J. gen. Virol. (I972), I6, I85-I97
I8 5
Printed in Great Britain
Biochemical Changes during Mixed Infections with
Bacteriophages T2 and T4
By N A H E E D
MAHMOOD
AND M A R Y R. L U N T
Microbiology Unit, Department of Biochemistry,
South Parks Road, Oxford, England
(Accepted Io April I972)
SUMMARY
Biochemical changes accompanying the partial genetic exclusion of wild-type
T2 genes in mixed infections with various mutants of phage T4 have been examined.
Total phage yields varied between I4 and 6o ~o of those observed after infections
by either wild-type phage alone. DNA synthesis was delayed, but then occurred at
rates close to those found with each wild-type phage strain. Among the progeny
virus, the frequencies of the excluded wild-type T2 genetic markers ranged between
0-o2 and o'45, and varied with the markers' positions on the genetic map. Measurement of the frequencies of markers among the progeny phage at t 5 to 6o min. after
infection showed that the weakly excluded T2 genes I and 2o increased slightly,
while the strongly excluded Tz genes 33 and 42 dropped sharply and then remained
constant. The strongly excluded T2 genes 42 and 56 formed low levels of their
respective products deoxy-CMP-hydroxymethylase and deoxy-CTP-ase in mixed
infections with the corresponding T4 amber mutants in a non-suppressor host.
The T2-induced enzymes thymidylate synthetase and lysozyme reached normal
levels during infections with the corresponding defective T4 strains. With mixed
infections in an endonuclease-I deficient host, [a2P]-labelled Tz phages yielded I4 ~o
of their D N A as acid-soluble products, while no breakdown of [32P]-labelled T4
DNA was detected. The possibility is discussed that partial exclusion results from
T4-induced nuclease action against T2 chromosomes followed by marker rescue of
T2 DNA fragments with intact T4 genomes.
INTRODUCTION
When a bacterium is simultaneously infected with two unrelated kinds of phage only one
virus type appears among the progeny. The phenomenon is known as exclusion. With
genetically related strains such as T2 and T4, recombination may occur and genetic markers
from both parents appear among the progeny, although markers of one parent, in this case
T4, predominate. This is known as partial exclusion. It is accompanied by lower yields of
phage than are observed with infections by T2 or T4 alone (Delbri)ck & Luria, I943;
Delbr~ick, I945; Streisinger & Weigle, I956; Adams, I959; Campbell, 1967).
Genetic aspects of the partial exclusion which occurs between phages T2 and T4 have
been extensively studied by Russell (I967) and de Groot (I966; I967a, b; Pees & de Groot,
I97o) who have largely used phage amber mutants which grow on suppressor (su +) and
not on su- strains of Escherichia coli. Their results, together with those of Stahl & Murray
(I966), show that the genetic map ofT2 closely resembles that ofT4 (Fig. I ; Edgar & Wood,
~966; Mosig, I97o), and that in mixed infections in su- hosts wild-type T2 can supply a
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I86
N. MAHMOOD
AND
M. R. L U N T
55
4 46 4 ~ " -
e
1
8
42
rl
20
37%
3 5 4 ~ _
63
Fig. I. Genetic map of bacteriophage T4 showing genes mentioned in this paper. The diagram is
based on that of Mosig (197o) in which positions are based on DNA distances. (SimplifiedFig. is
published with permission from the Chemical Rubber Co.)
function which cannot be provided by an amber mutant of T4. In addition genetic markers
from certain regions of the T2 genome appear much less frequently than others among the
phage progeny. In particular, the T2 genes 32, 56, 60 and the region between genes 4I and 55
are strongly excluded in crosses with T4, i.e. they appear with frequencies of about o-ooi to
0"05o among the progeny phage. Less strongly excluded regions appear with frequencies in
the range o.1 to 0"4 (Russell, 1967; de Groot I9672, b; Pees, I97o).
The mechanism of partial exclusion is not understood. In the experiments described here
we have examined some biochemical changes which occur during mixed infections with T2
and T4 particularly with a view to deciding whether T4-induced nucleases are involved in the
exclusion process.
