild type, designated r+, produces the smaller plaques because ofthe delayed lysis resulting from infection followed by superinfection ofthebacterialhostcellsby phage releasedfromthe first cellstolyse (4).

2'When

T-phage

r+ and r-

plaques

lie in

suffi-ciently close

proximity

on a petri

dish,

the r-plaque seems to beinhibited in its

development,

exhibitingaflat sideadjacent tothe r+

plaque

or

even an inverted crescent border.

In temperate phage X a mutant

showing

de-layed lysis has been observed (6). This paper describes a mutant of Salmonella

phage

P22,

infections of which showmanycharacteristics of lysis inhibition but, in contrast to the T-even phages, do not require superinfection.

MATERIALS AND METHODS

Bacteriophagestrains.Thephage used in the

follow-ing experimentswerewild-typeS. typhimuriumphage

P22,clearmutantC25, andLys-c2,a mutantderivative

of theC25 stock resultingfromultraviolet light

muta-genesis.TheC2 gene mutationwasdescribedpreviously

(12, 13). Note that, except where specifically

desig-nated, thephage used intheexperimentsweremutant

at the c2 gene; the double mutant Lys-c2- will be

referredto aLysr andthe singlec2- mutant asLys+.

Bacterial strains. S. typhimurium LT-2 and

deriva-tive mutant strains were used in all experiments. A

gal- mutant wasused as background in platings on

eosin methylene blue galactose-supplemented plates.

StrainC 527 is a histidine C amber mutant; strainC

527-1 is isogenic to strain C 527 but contains an

amber suppressor (17). Strain St/22 is resistant to

phage P22 but sensitivetovariant P22phages (see 19).

Media. Buffered saline contained: NaCl, 0.145 M;

KH2PO4, 0.022 M; Na2HPO4, 0.042 M; pH 7.0. M9

mediumsupplemented with Casamino Acids (M9CAA

andunsupplemented M9 medium (15) were used to

culture cells.Nutrient agar,Lbroth, E M B galactose

agar and soft agar for plating phage have been

de-scribed(12).

Ultravioletlightmutagenesis. The Lys-mutantwas

isolated after ultraviolet irradiation (Sylvania

germ-icidal 15-w lamp) of host bacteria and P22 Lys+

phage. S. typhimurium LT-2 strain 527-1 was

ir-radiated for 15 sec(3mlinasterile100-mmpetri dish

lid) at 90cm, which yields 10%U survival of

colony-formersonnutrient agar plates.Thephageweregiven

120secof irradiation at50-cm distance,yielding

ap-proximately 1% survival when plated on the

irra-diated bacteria (2, 16). TheLys- mutant was found

among isolates selected for small plaque size.

Determination oftime oflysis of undiluted infected

bacteria. S.typhimuriumLT-2inM9CAAmediumor

unsupplemented M9 was grown in log phase with

aeration to a concentration of approximately 108

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COHEN

bacteria/inl (Klett reading = 25, Xmax = 540 nm).

Thecells were centrifuged for 10 min at 4,300 X g,

and the pellet was washed with an 0.5 volume of

phosphate-buffered saline and spun again for 10 min

at 4,300 X g. The cells wereresuspended in an 0.1

volume ofphosphate-buffered saline, transferred to a

small aeration tube, and aerated at 37Cfor 15 min.

A 1-ml amount was then transferred into the

ice-chilled side arm of each of three Nephelo culture

flasks, infected with eithermutantLys-,Lys+,orboth

Lys-and Lys+ phage andretained in the cold saline an

additional 15min. The infectedcellswerewarmed by

addition to each flask of 9 ml ofprewarmed growth

medium and the flasks weretransferred intoashaker

water bath (37 C). Opticaldensity readings were

re-corded attimed intervals; asustained rapid decrease

inoptical density is indicative of cell lysis.

Duringaeration in saline, the cells exhaust internal

nutrients so that phage development begins

synchro-nously at the time nutrient medium is restored. To

exclude thepossibility that observed resultsmight be

due to thephosphate saline treatment,synchronization

of infection was accomplished by aerating the cells in

0.01 M NaCN or in unsupplemented M9 medium

lacking glucose. Results comparable to those reported

wereobtained.

One-step growth experiment. Bacteria were grown

in M9CAA medium to a concentration of

approxi-mately 108cells/ml, and 1-ml samplesweretransferred

to each of two aeration tubes. The cultures were

separately infected with Lys- or Lys+ phage at a

multiplicity of infection of5. After 5 minof

incuba-tion, two50-fold serial dilutionswerecarriedoutwith

prewarmed M9CAA, and the last culture was aerated.

