A defined in vitro system for packaging of bacteriophage T3 DNA

VIROLOGY

151,119-123

(1986)

A Defined in Vitro System for Packaging of Bacteriophage KAZUSHIGE HAMADA, HISAO FUJISAWA,’

AND

TEIICHI

T3 DNA

MINAGAWA

Department of Botany, Faculty of Science, Kyoto University, Kyoto 606, Japan Received October 21, 1985;accepted November 26, 1985 Using purified components, we have constructed an in vitro system for packaging of mature phage T3 DNA. In addition to mature T3 DNA, the system contained T3 proheads and the products of gene 18 (gp18) and gene 19 (gp19). The reaction required Mg’+, ATP, and polyvinyl alcohol. Spermidine was stimulatory but not absolutely required for the packaging reaction. Polyvinyl alcohol could be replaced by polyethylene glycol. The packaging efficiency decreased with decreasing molecular weight of the polymer, and low molecular weight polyols such as sucrose, sorbitol, and glycerol were inactive. The packaging reaction exhibited a sigmoidal relationship with respect to the concentration of ATP with the concentration for half maximal activity about 15 PM. A nonhydrolyzable ATP analog, adenosine 5’-0-(3-thiotriphosphate), inhibited the packaging reaction. 0 1986 Academic Press, Inc.

INTRODUCTION

During head assembly of bacteriophage T3, DNA is packaged into the prohead cavity with the aid of two noncapsid proteins, the product of gene 18 (gp18) and gp19 (Fujisawa and Yamagishi, 1981). To study the molecular mechanisms of the DNA packaging reaction, we have developed an in vitro system capable of packaging T3 DNA into infectious phage particles (Fujisawa et al, 1978). By dissection and reconstruction of in vitro packaging activity, we have been able to isolate and characterize a number of T3 proteins necessary for phage assembly (Fujisawa et al, 1980; Yamagishi et aL, 1980; Yamagishi et al., 1981; Matsuo-Kato et al, 1981). However, in vitro DNA packaging reactions are routinely run in crude extracts of phage-infected cells so that the involvement of host and phage functions cannot be determined precisely. Several host factors are required for phage assembly (Georgopoulos and Tilly, 1981). The early steps in DNA packaging require at least one host protein (re‘To whom dressed.

requests

for

reprints

should

be ad-

viewed by Feiss and Becker, 1983). To elucidate mechanisms involved in packaging, it is important to construct a packaging system composed of defined factors. Construction of such defined systems require reagent quantities of these factors. In the case of T&infected cells, gp18 and gp19 are present in small amounts, making their purification difficult (Hamada et cd., 1984). To alleviate these problems we have constructed recombinant vectors that overproduce gp18 and gp19 under the control of the leftward promoter (PL) of X in Escherichia coli and purified these proteins to homogeneity in large amounts from induced cells. The biological activities of the purified proteins were indistinguishable from those of the proteins prepared from infected cells (Hamada et aZ.,1986). In this paper, we describe a defined in vitro system, containing purified proheads, gp18 and gp19, which is active for packaging of mature T3 DNA. MATERIALS

AND

METHODS

Bacteria, plasmids, and phages, E. coli BB and ER22 were described previously (Ha-

119

0042-6822186$A00 Copyright All rights

0 1986 by Academic Press. Inc. of reproduction in any form reserved.

120

HAMADA,

FUJISAWA,

mada et aZ., 1984). ER22 (pKH1, pNT203) and ER22 (pKH2, pNT204) are transformants which overproduce gp18 and gp19, respectively, upon derepression of the PL promoter of X (Hamada et aZ., 1986). T3 amber mutants (the gene numbers are indicated in parentheses), are amNG69 (5), amHMlO1 (lo), and amHM90 (19) from our laboratory stocks. All mutants used in this study carried a mutation in the lysis gene (amNG220B, Miyazaki et aL, 1978). Multiple mutants were constructed by genetic crosses. Medium and buffers. M9 minimal medium supplemented with 0.5% casamino acids (M9A) was described previously (Fujisawa et aL, 1978). TM buffer is 30 mlM Tris-HCl (pH ‘7.4)-7 mM 2-mercaptoethanol. Chemicals. Polyvinyl alcohol 500 and polyethylene glycol were from Nakarai Chemicals. AdenosineB-O-(3-thiotriphosphate) was from Boehringer Mannheim Yamanouchi Company. Purification of proheads. Proheads were purified from cells infected with a 5- 19phage according to Nakasu et aZ.(1985). Purfwatim of gp18 and gp19. Gp18 and gp19 were purified to homogeneity from ER22 (pKH1, pNT203) and ER22 (pKH2, pNT204), respectively, as described in the accompanying paper (Hamada et aL, 1986). DNA packaging reaction A complete reaction mixture (40 ~1) contained 50 mM Tris-HCl (pH 7.4), 10% (w/v) polyvinyl alcohol (mol wt 22,000), 5 mM MgClz, 1 mM spermidine, 5 mM 2-mercaptoethanol, 50 mM NaCl, 0.1 mM ATP, 6.4 X 10” phage equivalent (peq) of mature T3 DNA, 1.1 X 10” peq of proheads, 8.8 pmol of gp18 and 1.2 pmol of gp19. The reaction mixture was incubated at 30” for 60 min for DNA packaging and the reaction was terminated by the addition of 2 ~1of 2 mg/ml of DNase I. After incubation at 30” for 20 min, the filled heads were converted to infectious particles by another incubation at 30” for 30 min with 8 ~1 of head acceptor extract containing tail and tail-fiber proteins. Phages were assayed by titration on E. coli BB.