METHODS
Bacteriophages. Phage T2(H) was originally obtained from Dr A. D. Hershey; wild-type
T4(D) was obtained from Professor N. Symonds, University of Sussex; rIi and amber
mutants of T4 were supplied by Dr S. Brenner, MRC Laboratory of Molecular Biology,
Cambridge, and Dr R. S. Edgar, California Institute of Technology. Stocks of phages T2,
T4 and T4rII mutants were obtained by lysis ofE. COIiBB and T4 amber mutants by lysis of
the amber suppressor (su +) strain of E. coli CR63. The ' e' mutant T4G59 was supplied by Dr
G. Streisinger, University of Oregon, and was grown and assayed according to the procedures described by Emrich (I968); the mutant cannot induce formation of the phage
specific endolysin, or lysozyme. The mutant T 4 t d m cannot induce phage thymidylate
syntbetase and was described by Simon & Tessman (1963); it was grown and its identity
checked by methods described by Wulff & Metzger (1963). Phage labelled with [a2p] in their
D N A were obtained by lysis of 25 ml. broth cultures of E. coli to which o-I to 0"25 m c
of carrier-free [a2P]-pbosphate was added 3o min. before infection, incorporation of [a2p]
into phage particles varied from about 4 x 106 to I-6 x I o 7disintegrations/min./I o1~particles.
Phage preparations were routinely purified by differential centrifuging, and radioactive phage
suspensions were also dialysed over 24 hr against 2 changes of IOOOvol. of o.Io M-NaCI+
o-o2 M-MgC12. Phage were assayed by the double layer method (Adams, 1959), using
appropriate resistant strains of E. coli to check purity.
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M i x e d infections with phages T2 and T4
I87
Bacteria. In addition to the strains already mentioned, the following were used: E.coliB3oo5
(thy-, ade-) as a restrictive host for T4tdIo; E. eoli o'II'(su+); both these strains were
obtained from D r S. Brenner. E. coli B. 4I which is deficient in the enzyme endonucleaseI was supplied by Dr H. Hoffmann-Berling, Heidelberg, Germany.
Media. Broth contained I o g . Oxoid tryptone, 5 g. NaC1/1. Phage absorption medium was
described by Hershey & Chase (I95Z); it was supplemented with DL-tryptophan (2 #g./ml.)
immediately before addition of phage. Phage dilution medium contained o.I I M-NaC1 + o-o×
M-MgCI~ + o.oI M tris-HCl, p H 7-2 + 0"07 ~o gelatin. M9 medium was prepared according to
S6chaud et al. (I965).
Radioactive materials. Carrier-free [3~P]-phosphate and [14C]-paraformaldehyde were
obtained from the Radiochemical Centre, Amersham, Bucks. The paraformaldehyde was
depolymerized by maintaining it at I ~o° with water in a sealed tube for 2 to 3 days.
Conversion of [32P]-labelledphage DNA to soluble fragments in mixed infections. One ml.
samples of the infected cultures (5 × Io8 bacteria) were placed in chilled tubes which contained o'o5 ml. of 5 M-HC104 and o'o5 ml. of bovine serum albumin (Armour; Io mg./ml.)
as carrier protein. After 3o min. at o ° the tubes were centrifuged, the supernatant fluids
removed, and each precipitate dissolved in ~ ml. of o.I M-NaOH; o.2 ml. samples of these
solutions and the original supernatant fluid were neutralized and separately dried down on
aluminium planchettes and their radioactivity measured in a low background counter
(Low betamat, Isotope Developments, Ltd). For volumes up to o'3 ml. the counts recorded
were directly proportional to volume; more than Iooo counts were recorded for all samples
and the standard error was usually not more than 3 %. The results were expressed as the
percentage of the total [32p] converted to acid-soluble form.
Growth and infection of bacteria. For all experiments bacteria were grown in broth to a
cell density of 5 × I o7 to I × IoS/ml., centrifuged, resuspended in absorption medium at a
density of I × Io°]ml. and starved with gentle aeration for 3omin. Appropriate phage
suspensions were then added, and after 5 n-fin, the infected bacteria were diluted into warm,
aerated broth.
Measurement of exclusion. Starved bacteria were infected with equal numbers of wild-type
T2 and T4, or mutant T4, at total multiplicities of 8 to 12 phages/bacterium. After 5 min.
unabsorbed phages were inactivated with T2 and T4 specific anti-sera, and the bacteria
diluted into broth. Samples were immediately assayed to measure initial infective centres.
After 6o min. the cultures were shaken with chloroform and the progeny phages assayed.
