Download Inhibition of T7 Development at High Concentrations of the Phage

115
J. gen. Virol. (1981), 53, 115-123
Printed in Great Britain
Inhibition of T7 Development at High Concentrations of the Phage
By L A R S
HAARR,*
HARALD
B. J E N S E N
AND D A G
E. H E L L A N D
Department of Biochemistry, University of Bergen,/t rstadveien 19, 5000 Bergen, Norway
(Accepted 13 October 1980)
SUMMARY
Escherichia coli B exposed to high doses of bacteriophage T7 did not lyse. A
similar effect was observed when the high dose was added within the first 7 rain after
primary infection. No viable phage was formed. D N A synthesis was inhibited rapidly
and the nucleoid structure was'absent. Protein synthesis was in general markedly
reduced and so were the activities of the phage-specific enzymes endolysin and D N A
polymerase. However, phage genes were transcribed both by the host R N A
polymerase and by the phage-specific enzyme. We suggest that inhibition of phage
development is due to structural alterations occurring in the cell wall/membrane such
that replication is inhibited.
INTRODUCTION
Development of T-bacteriophages in Escherichia coli is dependent upon the multiplicity of
infection (m.o.i.) and markedly influenced by superinfection. However, these effects are quite
different in T-even and T-odd systems. Lysis inhibition is observed after superinfection in a T2
or T4 system: lysis is markedly retarded and phage production continues (Doermann, 1948:
Bode, 1967). In contrast, superinfection under similar conditions (low m.o.i.) in a T7 system
does not result in lysis inhibition (Hershey, 1946). We observed that E. coli exposed to high
doses of T7 did not lyse. Similar effects were obtained when the high dose was added within a
limited period of time after primary infection. Therefore. was it phage production or the
process of cell lysis which was inhibited?
Development of T7 is normally restricted such that only one of two simultaneously
infecting mutants is replicated, and a superinfecting phage is excluded (Hirsch-Kauffmann et
al., 1976). We observed, however, that no detectable phage was formed after infection at high
m.o.i. Since such infection was abnormal, we prefer the term multiplicity of exposure (m.o.e.)
rather than m.o.i. The m.o.e, gives the average number of phage particles to which a single cell
is exposed.
One possible explanation for the cessation of phage development was inhibition of
replication, which is assumed to be associated with the cell wall/membrane (Siegel &
Schaechter, 1973). Ponta et al. (1975) reported inhibition of T7 production in E. coli carrying
the F + episome, presumably because of structural alterations of the celI wall/membrane.
Evidence that similar alterations occur at high m.o.e, was obtained from the observation that
leakage of ions increases (Ponta et al., 1976). Therefore. it would be of great interest to study
synthesis of nucleic acids and formation of the nucleoid structure (Helland, 1977) at high
m.o.e. Alternatively, leakage of ions could selectively affect transcription of some genes
(Summers & Siegel, 1970) such that the synthesis of essential proteins could be inhibited. To
study this, the pattern of phage-induced proteins was investigated by gel electrophoresis.
From our results we suggest that the lack of phage development is caused by structural
alterations occurring in the cell wall/membrane such that replication is inhibited.
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L. HAARR,
H. B. JENSEN
AND
D. E. HELLAND
METHODS
Bacteria and viruses. E. coli B was from B. D. Hall, and bacteriophage T7 L from F. W.
Studier.
Chemicals. The deoxyribonucleoside triphosphates were from Sigma. All radioactive
isotopes were products of The Radiochemical Centre, Amersham.
Growth of bacteria and infection. Unless specified, the bacteria were grown in tryptone
medium (Adams, 1959) and infected with CsCl-purified T7 (Studier, 1969) at 30 °C a: a
concentration of 5 × 108 cells/ml. Phage development was stopped at the specified time by the
addition of NaN 3 (final concentration 13 mM) and by keeping the temperature at 0 to 4 °C in
all subsequent steps until plating.
Measurement of enzyme activities. DNA polymerase (EC 2.7.7.7) activity was determined
as described by Grippo & Richardson (1971). Assay for T7 endolysin (EC 3.5.1.28) activity
was performed according to the turbidimetric method described by Kleppe et al. (1977).
