Copyright©1968 American SocietyforMicrobiology Printed in U.S.A.
BRUCE MOLHOLT' AND DEAN FRASER
Department ofMicrobiology,IndianaUniversity, Bloomington, Indianla47401
Receivedfor publication 11December 1967
Restriction of nonglucosylatedT2phage (T*2) as afunction of bacterial growth
statewasthesamefor endonucleaseI-containing and endonucleaseI-deficientstrains ofEscherichia coliB.Furthermore, E. colistrains withvarious levels ofrestriction
for T2 hadcomparableendonuclease Iactivities. ItwasalsofoundthataT4mutant
temperature-sensitive for gene 46 and 47functions wasfullyrestrictedat 42 C.It
activitiesareblockedbymutations in genes46 and47catalyzethe initialevent in
restriction ofnonglucosylated T-even phages.
bacteriophagesis unusual in that it contains
cytosine.These HMC residues are
infectedwith T2, T4, or T6
16, 26, 29).
nonglucosylatedwhether or not the host
This and the
successful infection ofEscherichia coli B. The DNA
partiallybroken down to
(10, 15).Like T2
one-third ofthe bacteria to which
It had been
suggestedthat endonuclease I
hydrolysisof T* DNA
(10, 22).However, recent
have shown that both endonuclease
'Present address: Department of Microbial
Genetics, Karolinska Institutet, Stockholm 60,
Ph.D.Thesis,Indiana Univ.,Bloomington, 1967)
are fully restrictive. In
addition,nonrestricting strainsarefoundtocontain normallevels of
en-donucleaseI (6, 24).Theaccompanying paper (6)
shows that exonucleases I, II, and III are also
found in normal levels in nonrestrictingbacteria.
The initiallesion inrestriction, therefore, does not appear to be catalyzed by any of the well-characterized deoxyribonucleases ofE. coli (19). We have also examined another set of nucleases
found in the T-even-infectedcell, those nucleases
controlled by T4 genes 46 and 47 which are
active in the breakdown of host DNA
wasfoundthatrestriction occurs intheabsence of
gene46 and 47function.
MATERIALS AND METHODS
Bacteria. The bacterial strains used in this study
arelistedin Table 1.
Bacteriophages. All T2 strains used were derivatives of an rI mutant of T2L. T*2 was made by several
cycles of growthonMT2002orMT1 102. The glucosyl
transferaseless mutant, T2gt, was isolated by plating
hydroxylamine-treated (28) T2 on a double
in-dicator composed of Rst- and Rst+ bacteria and
selectingturbidplaques (25). T4tsL109 and T4tsL86,
temperature-sensitive mutants of T4D in genes 46
and 47, respectively, were obtained from 0. Skold.
The double mutant T4tsL86L109 was isolated
ac-cordingtothe methodofWiberg (34). T4rII638 is a
deletionmutant for the entire rIlB cistron, obtained
from S. Benzer; T*4r11638 was grown on W4597.
OX174wasobtained from R.L.Sinsheimer.
Media. 3XD wasasdescribed in Fraserand Jerrel
(9); Dmediumwas one-thirdstrength 3XD. Lbroth
contained (grams per liter): tryptone (Difco), 10;
on November 11, 2019 by guest
TABLE 1. Bacterialstrains
colistrain Source (reference) Characteristicsa
Sinsheimer, his strain
galactose-negative mutant of
Arber, his strain
Bc251 (2) Nitrosoguanidine
MTI 101 Boyer (4)
Nitrosoguanidine mutagenesis of
Benzer, his strain B (3)
Nitrosoguanidine mutagenesis of MT1500
Hoffmann-Berling, hisstrain B41
Wild type is
low level of
high levelof restriction
galU--Rst- is the phenotype indicating absence of
restriction of nonglucosylated T-even phage.
Thegenotypesrstand galUrefertogenes control-lingrestriction and modification ofT-even phages.
Mutant rst-2maps nearhis in E.coli B(unpublished
data); galUis thegenefor UDPGsynthetase (31).
Thegeneinvolved in thesynthesisofendonuclease
I is designated dnsA.
yeast extract, 5; glucose, 1; and NaCl, 10; it was
adjusted to pH 7.0 with NaOH. Basal agar plates
were either L agar (L broth plus 1.5% agar) or
8; NaCl, 5; agar, 12. Soft agar wasthe same as
contained (gramsperliter):Na2HPO4, 2.9; KH2PO4,
1.5; NaCl, 4.0; K2SO4, 5.0; MgSO4, 0.12; CaCl2,
0.01; gelatin, 0.01; and chloramphenicol, 0.02. For
adsorption ofT4, 20 ,ug of L-tryptophanpermlwas
N-methyl-N'-nitro-N-nitrosoguanidine was obtained from Aldrich
Chemical Co., Milwaukee, Wis., and was used as
describedbyAdelberg, Mandel, and Chen (1). Restriction assays. A 0.2-ml amount of bacteria
at a concentration of108 to 109 cells perml was
in-cubated with 0.1 ml T*2 at 108 particles per ml for
10min at 37 C in 3XD orLbroth. Infectious centers
were then assayed, either by dilution into soft agar
seededwithE. coliB orby adding 2mlof B-seeded
soft agar directly to the adsorption tube. The yielder
frequency (ratio of infectious centers onagivenhost
toinfectious centersonMT2001) is usedas a
quanti-tativeexpression of restriction.