Exclusion was measured as the ratio of the number of wild-type plaques to the total number
of plaque-formers. This method was used even with amber mutants of T4, despite the fact
that the efficiency of plating of wild-type Tz(H) on the su + strain E. coli o - I I' is only o'5
relative to that on E. coli B. Since the progeny phage from T2 × T4am crosses must include
recombinants for all regions of the genomes not all phage with the am + genotype will
necessarily have the lower plating efficiency on strain o - I x ' . Exclusion was therefore
measured as the ratio of number of progeny plaques on E. coli B to the number formed on
E. coli O-II'.
Measurement of DNA synthesis by infected bacteria. The infected bacteria were diluted
into warm broth to give a cell density of 5 × IO8/ml. At intervals z ml. samples were withdrawn, chilled and precipitated with o'o5 ml. of 5 M-HCIO4 and o'5 mg. bovine serum
albumin as carrier prior to hydrolysis and determination of D N A deoxyribose according to
Burton (I956).
Protein estimations. The method of Lowry et al. (I95I) was used.
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N. M A H M O O D A N D M . R . L U N T
Enzyme assays
Deoxycytidylate hydroxymethylase. The tetrahydrofolic acid required for this and the
following assay was prepared from commercial folic acid (British Drug Houses, Ltd)
according to Jones, Guest & Woods (I96I). The enzyme assay was based on those of Flaks
& Cohen (I959) and Wiberg & Buchanan (I964). A total of o'35 ml. of enzyme incubation
mixture contained: l o # m o l e of potassium phosphate buffer, pH 7, I'25#mole-[14C] formaldehyde (o.5/~c/#mole), o.25#mole-tetrahydrofolic acid, 5#mole-MgSO4, 2"5
#mole-deoxycytidine monophosphate (d-CMP; Sigma Chemical Co.), 5 #mole-fl-mercaptoethanol and o-I to o.2 rag. protein from crude extracts. The mixtures were incubated at
37 ° for 4o min. The reaction was stopped by chilling the tubes and precipitating the proteins
with o'35 ml. of IO % (w/v) trichloroacetic acid. After 30 min. the precipitates were removed
and o'35 ml. portions of the clear supernatant fluids were heated (IOO°) for IO min. with
o'35 ml. of 1% (w/v) FeC13 solution in I N-HCl. Samples (o.I to o.2 ml.) of the heated
supernatant fluid were placed on glass fibre discs (GF]A, Whatman) with o.i to o.z ml. of
i N-HC1 and dried down under an infra-red lamp. The dried discs were placed in vials with
5 ml. of scintillation fluid (Butyl-PBD, Ciba, Duxford, Cambs. ; 7 g.]l. of toluene) and the
radioactivity was measured in a Beckman scintillation counter using the isoset for [1~C].
The standard error for the counts recorded was usually not more than 2 %. Enzyme activity
is expressed as t~mole of formaldehyde converted to acid-stable, non-volatile form in I hr]mg.
of protein.
Thymidylate synthetase activity. The assay procedure was identical to that used for d-CMP
hydroxymethylase except that deoxyuridylate (d-UMP Sigma Chemical Co.) was used
instead of d-CMP. (Flaks & Cohen, ~959)Assay of deoxycytidine triphosphatase. Five ml. samples of the infected culture (5 x lO8
bacteria/ml.) were chilled, centrifuged and resuspended in 2 ml. of tris-acetate buffer,
o.o2 M, pH 9"I. The suspensions were frozen, thawed and the bacteria broken by sonication.
Enzyme activity was measured with these extracts, using the method of Wiberg (I967).
Assay of lysozyme activity. Two ml. samples of infected cultures were chilled and the
bacteria broken by sonication. After centrifuging, the extracts were assayed by the method
of Greene & Korne (I967) using suspensions of E. coli B as substrate (Jacob & Fuerst,
I958).
RESULTS
Mixed infections: burst sizes and DNA synthesis
The burst sizes were measured, usually in E. coli B; in some instances the amber
suppressor strain E. coli CR63 was used to determine if differences in phage yield or exclusion
depended on whether all the T 4 genes could be expressed. The results are given in Table I.
As expected, the average burst sizes obtained from mixedly infected bacteria were always
lower than those from bacteria infected with either T2 or T4 wild-type phage alone. In
certain instances, namely when T4 amber mutants in genes 46, 47, or 2o were used in mixed
infections, the burst sizes were greater with the amber permissive host E. coli cR63 than when
E. coli B was used.
The results summarized in Table z indicate that in the mixed infections shown, D N A
synthesis was delayed, but then occurred at rates comparable to those in wild-type phage
infections.