Labelling of proteins and gel electrophoresis. E. coli was grown in M9 medium (Adams,
1959), portions of 12 ml were placed in Petri dishes (10 cm diam.) and exposed to u.v. light to
reduce host-specific protein synthesis (Hopper et ak, 1975). The cultures from all dishes were
mixed to form one batch and divided into portions which were incubated at 30 °C and
infected. Multiplicities of exposure are given in the figure legends and in the tables. At the
specified times, 2 ml of each culture was transferred to a test tube containing 30 #Ci
35S-methionine in 0.1 ml M9 medium and incubation continued for 3 min. The chase was
performed by addition of 1 ml 10% casamino acids and further incubation for 2 rain before
chilling on ice (Hopper et al., 1975). The cells were spun down, the pellet resuspended in 400
/al of a mixture containing 0.05 M-tris-HCt pH 6-8, 1% SDS, 1% fl-mercaptoethanol and
10% glycerol, and then boiled for 3 min.
Electrophoresis on slab gels was carried out as described by Marsden et al. (1976) using
5 % acrylamide in the stacking gel and a gradient of 5 to 12.5 % acrylamide in the running gel.
Proteins induced by herpes simplex virus type 2 in baby hamster kidney cells were labelled
with 35S-methionine and used as markers (Marsden et al., 1978). The gels were fixed,
destained and dried before autoradiography.
RESULTS
Effect of the m.o.e, on lysis by T7
In cells infected with m.o.e, of 5, lysis was initiated after approx. 22 rain. The absorbance
(turbidity) of the culture decreased markedly during the following minutes (Fig. 1). However,
when using a 10-fold higher m.o.e., a slow linear decrease in absorbance started at approx. 5
min and after 2 h had dropped by 30% (Fig. 1). This would suggest that neither 'tysis from
within' nor 'lysis from without' occurred, since both processes lead to clearing of the culture
(Delbrfick, 1940). Furthermore, 'lysis from without' at high m.o.i, transforms the rod-shaped
bacteria into spherical bodies (Delbrfick, 1940) which release their cellular contents into the
medium (Anderson & Doermann, 1952). In our experiments at high m.o.e., the number of
rod-shaped bacteria remained almost constant for at least 50 min, as shown in Fig. 2 (a),
whereas that of viable cells was markedly reduced within a few minutes. Labelled cellular
proteins were released into the medium during lysis at an m.o.e, of 5, but comparatively little
after infection at an m.o.e, of 55 (Fig. 2 b). In the following experiments lysis was measured by
decrease in the absorbance.
Fig. 3 shows that normal infection followed by a high m.o.e, a few min later had similar
effects on lysis as an initial high m.o.e. However~ the full effect of the second dose of phage
was obtained within the first half of the infection period only (e.g. at 6.5 rain). Addition of the
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117
Inhibition of T7 development
° 5
F
. . . . .
°2t
0-05
0-02
I
10
4
I
I
I
80
Time (rain)
I
L
120
Fig. 1. Effect of multiplicity of exposure on the turbidity of the culture. A culture of E. coli was divided
into three portions which were infected by T7 at an m.o.e, of either 1(O), 5 (A) or 50 (11) and incubated
further. The absorbance at 650 nm was measured at the specified times.
1 0 i-
,
~
,
,
811
,
100
r
10
(a)
(b)
E
4
~.a..-~
~ _ _ ~ o ~
80
~
=
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.~
8
E
~
6
×
:If
20
2I 'i
~
-
10
-
o
.-4 2
,<
-
2'0
3'0
4'0
Time (min)
5'0
10
20
30
4w0
Time (min)
5'0
Fig. 2. Effect of the multiplicity of exposure on the number of bacteria and on the release of cellular
proteins into the medium. (a) Two cultures of E. coli were infected by T7 at an m.o.e, of 5 (A) and 55
(IZ]) respectively. Samples of 1.5 ml were chilled rapidly in ice-cold test tubes containing NaN 3 (final
concentration 13 raM), and the number of rod-shaped bacteria were counted in a B/irker chamber using
a phase-contrast microscope. Surviving bacteria after infection at an m.o.c, of 55 (O) were measured
by plating on salt-free agar plates. (b) E. coli was grown in tryptone medium for two generations in the
presence of ~S-methionine (1-9 gCi/ml) and divided into two portions which were infected by T7 at an
m.o.e, of 5 (O) and 55 (A) respectively. Samples were harvested at different times as described
above, and the medium was separated from the bacteria by filtration (pore size 0-45/~m). A 300 ~1
amount of each sample of medium was precipitated with TCA, filtered and counted.
