gen. Virol. (t979), 45, 291-299
Printed in Great Britain
291
J.
In vitro Packaging
of Exogenous
By E L I S A B E T H
STROBEL
DNA
by Salmonella
Phage
P22
AND H O R S T S C H M I E G E R
Genetisches Institut der Universitdt Miinchen, Maria-Ward-Str. Ia, D-8ooo Miinchen 19,
Germany
(Accepted 3 May I979)
SUMMARY
We describe in vitro conditions for packaging of exogenous D N A of Salmonella
phage P22 which has terminally redundant, circularly permuted DNA. The
method is a modification of the Kaiser-Masuda procedure. The most important
aspect is to prepare all components (proheads, enzymes and concatemeric DNA)
in end- cells. The influence of several factors such as DNA- and Mg ~ + concentration and kinetics has been investigated.
INTRODUCTION
In order to study in more detail the packaging of phage P22 D N A into heads to form
mature phage particles, it was considered necessary to work out in vitro conditions for this
reaction. Although in vitro packaging of A-DNA works efficiently in the Kaiser-Masuda
system (Kaiser & Masuda, I973) we were not successful in applying this method reproducibly for P22 although variations in buffer and Mg ~ + concentration have been tried. Since
the D N A of P22 is a terminally redundant molecule, on which genetic markers are arranged in a circularly permuted manner reflecting the sequential cutting of head-ful-fragments from a D N A concatemer (Rhoades et al. I968; Tye et al. 1974), the substrate for
packaging, that is D N A of more than unit length, may be more sensitive to damage than
3,-DNA. This may explain why successful in vitro packaging has not been previously
described for other phages (such as T4 and PL) which also package their D N A sequentially
by the head-ful-mechanism (Streisinger et al. I967). Although Earnshaw & King (1978)
refer to the thesis by A. Poteete which describes packaging experiments using P22, the data
are not generally available and therefore the publication of our results may be useful.
We have attempted to find conditions which allow extraction of phage D N A mainly as
concatemers and which prevent further enzymic degradation. By constructing a donor
strain with suitable markers and working in a bacterial background without measurable
endonucleolytic activity, we are now able to package purified exogenous D N A in vitro with
fairly good and reproducible efficiencies using the Kaiser-Masuda system with some minor
changes in the concentration of Mg 2 + and buffer.
METItODS
Bacterial strains. The strains used were: DB 5575, cysAI348 hisC527 end-I (Susskind &
Botstein, 1975), kindly provided by E. G. Bade; su7o, cys~msu + (Raj et al. 1974); su7o(sie I),
su7 o lysogenic for P22 sieA- sieB- t S l 2 . I ts2. I, kindly provided by M. Levine.
Phages. Prohead and/or enzyme donors were: a m L I - a m N I o I 3 - a m H l o I cI-; am2,
2-amH2oo I3-amHzoI ci-; am5, 5 - a m N H 4 s-atoll69 I3-amHIox c F (this phage carries
two mutations in gene 5 in order to reduce back-mutations). D N A donor was 2-amH2oo
13-amHtoI immL. In this paper, terms such as am I and am2 are used to indicate the
relevant phage strain. Otherwise, symbols such as 'I-am' or 'I-amNIo' indicate specific
oo22-1317/79/oooo-3493 $o2.oo
~ I979 SGM
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E. S T R O B E L A N D H. S C H M I E G E R
genotypes as proposed by D. Botstein and used generally in the P22 literature. The basic
mutants used for construction of the phage strains by recombination were kindly provided
by D. Botstein, H. H. Prell and B. A. D. Stocker.
Media and solutions. NA, NB and saline have been described elsewhere (Schmieger, I968).
Reaction buffer for in vitro packaging is a modification of that described by Kaiser & Masuda
(I973) and contained 6 mM-tris-HCl (pH 7"4), 3 mM-fl-mercaptoethanol, ro mM-MgCI~, 1.5
mM-ATP and 6 mM-spermidine, if not stated otherwise.