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Mixed infections with phages T2 and I"4
18 9
T a b l e I. Average burst sizes in mixed infections between T2 and T4 phages
Cross
Average
burst size
Burst
size range
I68
180
lOO-24o
100-250
96
9I
65-1oo
62-IOO
B and CR63
B and CR63
30
I8
30
25-32
B
21
60-85
I4-31
3o-45
4o-6o
82 90
8o-94
85-96
40-60
5t-7o
73-86
4I-7I
76-I39
47-93
c~63
a
CR63
B
c~63
B and
B and
B and
B and
a
B
B
B
2I
25
20
3
3
4
I4
I2
21
8
4
3
2
T2 × T2
T4 x T4
Tz × T 4 amC76 (gene 42)
Tz × T4 amBz2 (gene 43)
T 2 × T 4 a m N 1 3 o ( g e n e 46)
T2
T2
T2
T2
T2
T2
x T 4 amNI 3o (gene 46)
x T4 amA456 (gene 47)
× T4 amA456 (gene 47)
x T4 am5o (gene 20)
x T4 am5o (gene zo)
x T 4 amA494 (gene I)
T2 x T4 amI34 (gene 33)
T2 x T4 rlI (deletion)
T2 x T 4 G59 (gene e)
T2 x T4 tdIo (gene td)
T2 x T4 amE56 (gene 56)
T2 x T4 amE5I (gene 56)
T2 × T4 amB85 (gene 56)
75
25
40
50
85
87
9I
50
65
8i
56
Io4
70
Bacterial host
E. e o l i
No. of
experiments
B
B
IO0
I OO
CR63
CR63
CR63
cR63
DNA synthesis in cultures of Escherichia coli B infected with wild-type Tz phage and
mutants o f T 4. Each culture contained infected bacteria at a cell density of 5 × IoS/ml.
T a b l e 2.
Time
after
infection
(rain.)
c--
T2
T4
o
Io
I5
2o
3o
60
26
47
59
70
88
I7o
28
46
54
67
92
18o
Amount of D N A synthesized
(/zmoles of DNA-deoxyribose]l. of culture)
*
T2 x T4
T2 x T 4
T2 x T4
am5o
am134
amC76
T2 x T4
gene 20
gene 33
gene 42
3o
35
44
6o
70
I4o
28
4I
44
5o
54
88
25
29
40
62
74
i2o
3o
30
32
44
64
i3o
T a b l e 3. Frequencies of different T2 genes in the progeny of mixed
infections with wild-type T2 and T4 mutant phages
Parental phage types
Gene tested
Fraction of wild-type
phage in progeny
T2 x T4 amA494
T2 × T4 am5o
T2 x T4 ami34
T2 × T4 amC76
T2 x T4 amBz2
T2 × T4 amA456
T2 × T4 amNI 3o
T2 × T4 amE5I
T2 × T4 amE56
T2 × T4 G59
T2 x T4 h +
I
2o
33
42
43
47
46
56
56
e
h+
o.22
0.35-o.40
o.o8-o.lo
o'o5-o'o8
o'o5-o'o8
oqo-o-I5
o.i2-o.2o
o'Io-oq6
o'o2-o'o9
o'42-o'45
o'3o-o'4o
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T2 x T4
amB22
gene 43
33
33
35
45
65
i4o
N. MAHMOOD AND M.R. LUNT
I90
Table 4. Mixed infections between wild-type T2 and T4 amN~ 3o
(gene 46): effect of temperature on exclusion
Temperature (°)
Host strain
30
37
3o; 37
R; B4I
B; B4I
CR63
Burst s i z e
Fraction of am +
plaques in progeny
I~2O
30
75
o-6o-o-70
o.2o
O'O6
Mixed infections: exclusion of different Tz genes
A series of mixed infection experiments were performed to ensure that the phage strains
used here showed the same exclusion behaviour observed by others. The results are summarized in Table 3. Mixed infections with T2 and T4amN~3o (gene 46) gave unexpected
results indicating that in non-permissive hosts the extent of exclusion was temperaturesensitive (Table 4)We unsuccessfully attempted to measure exclusion of the Tztd gene using the method of
Simon & Tessman (1963) to distinguish td and td + plaques; the method proved unsuitable
because wild-type T2 phages gave variable-sized plaques in the assay.