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L. HAARR~ H. B. J E N S E N AND D. E. H E L L A N D
118
1
,
,
,
~
,
0.2
c~
0.1
0.05
0-02
L
I
l
810
40
Time (rain)
I
120
Fig. 3. Effect on the turbidity of the cultures of the addition of a large dose of phage at different times
after infection.A culture ofE. coli was dividedinto four portions which were infected at a multiplicityof
5 and incubated further. Fifty phage particles/cell were added at either 6.5 rain (11), 15 rain (/x)or 30
rain (1~) after infection, and the absorbance measured at different times. The control (O) was not
superinfected,
Table 1. Production of phage at low and high m.o.e.*
Infectious centres (phage/ml × 10-~) at
A
g
M.o.e.
5
55
Fraction of the culture
Supernatant
Pellet
Supernatant
Pellet
6 rain
7.4
0-3
320
9.9
18 rain
8.1
8.3
260
7.0
30 rain
455
6.9
190
5.8
55 rain
515
6-8
260
6.7
120 rain
645
2.2
190
3.8
* Two cultures ofE. coli (5 × 108 cells/ml) were infected with T7 at an m.o.e, of 5 and 55 respectively.Samples
of 10 ml were chilled rapidly in ice-cold test tubes containing NaN 3 to give a final concentration of 13 mM.
Centrifugation was performed at 5000 g for 10 min (0 to 4 °C), and plaque-formingunits (p.f.u.) measured in
the supernatant. Each pellet was resuspended (0 to 4 °C) in 10 ml of a 70 m~-phosphate buffer (pH 7-l) containing 70 mM-NaCI and 0.1 M-MgSO4. Intracellular phage was released by the addition of chloroform, and the
number ofp.f.u, assayed.
dose at later times (e.g. at 15 rain) caused incomplete lysis. Once started, lysis was hardly
affected by the second dose of phage,
The absorbance increased slightly from the time of infection at low m.o.e, until a few
minutes before lysis. At high m.o.e, a slight decrease in absorbance started shortly after
infection. This seems to be consistent with the slight decrease in the n u m b e r of rod-shaped
bacteria (Fig. 2 a) and the slow release of cellular proteins (Fig. 2 b).
Inhibition o f phage production at high m.o.e.
Following infection at low m.o.e, there was an 80-fold increase in the n u m b e r of infectious
centres from 18 to 55 min (Table 1, supernatant fraction), Consistent with the observation
that the n u m b e r of phages adsorbed to the same cell increases by increasing the m.o.i.
(Hirsch-Kauffmann et al., 1976), the n u m b e r of phages in the pellet at 6 rain was higher at an
m.o.e, of 55 than of 5 (Table 1). Since the cells did not lyse under the former conditions (Fig,
2), phage production would increase the number of infectious centres in the pellet fraction,
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Inhibition of T7 develo 9rnent
40
,
,
,
,
i
,
,
,
,
10
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119
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I
(a)
&
(b)
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- -====~2____o
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.~ 10
8
l
,=
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60
Time (rain)
I
,
I
80
I
100
i
20
-
~
4 t0
~
'
60
t
80
Time (rain)
Fig. 4. R N A and DNA synthesis after infection at low and high multiplicity of exposure. (a) A culture
of bacteria was divided into two portions which were infected with either 5 (O) or 55 (Zx) T7
phages/cell. 3H-uracil was added 1 min later to a final concentration of 4 ,ug/ml (corresponding to 0.5
gCi/ml). Portions of 300 Itl were precipitated with TCA at the specified times, filtered and counted. (b)
Two portions of another culture were infected with T7 at an m.o.e, of either 5 (O) or 55 (&). A third
portion was infected with 5 T7 phages/cell initially, and 50 phages/cell added 6.5 rain later ([]).
14C-thymidine was added 1 min after infection to a final concentration of 5 gg/ml (corresponding to
0.12 gCi/ml). TCA precipitation and counting of radioactivity was performed as in (a).
However, Table 1 shows that this did not occur. At an m.o.e, of 55, the initial concentration of
phage in the supernatant was, as expected, about 300 x 108/ml, but there was no increase
after 6 min.