At 10-min intervals, 0.1-ml sampleswere withdrawn,

diluted in prechilled diluting broth, and plated for

infectivecenters.

Determination oftheeclipseperiod.S. typhimurium

LT-2, at aconcentrationof 108/ml, were infected with

Lys- orLys+ phage (multiplicity of infection = 5).

At the times indicated, 0.1-ml samples were

with-drawn into 4.9 ml ofice-chilled diluting broth, and

then 0.1 mlfrom these waswithdrawninto 4.7 mlof

diluting broth containing four drops of chloroform.

The tubes were shakenvigorously, allowed to stand for

30 minat roomtemperature,and platedforinfective

centers.

Endolysin assay. E. coliBcells, at a concentration

of4 X 108/ml, were sensitized by suspension in an

0.33volume of 0.1 M

ethylenediaminetetraacetate-1.0 M tris(hydroxymethyl)

aminomethane-hydro-chloridebuffer (pH 8.0) for5min at 0 C,followedby

centrifugation and resuspension to the original volume

in ice-chilled distilled water (11). Samples (1.5 ml)

were withdrawn from Lys-- and from Lys+-infected

cultures (108/ml)atvarioustimes during the infection

and transferred into ice-chilled nitrocellulose tubes

containing 0.15 ml of0.11 M NaCN. The samples were

sonically treated to disrupt the cells, and 0.15 ml of the

extract wasadded to 4.9 ml of sensitized E.coliB in a

Klett-Summerson colorimeter tube. The tube was

incubated at28C, andoptical density readings were

takenat 2-min intervals.

RESULTS

Inhibition oflysis. The results of plating mix-tures of phage Lys- with Lys+ phage (both are

clearmutants) are shown in Fig. 1.Proximity of thesmaller mutantplaquestothezoneoflysis of the larger phage plaques results in an apparent impairmentof thelysisof the latter. The effectis observedwhen thetwophageplaquesin

question

are closetogether. Thelarger plaque is

produced

by theLys+ phageandthe smaller bythe mutant

Lys- phage. The white zones surrounding the smaller plaques are not halos but are caused by bacterial growth.

Delayedlysis inliquid culture. The reduced size

of the mutant plaque suggests that the latent

period of the mutant is extended in time. The time of lysis of Lys--infected cells in liquid culture isobservedtobe

delayed

by25 to30 min. This delay period may extend in M9CAA to 40 min, and to more than 3 hr in unsupplemented M9 medium. Once lysis begins, the rate is the same for the Lys--infected cells as for

Lys+-infected cells. Thetime of

lysis

isdetermined

by

the genotype of the phage without apparent alterationof thetimingby superinfection. Thisis consistent with the observation that phage P22 normallyshows exclusion ofa secondarily infect-ing phage (14).

To determine whether Lys--infected cells, at

the higher cell concentration (108/ml), require moreoxygenthanwild-type infectedcells, Nephelo culture flaskswereadaptedtobubble pure

[image:2.485.265.452.422.619.2]

water-saturated oxygen into the

swirling liquid

in the

FIG. 1. Plaque morphology of Lys+andLys-phages

onmixedlyinfectedplates.

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DELAYED LYSIS WITH PHAGE P22

flask. Theincreased oxygen had no effect on the time oflysis: theLys+-infected cells lysed in the normal time period and the Lys--infected cells lysed some 40 min later.

Latent and eclipse periods. The results of a typical one-step growth experiment indicated that thelatent period, after 2,500-fold dilution of the infected cellsin M9CAA medium, was the same for Lys-- andLys+-infectedcells. Apparently the dilution allows lysis to occur which would be delayed in Lys-infectionsat a cell concentration of108/ml. The burst size calculated per infective center present after 10 min of the infection was 266 for the Lys+ and 520 for the Lys- phage infections, the yield of Lys- phage being approxi-mately twofold greater than the Lys+.

The eclipse period, which represents the time interval betweeninfectionandappearance of the first intracellular infective particles, is shown in Fig. 2. Theeclipseperiods ofLys- and Lys+ phage

10

lol

are comparable, even though lysis of the Lys-culture-as determined turbidimetrically-was delayed by 10min andwasless extensive.