Mixed infections: frequency of T2 genes at increasing times after infection
In T-even phage infections the rate of genetic recombination increases with time (Symonds,
I96Z; Frey & Melechen, I965). To test whether recombination might be affecting the
extent of appearance of T2 genes among progeny, the proportions of am and am + phages
were determined after lysing the bacteria with chloroform at different times after infection.
Crosses were performed between wild-type T2 and T4 amber mutants defective in two
strongly excluded genes 33 and 42, and between wild-type T2 and T4 mutants defective in
the weakly excluded genes I and 2o. The results are given in Table 5.
Mixed infections: expression of excluded genes
The amount of a particular gene product formed is expected to reflect the number of
functional copies of the relevant gene. I f partial exclusion is caused by the inactivation or
destruction of certain regions of the genome, then differences should exist in the amounts of
gene products made by weakly and strongly excluded T2 genes in mixed infections with T 4.
To test this we compared the amounts of an enzyme made on infection with T2 alone with
those in a mixed infection with a T4 mutant unable to supply the corresponding enzyme.
We chose the products of two genes e and td from regions known or expected to show weak
exclusion, and two from strongly excluded regions, namely genes 42 and 56.
Gene 42 product: synthesis of deoxycytidylate hydroxymethylase
The results in Fig. 2 show that there is a slight delay in appearance and a marked decrease
in the total amount of enzyme formed in a mixed infection compared with the rates and
levels found in infections with T2 or T4 alone. Estimation of thymidylate synthetase activities
in the same extracts showed that this enzyme was functioning normally even in the mixedly
infected bacteria where it reached levels similar to those shown in Fig. 5. Tests with mixed
extracts showed that the defective product formed by bacteria infected with T4amC76 did
not inhibit the activity of the normal T2-induced hydroxymethylase.
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M i x e d infections with phages T2 and T4
191
Table 5- Frequencies of wild-type T2 and T4 am genes at different times after infection. At the
times shown the infected cultures were lysed with chloroform and the phage released were
measured with Escherichia coli B and 0-I I' as indicator bacteria
Per ml. of culture
c
Time (min.)
after infection
,
T o t a l a m + as
p.f.u, on B
T o t a l a m as
(p.f.u. o n o--I I ' )
- - ( p . f . u . o n B)
Ratio
am +
~
as
am+ + am
p.f.u, o n B
p.f.u, o n O - I I '
c r o s s : T 2 w i l d - t y p e × T 4 a m 5 o ( g e n e 20)
15
20
25
30
35
40
6o
1 ' 8 × 106
3 ' 6 1 lO 7
I ' 9 X t09
5"7 x 109
2.o x l o TM
4"3 × lO1°
5"9 X IO 1°
4 " o x lO 6
o-3I
8.ox
3"5 X
I'I x
3"4 x
7"o x
9"0 X
o31
0"35
o'34
o-37
o'38
0"40
lO 7
I09
i o 10
i o 1°
IO 1°
IO 10
c r o s s ; T 2 w i l d - t y p e x T 4 a m A 4 9 4 ( g e n e I)
15
20
25
35
45
2 " 6 1 IO 7
1 . 5 1 lO s
6'7 x I0 s
4 ' 0 x 109
I ' 4 x IO1°
3 " 2 × IO 9
o'17
1 . 6 x l O 1°
0.20
4"5 x lO 1°
7'o X I 0 TM
0"23
0"23
50
2"3 × 10 I°
8 - 2 X 10 l°
0.22
60
2. 9 x 101°
9,0 x 10 TM
0-24
2"1 X IO 10
o.I 5
c r o s s : T 2 w i l d - t y p e x T 4 a m I 3 4 ( g e n e 33)
17
2O
22
25
27
30
60
1"3 x
7'0 x
I'5×
2.8 x
4"0 ×
6"5 x
1"7 x
io s
IO8
I09
lO 9
109
1o 9
IO1°
2.41
2. 3 x
I'I ×
2"3 ×
3"6 X
6.0 x
1.6x
10 8
IO 9
IO1°
101°
I010
lO I°
lO 11
0.35
0-23
0-12
O'11
O'10
o-lo
O-lO
c r o s s : T 2 w i l d - t y p e x T 4 a m C 7 6 ( g e n e 42)
15
20
25
30
35
40
6o
I'I X
I'2 X
6"4 x
2"4 ×
4"8 x
106
I0 s
IO8
IO 9
Io 9
2" 3 X
2' 7 x
4"6x
2. 9 ×
9'5 x
IO 6
108
1o 9
IO1°
i o 10
0"32
O-31
o-12
0-08
o-o 5
5,6x IO 9
l ' I X 1011
0"05
5"5 X 1 0 9
I"2 × 1011
o-o4
Gene 56 product: synthesis of deoxycytidine triphosphatase
The results in Fig. 3 show that in a mixed infection between wild-type T2 and T4amE5I
less enzyme is formed than in bacteria infected with wild-type T2 alone. Although the actual
levels of enzyme varied in different experiments, exactly similar results were obtained when
two other amber mutants in gene 56, namely T4amE56 and T4amB85 (kindly supplied by
Dr B. de Groot, Leiden) were used. Tests of enzyme activity in mixtures which contained
extracts of bacteria infected with T2 and extracts of bacteria infected with T4amE51 showed
that the T2 induced activity was unaffected by the product of the mutant T4 gene.