D N A Preparation. Strain DB 5575 was grown in NB to exponential phase, then infected
with the ' D N A - d o n o r ' phage (m.o.i. of 5 phages/cell) and incubated for 45 rain at 37 °C. The
culture was chilled and concentrated by centrifugation and resuspension in Io mM-tris-HCl
(pH 7"4)- D N A was extracted by careful rolling with an equal volume of buffer-saturated
phenol for IO min. After phase separation the DNA-containing aqueous phase was dialysed
against IO mM-tris-HCl (pH 7"4) and kept in liquid nitrogen. Just before use D N A was
thawed at 37 °C. The D N A concentration was determined by measuring the absorbance at
260 nm (I/zg/ml is 0"02 .42G0).
Preparation o f cell extracts. Exponentially-growing cultures of end- strain DB 5575 were
infected with the relevant prohead and/or enzyme donor phages (m.o.i. of 3) and incubated
at 37 °C for 60 rain if not stated otherwise. They were then chilled, concentrated by centrifugation and resuspension in I/2OO vol. reaction buffer without ATP and kept in liquid
nitrogen. Final lysis of the cells was achieved by freezing and thawing three times at 37 °C
just before use.
In vitro packaging procedure. Extracts were passed several times through a syringe
(needle no. i) to disrupt D N A mechanically so that it could not compete with exogenous
D N A in the packaging process. Then the crude extracts were centrifuged for 2 rain at
2500og and equal volumes (usually loo/zl) of the supernatants of two different extracts
were mixed: aml + a m 2 , a m l + a m 5 or a m 2 + a m 5 . These terms are used throughout the
paper indicating the mixture of individually prepared extracts of DB 5575 infected with the
relevant phage strains. Then, lo to 15/zg D N A and ATP (1"5 m i final concentration in
standard experiments) were added and the mixture incubated at 34 °C for 3 h and later
for 9o min, if not stated otherwise. The reaction was stopped by adding some drops of
chloroform. In some cases the remaining D N A was first digested with DNase (5o/zg/ml at
37 °C for 3o min) and then chloroform was added. Viable phages were assayed by plating
with su7o, permissive for all genotypes in the reaction, and with su7o(siel), which plates
phages with L-immunity only.
RESULTS AND DISCUSSION
General strategy
D N A donor
To be packaged, D N A has to fulfil two conditions: (1) It should be concatemeric, that is
it should be at least longer than the mature phage chromosome. Proof for this statement will
be given later. (2) The genetic marker carried by this D N A must allow selective detection of
in vitro produced phages (referred to here as 'in vitro phages'). The following features satisfy
the first requirement: (i) the ' D N A donor' carried an amber mutation in gene 2. Gene
product 2 (gp 2) is involved in the packaging process so that 2-am mutants cannot cut concatemeric D N A (Botstein et al. 1973); (ii) to avoid premature lysis of cells infected with the
' D N A - d o n o r ' phage and to accumulate the D N A concatemers the lysis defective double
mutant 2 am I 3 - a m was coJlstructed (Botstein et al. 1972); (iii) to reduce destruction of
the concatemers by bacterial nucleases strain DB 5575 was used as host for the ' D N A donor'.
This strain lacks any endonucleolytic activity on P22 D N A (Schumann & Bade, 1977).
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In vitro p a c k a g i n g o f P 2 2 D N A
293
Table I. Packaging o f exogenous phage D N A o f different origin in an amr + a m 2
extract mixture
DNA
None
Donor DNA (I-5/zg)
From end +-host
From end--host
From phage particles
(mature form)
P.f.u./ml on su7o(siel)
(in vitro phages)
< IO x
< IO t
I '5 × 1o4
< 1o I
The second condition, selectivity, was fulfilled by replacing the C-immunity region of the
P22 D N A donor by the corresponding section of the heteroimmune phage L (Bezdek &
Amati, I968 ). Therefore, this recombinant is able to grow on a su + strain lysogenic with
P22 siel- which is defective in superinfection exclusion systems A and B (Susskind et al.
197t). Phages with P22 immunity are inhibited in this indicator by the prophage repressor.
