Copyright ©1969 American Society forMicrobiology Printed inVol.4, No.U.S.A.3
Delayed
Lysis
with
aMutant
of
Salmonella
Bacteriophage
P22
LARRY W. COHEN
SeaverLaboratory ofBiology, PomonaCollege, Claremoiut, Ccaliforlnia91711
Receivedforpublication21 May 1969
A mutantofbacteriophage P22
(Lys-)
wasisolated which showsa plaquemor-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 insuffi-ciently close
proximity
on a petridish,
the r-plaque seems to beinhibited in itsdevelopment,
exhibitingaflat sideadjacent tothe r+
plaque
oreven an inverted crescent border.
In temperate phage X a mutant
showing
de-layed lysis has been observed (6). This paper describes a mutant of Salmonellaphage
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 isproduced
by theLys+ phageandthe smaller bythe mutantLys- 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 forLys+-infected cells. Thetime of
lysis
isdeterminedby
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 theFIG. 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 inasidearmNephelo
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
A80 ;
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
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[image:3.485.37.224.288.603.2] [image:3.485.245.436.436.565.2]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+ andLys-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 bacterialtiterespeciallyinminimalgrowth
medium, but shows normaltiming of lysiswhen the culture is sufficiently diluted. Infected cells deliberately superinfected after 10and 20minof infection werenot,however,delayedintheirlysis
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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[image:4.485.54.242.64.266.2]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 (thelysis-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
tolysis
inhibition,
whereas it istherare mutantform oftemperate
phages
X andP22 which shows
delayed lysis.
Inphage
T4infections,
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 ourlaboratory,
however, indicate that the Lys- mutation reduces theefficiency
of phage integration andlysogeny.
If selection in temperatephagesoperatesin favor of thecapacity
to
lysogenize,
thismight
accountfortherarity
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.
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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.
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