In our experiments the amounts of deoxy-CTP-ase formed in bacteria infected with either
T2 or T4 were always lower than those reported by Wiberg (1967). However, in his experments growing cultures, and in ours starved bacteria were infected with phage, and possibly
this may account for the differences observed.
r3
vlr~ I 6
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I92
N. M A H M O O D
I
I
I
A N D M. R. L U N T
I
I
1
I
I
6000 -
~5
7-
5000
~4
~ 4000
~ 3000
©
>'2
"+2000 -6 ]
0
0
0
5
10
15
Time after infection (rain.)
20
0
I
5
I
I
10
15
Time a~er infection (min.)
Fig. 2
I
20
Fig. 3
Fig. 2. Formation of d e o x y c y t i d y l a t e h y d r o x y m e t h y l a s e in cultures of Escheriehia c o l i n after
infection with p h a g e at m.o.i, of 4 to 6 for each strain. Enzyme activity is expressed as[~ moles of
p4C]-formaldehyde fixed/hr/mg, protein. © - - © , T2; A - - A , T4; O--O, T2 x T4amC76; A - - A ,
T4amC76.
Fig. 3. Formation of deoxycytidine triphosphatase in cultures of Escherichia coli ~ infected with
phage, at m.o.i, of four for each strain. Enzyme activity is expressed as n m o l e dCMP formed/hr]mg.
of protein. ( 3 - - 0 , T2; A - - A , T4; A - - ± , T4amE5I ; O--O, T2 x T4amE5I.
Products of genes e and td: synthesis of phage lysozyme and thymidylate synthetase
The results shown in Fig. 4 and 5 indicate that mixedly-infected bacteria formed these
enzymes in amounts typical of those produced in cultures infected with T2 alone.
Degradation of T2 DNA in mixed infections with T4
In these experiments the endonuclease-I-deficient strain E. coli B. 4 r was used as host
bacterium to avoid possible effects from superinfection breakdown (Fielding & Lunt,
I97o).
[a2P]-labelled T2 phage were mixed with an equal number of unlabelled T4 or T2 phage
and allowed to adsorb to starved bacteria at a total multiplicity of infection of eight; the
infected bacteria were diluted Io-fold out of absorption medium into warm broth and at
intervals ~ ml. samples were removed for measurement of acid-soluble [a~p] as described in
the Methods section. Similar experiments were done with [a2P]-labelled T4 and unlabelled
T 2 phage.
T4 phage which are mutant in genes 46 or 47 are unable to convert bacterial D N A to acidsoluble fragments. To test whether these genes might be involved in the solubilization of T2
D N A , infections were performed with [a2P]-Tz phage and each of the T4 mutants amN~3o
(gene 46) and areA456 (gene 47).
The result of one of these experiments is shown in Fig. 6. The results of experiments with
[a2P]-labelled T4 are not shown; in these between I and 2 ~0 of the total [a~p] appeared as
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Mixed infections with phages T2 and I"4
I
1
I
I
I
I93
I
80
t
70
O
60
15-
/
50
~" 40
-~ 1.0
30
o
2O •
~'
--
0.5
.
10
0
0
I
I
I
I
t
I
10
20
30
40
50
60
0
Time after infection (rain.)
Fig. 4
0
5
10
15
Time after infection (min.)
Fig. 5
20
Fig. 4. Formation of endolysin in cultures of Escherichia coli B infected with phage at m.o A. of 4 to 6
for each strain. Enzyme activity is expressed as drop in E45o of o.ol/min./mg, of protein. O O,!T2;
A - - A , T4; A - - A , T4.G59; ~ O , T2 x T4.G59.