Therefore, even rare packaging events of exogenous D N A should be selectively detected.
The complete genotype of the ' D N A donor' which we constructed by recombination was
therefore z-am 13-amLC~22L
Prohead and enzyme donors
The system in which packaging of exogenous D N A should occur must contain precursors
of mature phage heads, the proheads, all gene products involved in phage maturation and an
energy source.
Proheads to be filled with D N A accumulate in cells infected with P22 mutants defective
in genes necessary for packaging, like genes i or 2, and can be found in their extracts
(Botstein et al. I973). Our x-am or 2-am mutants used as prohead donors were defective in
lysis like the D N A donor by the I3-am mutation in order to accumulate proheads during
prolonged incubation. (Although we have not tried extracts of non-lysis-defective prohead
donors the usefulness of prohead accumulation by lysis inhibition may be shown in a later
section.) However, since the functions of gp 2 and gp I are needed for packaging also, these
gene products had to be present in the system. This was achieved by mixing extracts of cells
which were separately infected with I-am and 2-am mutants thus producing an in vitro
complementing system. In some experiments z-am and a-am extracts were combined with
s-am extract. The 5-am-infected cells can also supply all structures required except proheads
since gp 5 is the structural protein for the phage head.
Necessity o f concatemeric D N A
Extracts of amx- and amz-infected cells of the e n d strain DB 5575 were prepared as
described in Methods and equal volumes were mixed. Comparable amounts of D N A
0"5/zg/loo/~1 in the experiment described here) prepared from ' D N A donor'-infected cells
and mature D N A extracted from phage particles of the D N A donor were added. It turned
out that only cell-borne D N A yielded in vitro phages (Table I). Even increasing the mature
D N A up to 7/~g/loo #1 did not yield phages. From this we concluded that an oversized
substrate is necessary for in vitro packaging of P2z DNA. This interpretation is supported by
the observation that in packaging experiments with D N A from phage-infected end + strains
no in vitro phages were produced. Size determination of all-labelled D N A from a ' D N A
donor'-infected end + culture in sucrose gradients revealed that replicating phage D N A
has been degraded very quickly despite nuclease-inhibiting precautions, such as E D T A and
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294
E. S T R O B E L
I
I
A N D H. S C H M 1 E G E R
I
[
I
I
I'
I
e~ro~o~ o
10 3
I
I
•
/~q
f
-~10 2
10 ~
I
1
20
[
I
40
I
I
I
I
60
80
Time (rain}
I
100
/,L__.
180
Fig. I. IOO #1 extract of aml-infected DB 5575 cells were mixed with IOO #1 of extract from am5infected cells. 15#g D N A of 2-am I3 am immL-infected DB 5575 were added. The reaction
mixture was incubated at 34 °C. At different times, samples were taken, treated with chloroform
and the p.f.u, with L-immunity were assayed on indicator strain su7o(sieO.
low temperature (data not shown). Therefore, all experiments described in this paper were
performed with the end- mutant. However, agarose gel electrophoresis of D N A of phageinfected end- cells showed that such preparations also contained degraded material, the
quantity of which varied between different batches. Because of this variability in the fraction
of concatemeric D N A suitable for packaging, we did not make efforts to determine the total
phage D N A content in extracts by isotopic labelling. It is one of the reasons for the great
variation of phage yields in different experiments. The efficiency of in vitro packaging also
varied between different extract preparations. Therefore, absolute data from different experiments presented should not be compared.
To demonstrate that the phages found in such experiments are really in vitro products
containing exogenous DNA, we determined their genotype by marker rescue on a set of
Salmonella strains which carry P2z prophages with well defined deletions (Chan & Botstein,
I972 ). In a typical experiment in which donor D N A was added to an a m [ + a m 5 extract
mixture, 18 out of 2o plaques had the ' D N A donor' genotype z-am, one was 5-am and one
was wild type (resulting from back-mutation or more probably, by recombination with the
siez prophage of the indicator strain). This proves clearly that we are indeed dealing with
in vitro matured phages.