To investigate whether the observed inhibition of phage production stems from the
maturation process or an earlier stage of phage development, e.g. synthesis of macromolecules, the synthesis of nucleic acids and proteins was measured.
Effects of high m.o.e, on the synthesis of nucleic acids
Synthesis of RNA and D N A was determined by incorporation of 3H-uracil and
~4C-thymidine respectively into acid-insoluble material. R N A synthesis was virtually
unaffected by a high m.o.e. (Fig. 4 a). In contrast, a marked reduction of D N A synthesis was
observed commencing approx. 5 rain after infection at high m.o.e. (Fig. 4 b). When infection
was initiated at low m.o.e, a high dose of phage added a few min later reduced D N A synthesis
rapidly (Fig. 4 b).
Effects of high m.o.e, on protein synthesis and enzyme activities
In general, protein synthesis was measured by incorporation of ~S-methionine into
acid-insoluble material. Following infection at a high m.o.e., synthesis was reduced approx.
eightfold at 10 min and 15-fold at 15 min relative to controls. Addition of a high dose of
phage (m.o.e. 55) at either 6 or I l rain after infection at low m.o.e, had similar results (results
not shown). Reduction of protein synthesis was also reflected in the enzyme activities. Thus,
the phage-specific D N A polymerase activity was reduced by a factor of 2, and that of
endolysin by a factor of 10 (Table 2).
Synthesis of the individual phage-induced proteins was analysed by slab-gel electrophoresis
following pulse-labelling with 35S-methionine from 7 to 10, 12 to 15 or 22 to 25 rain after
infection. The same amount of radioactive material was applied in each slot so that the relative
amounts of the individual proteins could be compared directly. The main conclusion from the
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120
L. H A A R R ,
H. B. J E N S E N
AND
D. E. HELLAND
Table 2. DNA polymerase attd endolysin activities at low and high m.o.e.*
Enzyme activity/rag protein of crude extract at
Enzyme
M.o.e.
3 rain
6 min
12 rain
20 rain
25 rain
30 min
45 min
5
55
5
55
301
364
-
5.8
4.8
1393
660
91
9.6
1444
185
29
275
26
-
835
29
439
21
DNA polymerase
Endolysin
* Infected cells were crushed in a French press, centrifuged at 12000 g for 10 rain, and D N A polymerase
activity (acid-insoluble ct/min) measured in the supernatant. The endolysin activity was measured immediately
after thawing infected cells. Units x 10 -2 (Kleppe e t a & 1977) are used,
(a)
Apparent
tool. wt.
x I0 -3
1
~ ,
~
157
"
2
, ....
3
1
2
(c)
3
l
,., .
o~ .....
**<, ~ . :
2 3 1 2 3
. . . . . . . .
.
.
.
.
"~
, m ,.~ ~,~ ~
-
~
3, 1 IBliBimilBidljB!il
;
14-5 °
.
(d)
'
Gene
product
~
~
,~.~ ~ . , . . m , ~
84
42.5
39-5
(b)
.
.....
.
"
. . . . .
.
.
.
:
'-
L~.. ~ , ~ . . - ~ ......
._
,,~
~
q"
~
..,r
.J'~
.
16
12
10
9"3%
1l
14
1.7
0,3
Fig. 5. Autoradiograph of polypeptides induced by T7 at low and high multiplicities of exposure. The
numbers of phages/cell were (a) 5: (b) 55; (e) 5 initially and 55 from 6.5 rain; (d) 5 initially and 55
from 11 rain. Pulse-labelling was performed at: 1, 7 to 10 min: 2, 12 to 15 rain; 3, 22 to 25 rain.
results (Fig. 5} is that early proteins (e.g. from genes 1 and 1.3), class II proteins (e.g, from
genes 3,5 and 6) and structural phage proteins (e.g. from genes 10, 14 and 16) were
synthesized under all conditions. Thus, transcription at high m.o.e, is not limited to one or two
classes of genes.
Although all the main types of phage proteins were detected, the pattern of synthesis was
apparently altered to some extent as compared to that of the control. Whilst synthesis of some
early proteins (e.g. R N A polymerase, gene 1) was apparently unaffected, that of the 0.3
protein seemed to be shut-off earlier. The normal decrease in the gene 6 protein from 15 to 25
rain was neither observed after infection at high m.o.e., nor when infection started at low
m.o.e, and a high dose of phage was added shortly afterwards (Fig. 5 b, c), Under these
conditions the synthesis of several late proteins, e.g. from genes 12, 16 and 19 was delayed.