Preparation ofqX174DNA. The hot phenol
tech-nique of Guthrie and Sinsheimer (14) was used to
OX174DNAfrom phage grownon
MT2001 in 3XD medium. Routinely, 108 infectious
DNA molecules per ml were obtained from
stocks at 101l phage per ml.
Endonuclease Ipreparation. The enzyme was
pre-paredasthecrude supernatant fractionafter
centrifu-gation ofspheroplasts made by the
lysozyme-ethylene-diaminetetraacetate (EDTA) method of Guthrie and
Sinsheimer (14). EndonucleaseIis released
quantita-tively by this treatment (5, 23). Spheroplast
super-natant fluids were dialyzed in the cold against two
changes of 100 volumes 0.15 M
tris(hydroxymethyl)-aminomethane(Tris)-buffered saline (pH 8) toremove
sucrose and EDTA.
Endonuclease I assay. Assay specificity depends
upon the inhibition of endonuclease I by soluble
ribonucleic acid (20) and loss of
in-fectivity through endonucleolytic conversion of
circles to linear molecules (8). Crude extracts
dis-played no activity against
pre-treated with ribonuclease I (see control curve, Fig.
3).Forthe assay, 0.03 ml of 10mg/mlribonucleaseI
(Worthington Biochemicals Corp., Freehold, N. J.)
was incubated with 3 ml of dialyzed spheroplast
supernatantfluid for 30 min at37C. Of the resultant
preparation, 0.2 ml was added to 1.8 ml of
(101"phage equivalents per ml) at 37C, and
0.4-ml portions were removed at timed intervals to
0.4 ml of MT2001 spheroplasts. After 10-min
in-cubation at 37C, 3.2 ml ofprewarmed PAM (14)
wasadded, incubationwas continued for 5 min, and the resulting infectious centers were assayed on
MT2001. Controls consisted of (i) untreated
DNA plus spheroplasts, (ii)
un-treated spheroplast supernatant fluid plus
ribonu-clease Iplus spheroplasts.
Breakdown of ultraviolet-irradiated 32P-labeled
T*2 DNA. MT1102 grown in Tris-glucose medium
(18) supplemented with 2.5 juc/ml of inorganic 32p
(Nuclear Science and Engineering, Pittsburgh, Pa.)
wasinfected with T2. The crude T*2lysatewas
chro-matography and was irradiated at 40 cm from a
Westinghouse Sterilamp for 4 mintogive 100 lethal
hits. Thephagewerethenadsorbedto3ml of MT1100
orMT1101 in T2adsorption bufferat aninput ratio
of 1.0. After3min,theinfected cellsweresedimented,
washed in cold adsorption buffer (without
chloram-phenicol),andresuspended in 1 ml of coldadsorption
buffer (without chloramphenicol). The suspension
was broughtto 37 C anddilutedwith 2ml of37 C
3XD (time-zero). Samples (0.5 ml) were withdrawn
on November 11, 2019 by guest
attimedintervals, dilutedin 2 ml ofcoldDmedium,
andcentrifuged at 7,000 X g for 15 min;the
super-natant fractions were assayed for acid-soluble 32P
Samples were counted inaPackard model 3003liquid
Luria(21) has shown that the
fraction ofT*2 restricted upon
Rst+ strain depends onthe
physiologicalstate of the host. The
dependence of restrictionin MT1 100 on
Fig.1.Restriction of T*2 decreases
completerestriction is restored
regain exponential growth.This loss
of restriction forT*2
suggested the involvement of
10 ir z Li
I.-() 107 0
0 2 4 6 8
HO U R S, 3 7C
Lij 3 -10-
FIG. 1. Effect of aging on restriction ofMTl100 for T*2. An exponential culture ofMTII00 was
di-lutedto107cells/mlin3XDat37C, and2-ml samples
were withdrawn at various timesfordetermination of
total cellcountandyielderfrequency (infectiouscenters
perinfected cell) for108T*2perml.At6 hr(stationary phase), the culturewasdiluted20-fold into fresh 3XD
at37 C(broken line).Symbols: 0 = totalcellcount;
0 =yielderfrequencyfor T*2.