Time required for the packaging reaction
To examine the effect of time on packaging we followed the kinetics of phage production.
An a m I + a m 5 extract mixture was sampled various times after adding the exogenous D N A
and aliquots were diluted, treated with chloroform and p.f.u, assayed on su7o(siel). At
about 4o min after addition of D N A a plateau was reached, indicating that at this time all
phages capable of in vitro maturation under these conditions had been formed (Fig. i).
Packaging is a very fast reaction, since 5O~o of all phages are already completed within the
first Io min. Nevertheless, in standard experiments we incubated for 8o min (at the beginning 3 h).
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In vitro packaging of P22 DNA
295
l l l l k F l l l
o
4
×
2
(a)
10 ~
Q
O
,¢
104
10 3
-
k•
0
I
1
I
I
I
I
1
1
20
40
60
80
100
DNA (ug)
Fig. 2. Reaction mixtures of extracts from a m l - and am5-infected DB 5575 cells were mixed and
different concentrations of d o n o r D N A were added. Incubation was at 34 °C for 3 h. (a) Yield
of p.f.u.//tg D N A as calculated from the data presented in (b). (b) Control A : 20 #g D N A , digested
with DNase I. Control B : 2o/zg intact D N A , mixture without ATP.
Dependence on DNA concentration
It would be expected that the number of in vitro phages would increase with increasing
input of exogenous DNA. To test this we added increasing amounts of D N A to constant
volumes of different extract mixtures. Figure 2 shows the result obtained with an amI + a m 5
mixture. There was a proportionally increasing yield of p.f.u, up to Ioo/tg D N A except for
the value at 50/zg which was, for unknown reasons, higher than expected. The fact that this
is not the saturating DNA concentration is apparent from Fig. 2(a) in which the Ioo/~g D N A
value was again in the range of proportionality but the 5o/zg value exceeded it. As a control,
one sample was prepared with 2o/zg D N A previously digested with DNase I (5o/zg/ml;
to min at 37 °C). Point A in Fig. 2(b) shows that the yield of p.f.u, was at the level of the
control without DNA. The same value was observed for a control with 2o #g intact D N A
when ATP was omitted from the system (point B in Fig. 2b).
Effect of time o] extract preparation
Preliminary experiments showed the importance of the length of time that infected cells
were incubated before preparing extracts. Since the infecting phage mutants were lysis
defective it was to be expected that with increasing time after infection the amount of
proheads and other products necessary for the packaging reaction increased progressively.
Table 2 shows that for different extract combinations this prediction is fulfilled. The
general procedure was to incubate the differently-infected cells of DB 5575 for the times
indicated and then to lyse them. Equal volumes of the differently-infected extracts with the
same incubation time were mixed and DNA added to each mixture. Packaging was allowed
for 3 h or 9o min at 34 °C (indicated at the relevant experiment) and then the number of p.f.u.
was assayed. From these experiments it is clear that it is very important to lyse the cells
infected with the head and enzyme donors as late as possible in order to allow accumulation
of all products required for packaging.
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296
E. S T R O B E L A N D H. S C H M I E G E R
T a b l e 2. Influence of time for extract preparation
Time of lysis
(min p.i.)
P.f.u./ml on su7o(sieI)
(in vitro phages)
E x p t I*
30
I'O X IO a
Expt II
50
75
6O
75
4o
3'0 x
I "4 x
6'2 x
6'9 x
7"5 ×
70
2"7 x 10 4
45
75
I"5 X 104
Expt III
Expt IV
lo 4
105
I0 4
1o5
lo2
I "5/" 105
* Expt I was am] + a m z plus 2o/zg D N A ; Expt II was aml +am2 plus 14 #g D N A ; Expt III was am I -~ am2
plus 15 #g D N A ; Expt IV was amI +am5 plus l o # g DNA.