Fig. 5. Formation of thymidylate synthetase in cultures of Escherichia coli B infected with phage at
m.o.i, of 4 to 6 each strain. Activities are expressed as #moles of [14C]-formaldehyde fixed/hr/mg.
of protein. O - - O , T2; A - - A , T4: A--/k, T4tdIo; 0 - - 0 , T z x T 4 t d I o ; [] [], T 4 x T 4 t d I o .
acid-soluble material at the start and did not increase during the infection. When bacteria
were infected with [a~P]-T2 and T4amNI3o the amount of acid-soluble [a2p] formed was
slightly less than that with wild-type T4 and [a2P]-T2 phages.
DISCUSSION
The pattern of exclusion observed in our experiments agrees with that found by Russell
0967) and de Groot (1966; I967a, b). Our results differ from theirs in that we obtain consistently higher frequencies for the T2 wild type genes among the progeny phages. The reason
for this is not known, but may be related to our use of a different T2 strain, or to differences
in the conditions of infection, for example our use of starved bacteria.
The amounts of D N A synthesized in mixedly infected bacteria were less than those formed
in cultures infected with either T2 or T4 wild type phage alone, although even in mixed
infections the amounts of D N A made were greater than expected from the corresponding
phage yields. The low yields may partly result from poor complementation between phage
proteins made by the two parental types. This has been shown to occur with the tail-fibre
proteins of T2 and T4 (Stahl & Murray, I966; Russell, 1967) and which, with other unfavourable protein interactions, might contribute to the low yields in all of the crosses
described here.
The simplest explanation for the unequal recoveries of the wild-type T2 genetic markers
13-2
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I94
N. M A H M O O D
I
I
A N D M. R. L U N T
I
I
I
I
12
IO
i
~
8
6
4
0
0
__f/~/~---------~._~
I
I
I
I
f
I
10
20
30
40
50
60
Time after infection (rain.)
Fig. 6. Degradation of [32P]-labelled T2 DNA in cultures of Escherichia coil B. 4[. The infected
cultures contained bacteria at a cell density of 5 x I@/ml. and a total of about ]o5counts/min./ml.
of [32p]. The sums of the [3~P]-counts recovered in the soluble and pellet fractions for each sample
were within 5 G of the total counts known to be present. ©--©, [3~P]-T2 x T2 ; 0 - - 0 , [3aP]-T2 x T4;
/~--A, [32p]-T2x T4amA456 (gene 47).
is that there is some T4-induced attack on selected regions of the T2 genome which are
thereby made less available for genetic transcription or for transmission to progeny phage.
Masamune 0968) showed that in mixed infections with wild-type phages, at 4 min. after
infection the T2 D N A was present as high mol. wt fragments which sedimented more slowly
than whole T2 D N A , while the parental T4 D N A was recovered intact. We observed some
conversion of [32p]-labelled T2 D N A but not T4 D N A to acid-soluble fragments in mixedly
infected bacteria, re-incorporation of nucleotides into new virus D N A possibly accounting
for the relatively small amount of acid-soluble products found (c.f. Wiberg, ]966). The
diminished T2 D N A breakdown observed with a mutant of T4 defective in gene 47 suggests
that a DNase coded for by this gene may be involved. An interpretation of Masamune's
and our results is that initially a few breaks are introduced into the T2 genome and that
subsequently non-specific nucleases convert the damaged D N A to acid-soluble fragments.
Selective inactivation of specific regions of the T2 genome is also indicated by our finding
that the strongly excluded T2 genes 42 and 56 form only low levels of their respective products, namely deoxy-CMP-hydroxymethylase and deoxy-CTP-ase, in mixed infections with
the corresponding T4 amber mutants in an su- host. By contrast the T2 ' e ' gene is not
strongly excluded and Tz-induced lysozyme reached normal levels in mixed infections with a
T 4 ' e ' mutant. In addition bacteria infected with T2 and T4tdIo produced almost as much
thymidylate synthetase as bacteria infected with T2 alone. Our attempts to measure exclusion of the Tztd gene were not successful. However, the T4td gene lies between genes 32 and
63 (Edgar & Wood, I966) which are well separated in terms of D N A distances (Mosig, I97o ),
and Russell 0967) found that although T2 gene 32 was strongly excluded, Tz gene 63
survived about as well as gene ' e ' in mixed infection with T4. On genetic grounds it is
therefore possible that the Tztd gene lies in a region which escapes the exclusion effect
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Mixed infections with phages T2 and T4
195
operating near gene 32. The formation of normal amounts of T2td gene product in mixed
infections appears consistent with this.