These effects of the m.o.e, on the protein synthesis need to be investigated further.
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Inhibition o f T 7 d e v e l o p m e n t
121
Effects o f m.o.e, on the nueleoid structure
DNA in uninfected E. eoli has a condensed and folded tertiary structure (Stonington &
Pettijohn, 1971; Pettijohn & Hecht, 1973; Worcel et al., 1973). Nucleoids containing phage
DNA are present after infection with T4 or T7 (Hamilton & Pettijohn, 1976; Helland, 1977).
Following infection by T7 at high m.o.e, the nucleoid was undetectable.
DISCUSSION
Exposure of E. coli to high doses of T7 inhibits phage development (Table 1). This is
different from superinfection exclusion which allows development of the primary infecting
phage exclusively (Hirsch-Kauffmann et al., 1976), and also from lysis inhibition in
T-even-infected cells in which phage production continues (Doermann, 1948: Bode, 1967).
Several of the processes involved in T7 development are inhibited at high m.o.e. The
phage-specific DNA polymerase activity and DNA synthesis were both reduced (Table 2,
Fig. 4 b), and the nucleoid structure was absent. Protein synthesis in general was several-fold
lower than in the control. However, separation of the individual phage proteins in a
polyacrylamide gel (Fig. 5) showed that RNA synthesis was not restricted to the genes
transcribed by the host RNA polymerase (Summers & Siegel, 1970), and that both sets of
genes normally transcribed by the phage-specific enzyme (Summers & Siegel, 1970) were
expressed.
Incorporation of uracil into RNA was almost unaffected at high m.o.e. (Fig. 4 a). but the
total amount of T7-specific RNA polymerase was reduced, as evidenced from the general
inhibition of protein synthesis and the pattern of individual phage proteins (Fig. 5). Different
RNA species could be made at high and low m.o.e, but this could only be analysed by
hybridization-competition experiments.
The reason for the inhibition of phage development at high m.o.e, is not obvious. We have
to consider several possibilities. For example, increase in the intracellular amount of internal
phage proteins cannot be excluded. However, it is unlikely that the intracellular concentration
of other phage components are subject to considerable variation since DNA of the
superinfecting phage is not injected into the cell and phage coats remain outside the cell wall
(Benbasat et al., 1978).
Leakage of K + ions increases at high m.o.i. (Ponta et al., 1976). However. such a leakage
cannot alone explain our observations for the following reasons. A low internal K +
concentration would inhibit the activity of host RNA polymerase on the class I phage genes
and stimulate the phage RNA polymerase to transcribe class II and class III genes (Summers
& Siegel, 1930). This, in turn, would affect the total protein synthesis from these messengers,
whereas Fig. 5 shows that all classes of genes are transcribed. Although the synthesis of late
proteins was delayed at high m.o.e., there was a significant increase from 15 to 25 rain (e.g.
gene product 16, Fig. 5 b, c), suggesting that the relative amount of these proteins would be
close to normal after further infection. Leakage of other ions, e.g. Mg 2+, could produce effects
other than those expected from low K + concentration (Chamberlin & Ring, 1973).
Increased leakage of ions at high doses of T7 (Ponta et al., 1976) suggests that structural
changes occur in the cell wall/membrane. Consistent with this, fluorescence studies indicate
that phages induce conformational membrane changes in the host (Hantke & Braun, 1974).
Evidence that replication is membrane-associated has been presented by several authors, e.g.
Siegel & Schaechter (1973). DNA has a condensed tertiary structure which is attached to the
cell membrane (Kleppe et al., 1979). In addition, there are several reports on membraneassociated protein synthesis (Schlessinger, 1963; Hendler & Tani, 1964: Tani & Hendler,
1964: Moore & Umbreit, 1965). It seems likely, therefore, that membrane changes induced by
high doses of T7 can affect replication (Fig. 4 b), the nucleoid structure and protein synthesis,
and thus inhibit phage development (Table 1). At present, we do not know whether this
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L. HAARR,
H. B. JENSEN
AND
D. E. HELLAND
inhibition is due to the continuous presence of large amounts of phage, or whether the damage
is complete and irreversible after a limited time of exposure. Experiments to study this are in
progress.
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