Lysozyme synthesis during Lys- and Lys+ infections. The delay in lysis observed in the dense liquid cultures ofLys--infected cells could be due to delay in the synthesis of endolytic enzyme. Cells were grown in M9CAA medium; one culture was infected with Lys- phage and theother withLys+phage, each at a multiplicity of20. Klett readings were taken atintervals to determine the time of lysis, and samples were withdrawn and assayed for lysozyme. Lysis began in theLys+-infectedcells at 45 min and in the Lys--infected cells at approximately 65 min of the infection. The results of the lysozyme assays (28 C) are presented in Fig. 3A and 3B. Samples taken at 30minof theinfectionshowed higher enzyme activity inLys--infected cellsthan inLys+,but by 40 min theactivitiesappearedto

be about the same.

Double infection ofbacteriawithLys- andLys+ phage. Simultaneous infection with Lys- and Lys+mutant phageproducedmostinterestingand unexpected results. The cells grown on M9CAA medium, washed, and aerated in phosphate-buffered saline were infected with Lys+ phage

(multiplicity

ofinfection = 20),Lys-phage (mul-tiplicity ofinfection = 20),orbothphage (multi-plicity ofinfection = 10foreach). After allowing 5 min for attachment, prewarmed M9CAA me-diumwasaddedtothecells inasidearm

Nephelo

cultureflask (Fig. 4). TheLys- mutation appar-ently isnotonlydominant,but, in mixed infection withLys+ phage,

lysis

isinhibitedeven morethan

~~-

35-30 25t

20

15

10

5

z

Hi

y

k

N

9 b,-0

C

0 20 40 60 80

TIMEAFTER INFECTION(min)

100

r

A

80 ;

60-40

-20

0

0 2 4 6 8 10 12 14

INCUBATION

K 0 2 4 6 8 10 12 14 TIME (minutes)

6 FIG. 3. Lysozymeactivities insamples taken at

vari-10 O 3 4 5 1 7 oustimesduringLys-andLys+ infectionsandassayed

onsensitized E. coliBat28 C. (A) lysozymeassaysof

TIME AFTER INFECTION (Minutes) Lys+-infected cells; (B) assays of Lys--infectedcells.

FiG. 2. Intracellular development of phage particles 0, Sensitized cells with no cell extract; *, assay of

inLys- (-) andLys+ (0) infections. Opticaldensity cellextractsfrom samplestakenat10 min; A,assayof

readings and chloroform-lysed plating samples ofthe 20-minsamples;A,assayof30-min samples; Aassay

culturesweretakenatthe timesindicated. of 40-min samples; 0),assayof 50-min samples.

VOL. 4,1969

211

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COHEN

30

25

(I)

z

J

20

15

10

O 10 20 30 40 50 60 70 80 90 100 110

TIME AFTER INFECTION (minutes)

FIG.4. Double infection of S. typhimurium in

M9CAA withLys-phage (0)andLys+ phage(0) or

with Lys- and Lys+ phage (A). Multiplicities of

in-fection totaled 20ineach infection.

in Lys--infected bacteria. When the infections weresynchronized by allowingattachmentin the presence of 0.01 M NaCN followed by dilution

into M9CAA medium, the resultswerethesame.

However, when unsupplemented M9 salts

me-dium was used instead of M9CAA, the results

indicated that the Lys- mutation in doubly

in-fected cells is still dominant in that lysis is

de-layed. However, the lysis of the cells infected

solely withLys- phage isdelayed forsome 3hr.

When L broth was added to the unlysed

Lys--infected cells to a concentration (v/v) of2.5%,

lysis followed in15to20min (3).

Is theLys amutant ofphageP22? Strainsof

S. typhimuriumhave beenshown toyielda

num-ber ofP22-relatedphages (18, 19) which canbe

distinguishedfrom P22onthe basisof hostrange and morphological, physical, andimmunological differences. The following observations lead to

the conclusion thattheLys-isolate is a mutant

of P22:wefound it identicaltoP22phageunder

an electron microscope (1). The bacterial strain

which yielded the Lys- phage was treated with

ultraviolet light followed by plating ofthe

ultra-violet-treated bacteria; this treatment gave

con-fluentbacterialgrowthoverthe surface of nutrient

agar plates (39 plates having approximately 108

bacteria per plate) without yielding any phage.

This mutant will form plaques on the bacterial

background on which it was isolated. The

mu-tants isolated will not plate on cells already

lysogenic for phage P22 nor on strain St/22.