The data presented in Table 5 show that the strongly excluded am + T2 genes 33 and 42
contribute to the yield of progeny phage at about one-tenth the rate of the corresponding
T4 am genes. This results in a sharp drop in the frequency of the T2 am + markers among the
progeny, mainly during the early period of maximum DNA synthesis. By contrast, the
weakly excluded T2 am + genes i and 2o increase among the progeny phage at rates similar
to those of the T4 am markers; the frequencies of these T2 genes therefore remain almost
constant throughout the infections, although they are always present in lower amounts than
the corresponding T4 genes.
An interpretation of these results is that events required for the rescue of T2 genes occur
relatively early in infection and that subsequent changes in the frequencies of T2 markers
depend on the balance between their rescue, replication and withdrawal from the intracellular DNA pool as phage maturation occurs. Thus T2 genes which are inefficiently
rescued may be effectively diluted out as DNA replication proceeds.
There is some support for this explanation since if T4-induced exclusion causes breaks in
T2 chromosomes, the resulting intracellular collections of genomes resemble those studied
by Mosig and her collaborators. They have shown that after mixed infections with wild-type
T 4 partial genomes and whole T4 genomes carrying amber mutations, the yields of am +
markers are smaller than when normal size chromosomes provide the am + genes (Mosig &
Werner, 1969). In single infections, incomplete T4 genomes replicate autonomously only
when they possess an initiation site near gene 43, and then they replicate only once and to
those ends of their DNA molecules which lie clockwise from gene 43. In mixed infections
with normal phage, repeated replication of markers from partial genomes requires their
recombination into intact T 4 chromosomes (Marsh, Breshkin & Mosig, I971). Therefore in
our experiments, if T2 DNA replication also initiates close to gene 43, the strong exclusion
effects near this site must require all T2 markers, before they can replicate, to be integrated
with T4 chromosomes. Then T2 genes (e.g. I and 20) distant from exclusion sensitive sites
would be most readily rescued, and those near excluded sites (e.g. 42 and 33) would be at
the ends of T2 DNA fragments and less favoured for rescue by recombination.
The biochemical mechanisms responsible for exclusion remain unknown. More than one
T4-induced function appears to be responsible since Pees (I97O) has shown that a mutant of
T4 is unable to exclude gene 56 of T2 although the other sensitive sites in the T2 genome are
excluded normally. This result also shows that there are differences between the exclusionsensitive sites in the T2 genome. Our results and those of Masamune (1968) indicate that
DNases could be involved, but if DNase action is primarily responsible, the unequal extents
of exclusion of different T2 genes require the action of site-specific nucleases capable of
recognizing relatively short nucleotide sequences. There is no direct evidence that such
enzymes are involved in T2-T4 exclusion, although nucleases of this type are known and
appear to be responsible for restriction of 'foreign' DNA by E. coli (Linn & Arber,
1968; Arber & Linn, 1969; Meselson & Yuan, I968 ) and Haernophilus influenzae (Smith &
Wilcox, 197o). Similar phenomena include the restriction of T-even phages which lack
glucose on their DNA (Shedlovsky & Brenner, I963; Symonds, et al. 1963; Hattman,
Revel & Luria, I966 ), and the phage T5-induced breakdown of the DNA of heterologous
super-infecting phages (Fielding & Lunt, 197o). In the case of the closely related phages
Tz and T4 the exclusion is only partiaI, probably because genetic recombination permits
rescue and subsequent replication of undamaged regions of the T2 genome.
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~96
N. M A H M O O D A N D M. R. L U N T
W e a r e g r a t e f u l t o D r R . L. R u s s e l l , C a l i f o r n i a I n s t i t u t e o f T e c h n o l o g y , f o r p e r m i s s i o n t o
r e a d his P h . D . t h e s i s w h i c h d e s c r i b e s e x t e n s i v e g e n e t i c e x p e r i m e n t s o n T 2 - T 4 e x c l u s i o n a n d
contains conclusions similar to t h o s e p r e s e n t e d here. N. M. t h a n k s the British Council for a
scholarship. The w o r k was s u p p o r t e d by the Medical Research Council.
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