since the specificactivity of this enzyme decreases markedly in the
Endonuclease I levels of four strainsof E. coli, each with a unique level of restriction for T*2, wereexaminedtodetremine whetherasignificant correlation does indeed exist between this enzyme and restriction. Since restriction
dependsupon growth state (Fig. 1), yielder frequencies of these fourstrains arepresentedas afunction ofgrowth state (Fig. 2). The C strain MT2001 is pheno-typically Rst- whereas B strains MT1500, MT1501, and MT1502 are Rst+, but
withthree distinct patterns of restriction which vary over
endo-nuclease I were the
restricting factor,then a hierachy
levels,MT1502 > MT1500 > MT1501 > MT2001,
wouldbe expected. No such
Endonuclease I-deficient mutants have been foundtoretain restriction for
(6, 24, 30). However, these
of theenzyme (J. Eigner and H.
communications),and it is
possiblethat if this
strategically placedin the cell
could still effect
restriction.To test this
by growthstate in an
(MT1540)was studied. If
endonucleaseIwas acriticalfactor in
depletionof the residualamountof enzymewould
LLi -ii LLi
01 2 3 4 5 6 7
HOURS AT 3 7C
FIG. 2. Effect of agingonrestrictionoffourstrains
of Escherichia colifor T*2. Yielderfrequencies were
determinedby assaying infectiouscenterson MTIIO0.
Attime-zero, each strain was dilutedto 10' cells/ml.
Symbols: A = MT2001; 0 = MT1501; A =
MT1500; 0 = MT1502.
on November 11, 2019 by guest
2 5 10
EXPOSURE TO ENDO I
FIG. 3. EndonucleaseIassay ofhosts with various capacitiesforrestriction. Endonuclease Iwasreleased from thefourstrainsof Fig. 2andassayedas ribonu-clease-releasedactivity against infectious
Symbols: X = controlcurve,MT1500endonuclease I
without ribonuclease pretreatment; A = MT2001;
0 = MT1501; A = MT1500; 0 = MT1502.
be expected to occur rather early in stationary
phase dnsA-cells, resulting inan earlier loss of
restriction than seen in aging dnsA+ bacteria.
However, cultures of dnsA- and dnsA+ cellswere
it upondilution, with identicalkinetics (data not
shown). It was concluded that the level of
endo-nuclease I is unimportant in restriction of
T-even-induced nucleases. Two experiments
havebeen conductedtoassessthe role of
T-even-induced nucleases inrestriction, since restriction
could bea"self-digestion" occurringwhen T-even
DNA has lost its protection (glucosylation)
against enzymes active in degrading host DNA.
The first experiment examined breakdown of
32P-labeled T*2 DNA which had been heavily
irradiated with ultravioletlight.Eachparticlewas
subjected toanaverage of 100 lethal hitsto
pre-clude phage-specific enzyme synthesis during
infection. Figure 4 shows that theDNA of such
particles wasbroken down to acid-soluble
prod-ucts upon infection of MT1100
much less breakdown occurred with the rst-mutant MT1101. These results indicate that
phage-induced enzymes are not involved in the
breakdown ofT* DNA.
Genes 46 and 47 inphageT4control nucleases
which catalyze thebreakdown of bacterial DNA
involvement in restriction of the nucleases
0 2 5 10
M N S POSTINFECTION
FIG. 4. Breakdown of ultraviolet-inactivated T*2.
MTII00 (rst+) and MTII0O (rst-) were infectedat
time-zero with 32P-labeled T*2 which had sustained
over100lethal ultraviolethits. Symbols:* =MTI100;
O = MTI101.
temperature-sensitivemutant,T4tsL86L109,wasisolatedfrom acrossbetween T4tsL86 (ts mutationingene47)
and T4tsL109 (ts mutation in gene 46). The
galU- host, W4597, resultinginthe formation of T*4tsL86L109. If the nucleases controlled by
genes46and 47areactive in restriction, the
non-glucosylated double ts mutant should not be
restricted on a Rst+ strain at the nonpermissive
temperature. It is shown in Table 2that
restric-tion occurred normally in strain MT1100 at 42
C. Thus, we concluded that the nucleases
con-trolled by genes 46 and 47 are not involved in
restriction of nonglucosylated T-even phages.
The findings that dnsA- mutants ofE. coli B
and K-12 restrictnonglucosylated T-evenphages
(6, 24, 30; this report) and thatrst-mutants
con-tain wild-type levels of endonuclease I (6, 24;
this report) render unlikely the involvement of
endonuclease I in theprocess of restriction. The
residuum of endonuclease I in dnsA- mutants
does not appeartocatalyze restriction, sincethe
kinetics of restriction loss upon entering
stationary phase was foundto be thesame for
None of the three well-characterized
exonu-cleases ofE. coli (19) can be implicated in the
primarycontrol of restriction (6, 24). However,
further degrade DNA after restriction has
occurred. Such digestion may beresponsible for
the loss in early enzyme-synthesizing capacity
seen inT2gt-infected cells in the absence of pro-teinsynthesis (17).