Antiserum prepared against P22 Lys+c2- phage gave essentially identical K values with P22 Lys+c+ and Lys-c2- phage (Lys+c+, 7,720

min-;

Lys-c2, 7,860 min-'), indicating that, on the basis of rates of inactivation in the presence of anti-serum, the Lys-c2- phage is indistinguishable from wild type. These data were obtained from experiments in which Lys+c+ and Lys-c2- phage (distinguishable on the basis of plaque mor-phology) were inactivated together in the same reactionwith antiserum. The Lys+c+ and

Lys-c-2

phage heat inactivate (78 C) at identical rates and show identical kinetics of attachment to sensitive bacteria (98 % within 4 min).

DISCUSSION

A number of observations indicate that the Lys- phage is indeed a mutant derived from a Lys+ P22 and not a P22-related phage (18, 19): thephage areidentical to P22 under an electron microscope and are serologically indistinguish-able from P22. Heat inactivation data, kinetic studiesof attachment,and the absenceofplating ability on Salmonella lysogenic for P22 or on

strain St/22 are also in agreement with the con-clusion. It is unlikely that the Lys- phage was

already present in theultraviolet-treated bacteria

as a prophage; ultraviolet light administered to

the Salmonella LT-2 strain 527-1 (on which the mutant was isolated and does plate) did not

produce plaques. Furthermore, the Lys-mutant

isaclearplaqueformer which wouldnotnormally be expected had it been a prophage in the host cells.

The Lys- mutant of Salmonella phage P22 exhibits some of the growth characteristics of phages that are subject to lysis inhibition. It

apparently inhibits normal P22 phage develop-ment when the two plaques arejuxtaposed on a

petri plate, showsdelayedlysis in

liquid

cultures ofhigh bacterialtiterespeciallyinminimal

growth

medium, but shows normaltiming of lysiswhen the culture is sufficiently diluted. Infected cells deliberately superinfected after 10and 20minof infection werenot,however,delayedintheir

lysis

ifdiluted. It appears, therefore, that dilution it-self enables lysis to proceed at normal times. Perhaps delayed lysis in concentrated Lys--infected cellsuspensions is due tothe accumula-tionofalysozymeantagonistwhich,in thediluted cultures,isattoolowaconcentrationtobe effec-tive. Attemptstoshowthe presence in theculture fluid of a substance capable of inhibiting lysis have not yet been successful.

Intracellular phage development proceeds normally in the Lys- mutant infections, and endolyticactivity,asassayedon sensitizedE. coli

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DELAYED LYSIS P22

B, is the sameorevenhigher than normalatall stagesoftheinfection. Harris et al. (8) described mutantsofphageX which produce lysozymebut donotlyse,and Emrich (5) describeda"spackle" (S-) mutantof phage T4 which lyses eventhough no lysozyme is produced. It is clear that the presence of lysozyme is insufficient to induce lysis; lysis is strongly influenced by at least one

other phage-induced product (6, 7). A normal function of a second gene product, perhaps the S+ gene product described by Emrich, may be consideredasantagonistictolysozyme, stabilizing thecell membraneor wall, orboth, until late in theinfection. Since the S+ in T4 isdominant,it is presumed to produce the active product. Em-rich's mutant T4 S- is abnormally sensitive to

lysis from without and, like r-, itis not subject to lysis inhibition. This suggests that lysis in-hibitionmightbe the result ofstimulated produc-tionoftheS+ geneproduct.Inthe T-even system, the r+ locus could be considered to control the rate ofproduction of S+ substance.

Speculating

further,

the P22 Lys- mutantand certainmutantsofX (8) wouldbeover-producers of that stabilizing factor. Indeed, double infec-tions with Lys-andLys+ mutantphage indicate that the Lys- mutation is dominant, and Harris reported that infection of induced wild-type X lysogens with mutant ts9B (the

lysis-inhibited

X mutant) results in delayed lysis as iflysis were impeded bythe ts9B gene product.

The phage type most prevalent, and hence classified aswild type among the virulentT-even phages,is

subject

to

lysis

inhibition,

whereas it is

therare mutantform oftemperate

phages

X and

P22 which shows

delayed lysis.

In

phage

T4

infections,

delayed lysis

is associated with much higher yields of phage per infected bacterium. This higher yield is observed in infections with the Lys-mutantofP22,

especially

ininfections in unsupplemented M9 medium.

Preliminary

ob-servations in our

laboratory,

however, indicate that the Lys- mutation reduces the

efficiency

of phage integration and

lysogeny.