104
(a)
r ; ij
i
~
i
/"
(b)
j-
/
103
i
i
i
/e//°-°
~- 102
10 )
i
0,01
1
I
Concn am5 extract
0
i
O.OI
i
i
0.1
I
I
I
Conch am2 extract
Fig. 3. 6o#I extract of am2-infected cells were mixed with the same volume of am5 extract,
19 #g DNA were added and the reaction mixture incubated for 90 min at 34 °C. Chloroform was
then added and p.f.u, assayed on su7o(sieI). (a) The concentration of am2 extract was kept constant (concentration of original extract= I) and the am5 extract was diluted to the concentration
indicated on the abscissa. (b) The concentration of am5 extract was kept constant and the am2
extract was diluted before it was added.
Influence of extract ratios
A s d e s c r i b e d a b o v e the s t a n d a r d p a c k i n g m i x t u r e consists o f t w o different cell e x t r a c t s o n e
o f t h e m b e i n g a p r o h e a d ( a n d e n z y m e ) d o n o r , the o t h e r c o n t r i b u t e s t h e c o m p o n e n t m i s s i n g
in the p r o h e a d e x t r a c t in a d d i t i o n to m o s t o f the o t h e r c o m p o n e n t s necessary. In o r d e r to
get o p t i m a l c o n d i t i o n s for p a c k a g i n g we s t u d i e d the influence o f the r a t i o o f b o t h e x t r a c t s
in different m i x t u r e s . T h e d a t a are s h o w n f o r the a m 2 + a m 5 c o m b i n a t i o n . In o n e e x p e r i m e n t a l
set the a m z e x t r a c t was k e p t c o n s t a n t by u s i n g e q u a l v o l u m e s o f u n d i l u t e d e x t r a c t . T h e
c o n c e n t r a t i o n o f t h e a m 5 e x t r a c t was v a r i e d by d i l u t i n g it in a s s e m b l y buffer a n d a d d i n g e q u a l
v o l u m e s to t h e r e a c t i o n m i x t u r e . D N A was a d d e d to e a c h s a m p l e 0 9 # g / I 5 ° #1). I n a n o t h e r
set the a m 5 e x t r a c t was k e p t c o n s t a n t a n d the a m z e x t r a c t was varied. F i g u r e 3 a s h o w s
t h a t t h e r e is a p r o p o r t i o n a l increase o f in vitro p h a g e yield w i t h i n c r e a s i n g c o n c e n t r a t i o n o f
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In vitro packaging o f P22 DATA
10 5
297
. . . . . . . . .
10 4
,~
10 3
0
2
4
6
8
ATP (rnM)
Fig. 4. am2 and am5 extracts in different buffer (tris-HC1) concentrations were mixed, 14 #g D N A
added and the A T P concentrations adjusted to the final concentration indicated on the abscissa.
The buffer concentrations at p H 7"4 were: A, 6 mN; B, 25 mN; C, loo raM.
am5 extract which does not come to saturation within the range of the experiment. This
means that the amount of proheads conferred by the am2 extract is not yet exhausted even
by the highest concentration of am5 extract. This result is confirmed by Fig. 3 b where the
amount of proheads has been varied. We observe that even one half of the proheads is
sufficient to yield maximal phage numbers. Thus, we may conclude that in this system the
amount of proheads is not the limiting factor, but one of the other components contributed
by the am5 extract. Otherwise, this experiment shows that there is not too much excess in the
amount of proheads since already the I • 5 dilution causes a significantly lower phage yield.
This demonstrates that accumulation of proheads in the infected cells by lysis inhibition
due to the 13-am mutation is justified.
Influence of A TP concentration
It has been shown in Fig. 2 (point B) that ATP is necessary for efficient packaging. To
determine the optimal concentrations of ATP for our system we performed packaging
experiments with several extract combinations under standard conditions, but with different
ATP concentrations. Curve A in Fig. 4 shows the result obtained with an a m 2 + a m 5
mixture which is representative also of other extract combinations. The ATP optimum
was at I. 5 mM, thus being identical with the concentration in the Kaiser-Masuda system.