It ispossiblethat T-evenphageshaveadapted
on November 11, 2019 by guest
TABLE 2. Relation ofgente46and47function to restrictionla
Glcsl-46and 47 +
Phagestrain tion nucleases OnMT1101(rst-) OnNIT1100 (rst+)
At3OC At42 C At30 C At42 C
T4tsL86L109... + 1 1 1 1
T*4r1I638...+... 10-' 10-1 3 X 10-' 6 X 10-'
T*4tsL86L109 . - - 10-1 10-1 8 X 10-' 9 X 10-'
aT4strains were adsorbedto 2 X 109cells/ml inT2adsorption buffer plus
;g ofL-typtophanper mlat amultiplicity of 0.1 for 10min at 30or 42 C. The cells were then
resuspended at 2 X 108 per ml in 3XD at 30 or 42 C, and infectious centers were assayedon MT1100
after a further 20 min of incubation. The yielder frequency is the number of infectious centers formed
per infected cell. Burst sizes of both T4tsL86L109 and T*4tsL86L109 were much smaller onboth MT1100
and MT1101 at42Cthan at30C.
toutilize therestricting factortocatalyze the high recombination rate characteristic of T-even
Accordingtothis model, upon
bacteria, parental T*DNA would look like progeny DNA
accompanyingrecombination. However, this
possibilityappears to be
unlikelysince the rate
of recombination forT-even phages is the samein Rst+and Rst- hosts
breakdown ofT-evenDNA maybea
result,rather than thecause, ofrestriction.
nonglucosylatedT-even DNA as acid-soluble material has
been foundtoaccompanyrestriction in E. coliB
(10, 15), T*6DNA maynotbe
down upon restricted infection of E. coli BB
communication)found that T2is
excluded withoutdetectable breakdown of T2DNA
bycertain strains of E. coli
DNA was made
breakdown ofT*2 DNA is
capacity of phageDNA
this conclusion is at best
indirect,since DNA breakdown and
restrictionmay not be
Evidence that the phage-induced nucleases controlled
bygenes46and47 are notinvolved in
fromthe present studies in which gene 46 and 47products were
inactivated. However, the experiments reported
here donotruleoutcontrol ofrestriction by other enzymes synthesizedduring T4infection. Warren and Bose (33) have demonstrated that T4 prob-ably induces endonucleases that are active in degrading host DNA prior to exonucleolytic cleavage by the gene 46 and 47 controlled nu-cleases. Similar observations have been made by Kutter and Wiberg (personal communication). Since T4 mutants
lackingendonucleolytic activ-ity have not been
identified,the specific effect of these enzymes on restriction has not been as-sessed. Inaddition to the ability to induce endo-and exonucleases
activeagainst host DNA, T4 appears to possess a gene (ex) that controls breakdown of T2 DNA during T2-T4 mixed infection
100% glucosylated,T2 is not
excludedby the T4 ex gene product,
T*4 isunlike T*2 in that it
failstoundergo multi-plicity activation on rst+ hosts (15). Also, T*4 is generally morerestricted than is T*2 by rst-
frequency(0.1) of T*4 on MTllOl]. These observations
implicatea T4-induced product, perhaps
ofthe ex gene, which acts to restrict T*4, in addition to those restricting fac-tors active during T*2
byT*4 on Shigella is dueto the loss of
synthesisofsome phage pro-teins on
specificallythose which shut off host
Nevertheless, it is presumed that restriction is
a host-controlled process since changes in the
ofthe host cell and loss of gene rst
drasticallyalter the restrictive response ofE. coliB for
phage, and since the breakdown of T* phage which
accompaniesrestriction occurs in the ab-sence
on November 11, 2019 by guest
Weacknowledge the receipt ofbacteria and phage
specifically for these experiments from W. Arber, H. Boyer, T. Fukasawa, H. Hoffmann-Berling, and
0. Skold. We also thank W. Arber, J. Eigner, H.
Hoffmann-Berling, andJ.S.Wiberg for making
avail-able details oftheir work prior to publication, and
W.Arber, G. Bertani, J. Eigner, and S. Linn for their
critical reading of themanuscript.
These studies werepartially completed at Indiana
University through the support of Microbiology
Training Grant 5T1-GM-503 (to B.M.) and Grant 5
ROI-CA-02772-11 (to D.F.) from the U.S. Public
Health Service. Part of thesestudies were also
sup-ported by apostdoctoral fellowship, I-F2-AI-16,812,
from the U.S.Public Health Service.
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