If selection in temperatephagesoperatesin favor of the

capacity

to

lysogenize,

this

might

accountforthe

rarity

of lysis-inhibited types among temperate phages X and P22.

ACKNOWLEDGMENTS

I am indebted to Mary Showers for her able and dedicated assistanceduring the conduct of the experiments, to GerhardOtt for his fine photography, to FredEiserling (UCLA) for electron microscopy of the mutant phage, and to N. Yamamoto and D. Berkowitz for their generouscontributions of S.typhimnuriumLT-2 strainSt/22 andstrains527 and527-1.

This work was supported by Public Health Services grant Al 08671 from the National Institute of Allergy and Infectious Dis-eases.

LITERATURE CITED

1. Anderson, T. F. 1961. On the fine structure of the temperate bacteriophagesP1, P2 and P22. Proc. European Regional Conf. Electron Microscopy, Delft. Almqvist and Wiksell, Uppsala.

2. Campbell, A. 1961. Sensitive mutants of bacteriophage X. Virology 14:22-32.

3. Cohen, L. W. 1969. Delayed lysis, withSalmoniella bacterio-phage P22: induction oflysis byaddition of cysteine or histidine to the growth medium. J. Virol. 4-214-218. 4. Doermann, A. H.1948. Lysis and lysis inhibition with

Escheri-chia colibacteriophage.J. Bacteriol. 55:257-276. 5. Emrich, J. 1968. Lysis of T4 infected bacteria in the absence of

lysozyme.Virology 35:158-165.

6. Groman,N.B., and G.Suzuki.1962.Relation of endolysin to lysis by lambda bacteriophages. J. Bacteriol. 84:596-597. 7. Groman,N. B.,andG.Suzuki. 1963. Quantitative study of

endolysinsynthesis during reproduction of lambda phages. J.Bacteriol. 86:187-194.

8. Harris, A. W., D. W. A. Mount, C. R. Fuerst, and L. Simino-vitch. 1967. Mutations in bacteriophage lambda affecting host celllysis. Virology 32:553-569.

9. Hershey,A.D.1946a. Mutation of bacteriophage with respect

totype ofplaque. Genetics31:620-640.

10.Hershey, A. D.1946b. Spontaneous mutations of bacterial vi-ruses.ColdSpringHarbor Symp. Quant. Biol. 11:67-77. 1 t.Jacob, F., C. R. Fuerst, and E. L. Wollman. 1957. Recherches

surlesbact6rieslysogenes defectives.II. Les types physio-logiqueshiesauxmutations du prophage. Ann. Inst.

Pas-teur93:724-753.

12.Levine,M. 1957.Mutations in the temperate phage P22 and lysogeny in Salmonella. Virology3:22-41.

13. Levine, M., and R. Curtiss. 1961. Genetic fine structure of the Cregionofthelinkagemapofphage P22.Genetics 46:1573-1580.

14. Rao, R.N. 1968. Bacteriophage P22 controlled exclusion in Salmonellatyphimurium.J.Mol. Biol.35:607-622. 15. Smith,H.O., and M. Levine. 1964. Two sequential repressions

of DNAsynthesisin theestablishmentoflysogenyby phage P22 anditsmutants.Proc. Nat. Acad. Sci. U.S.A. 52:356-363.

16. Weigle, J. 1953. Induction of mutations in bacterial virus. Proc.Nat. Acad.Sci. U.S.A. 39:628-636.

17. Whitfield,H. J., R.G. Martin, and B. N. Ames. 1966. Classi-ficationofaminotransferase (Cgene) mutants inthe histi-dineoperon.J. Mol.Biol.21:335-355.

18. Yamamoto, N., and M. L. Weir. 1966. Genetic relationships between serologically unrelated bacteriophages P22 and P22lb. Virology 28:168-169.

19. Young,B.G., P. E. Hartman, and E. N. Moudrianakis. 1966. Some phages released from P22 infected Salmonella. Vi-rology 28:249-264.

VOL. 4,

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Figure

FIG. 1.on mixedly Plaque morphology of Lys+ and Lys- phages infected plates.
FIG. 3.Lys+-infectedcellonous20-minof0, sensitized 40-min Lysozyme activities in samples taken at vari- times during Lys- and Lys+ infections and assayed E
FIG. 4.fectionwithM9CAA DoubleinfectionofS.typhimuriumin with Lys- phage (0) and Lys+ phage (0) or Lys- and Lys+ phage(A).Multiplicities of in- totaled 20 in each infection.

References

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