However, at higher concentrations we observed precipitation in the extracts due to decrease
of the pH value, a situation which might prohibit further increase of phage yield at higher
ATP concentrations. To overcome this we repeated these experiments with higher buffer
concentrations (curve B: 25 mM; curve C: I OO mu). It was possible to obtain precipitation
only at high ATP concentrations or to avoid them completely. However, we observed no
further increase in the production of in vitro phages.
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298
E. STROBEL
I
I
AND
I
I
I
H.
I
I
SCHMIEGER
I
I
I
I
I
10 3
to:
/
-->
•
10 ~
I
I
I
20
I
40
I
I
I
I
60
80
MgC12(ram)
I
)
100
I
I
120
Fig. 5. Reaction mixtures of extracts from am1- and am5-infected DB 5575 and 25 #g D N A were
prepared and assayed as described in Fig. i. MgC12 was present at the concentrations indicated.
f
r
[
I
10~
e,-*°/°-°\e~o
_
zk
IO ~
0
0
,c 102
I0 ~
I0
20
Spermidine I m~,l)
30
40
Fig. 6. am2 and am5 extracts were mixed, 14 #g D N A added and spermidine adjusted to the final
concentration given in the graph. Incubation took place for 90 ruin at 34 °C. After chloroform
treatment p.f.u, were assayed on su7o(sieO.
D e p e n d e n c e on the M g 2 4- c o n c e n t r a t i o n
S i n c e e n d o n u c l e a s e a c t i v i t i e s a r e i n v o l v e d in t h e p a c k a g i n g o f P 2 2 D N A m a g n e s i u m
m u s t p l a y a n i m p o r t a n t role as a c o f a c t o r . I n o r d e r t o o p t i m i z e o u r s y s t e m t h e p a c k a g i n g
efficiency o f e x o g e n o u s D N A w a s m e a s u r e d in a m I + a m 5 e x t r a c t m i x t u r e s w i t h i n c r e a s i n g
M g ~ + c o n c e n t r a t i o n s . F i g u r e 5 s h o w s t h a t p a c k a g i n g o c c u r r e d o v e r a w i d e r a n g e o f M g 2+
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In vitro packaging of P22 DNA
concentration, but that there was a maximum at
t h a n t h a t u s e d b y K a i s e r & M a s u d a (1973) f o r t h e
e x p e r i m e n t w i t h o u t m a g n e s i u m b u t w i t h E D T A (7
shows the absolute requirement of the reaction for
299
a b o u t 4 0 mM. T h i s is f o u r t i m e s h i g h e r
in vitro p a c k a g i n g o f ~ t - D N A . A c o n t r o l
mM) w h i c h d i d n o t yield p h a g e p a r t i c l e s ,
magnesium.
Influence o f spermidine concentration
P o l y a m i n e s p l a y a n i m p o r t a n t role in t h e c o n d e n s a t i o n o f D N A d u r i n g p h a g e m a t u r a t i o n .
T h e r e f o r e s p e r m i d i n e is a n e s s e n t i a l i n g r e d i e n t in in vitro p a c k a g i n g s y s t e m s like t h a t o f
K a i s e r & M a s u d a 0 9 7 3 ) . T o o p t i m i z e its c o n c e n t r a t i o n f o r p a c k a g i n g o f P 2 2 D N A w e
p e r f o r m e d a s t a n d a r d e x p e r i m e n t in a n a m 2 + a m 5
mixture varying the spermidine conc e n t r a t i o n f r o m o t o 4o mM. A s c a n b e seen in Fig. 6, 6 mM w a s o p t i m a l f o r o u r s y s t e m , a s
well as f o r t h e K a i s e r - M a s u d a s y s t e m .
We thank the Deutsche Forschungsgemeinschaft
for supporting this work.
Note addedin proof. After acceptance of this manuscript the work of A. R. Poteete & D. Botstein
(1979), "Encapsulation of Phage P22 DNA in vitro", was published in Virology 95, 55o-564.
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function. Virology 49, 268-282.
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CHAN, R. K. & BOTSTEIN, O. (1972). Genetics of bacteriophage P22. |. Isolation of prophage deletions which
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protein. Journal of Molecular Biology 126, 721-747.
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(Received 29 September I978 )
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