AND

MINAGAWA

Preparation of head acceptor extract. The head acceptor extract was prepared as follows. E. coli BB was grown to 5 X 10’ cells/ ml in 500 ml M9A medium at 37” and infected with 5- lo- phage at a multiplicity of 7 at 30”. Twenty-five minutes after infection, the culture was chilled and cells were sedimented, washed with 0.15M NaCl-TM buffer, and resuspended in 3.5 ml of TM buffer. The mixture was treated with lysozyme (0.5 mg/ml) for 60 min in an ice bath and, after freezing-thawing, centrifuged at 38,000rpm in a Hitachi RP40 rotor for 120 min at 2”. The supernatant was dialyzed against 100 ml of 50% (v/v) glycerol100 mMNaCl-10 mMMgClz-TM buffer and stored at -20”. RESULTS

In vivo and in vitro studies of T3 assembly have shown that proheads interact with gp18, gp19, and DNA during DNA packaging. To determine whether or not these factors are sufficient for DNA packaging, mature T3 DNA purified from phage particles was incubated with purified proheads, gp18 and gp19, for 60 min at 30” and, after treatment with DNase, the filled heads were assayed by complementation of a head acceptor extract containing tail and tail-fiber proteins. The results shown in Table 1 indicate that mature DNA is packaged efficiently into purified proheads in the presence of purified gp18 and gp19 as judged by conversion to infectious phage after incubation with head acceptor extract. In addition to DNA and the purified phage proteins, ATP, polyvinyl alcohol and Mg2+ were required for the packaging reaction. The concentration of ATP for half maximal packaging was about 15 piV and the kinetics was clearly sigmoidal (Fig. 1). Higher concentrations of ATP were inhibitory to the packaging reaction. A nonhydrolyzable ATP analog, adenosine 5-O(3-thiotriphosphate) (ATP-Y-S), inhibited the packaging reaction (Table 1). The requirement for polyvinyl alcohol could be replaced by polyethylene glycol and the stimulatory effect of polyethlene glycol de-

IN VITRO SYSTEM

DEFINED TABLE

1

REQUIREMENTS FOR DNA PACKAGING BY in Vitro SYSTEM COMPOSEDOF PURIFIED T3 GENE PRODUCTS

Condition 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Complete” -DNA -Prohead -GPlS -GP19 -ATP -Polyvinyl alcohol -MgC12 + 10 mM EDTA -Spermidine -2-Mercaptoethanol + 10 mM N-ethylmaleimide +20 fl ATP-y-S* +200 pM ATP-y-S* -ATP + 20 pM ATP-7-S SDNase I, head acceptor extractC +Head acceptor extractd -T3DNA + T7DNA

Phage production (PFU/ml) 95 x 106 150 <50 298 X l@ <50 <50 <50 11 x 16 31 x 106 110 x 108 483x1@ <50 <50 150 31 x lb 722 x lo6

“The complete reaction mixture and DNA packaging reaction, followed by complementation for phage assembly, are described under Materials and Methods. * Concentration of ATP was reduced to 20 pM. ’ DNase I and head acceptor extract were added to the complete reaction mixture at the start of DNA packaging reaction. d Head acceptor extract was added to the complete reaction mixture at start of DNA packaging reaction.

creased with decrease in the molecular weight of the polymer (Table 2). Polyols with low molecular weights such as sucrose, sorbitol, and glycerol (lO%)were inactive (data not shown). Spermidine, while stimulating the packaging reaction, was not absolutely required (Table 1). Addition of the head acceptor extract at the start of the reaction inhibited DNA packaging, probably because the extract should contain DNases. As conversion of mature DNA to a concatemeric form does not occur under the present reaction condition (data not shown), mature DNA should be packaged directly. Consequently, mature T7 DNA as

FOR PACKAGING

121

T3 DNA

well as T3 DNA was packaged in this in vitro system (Table 1). DISCUSSION

The results presented in this report clearly show that mature T3 DNA can be packaged into the head in a defined system composed of purified proheads, gp18 and gp19. No other phage and host proteins are required for the packaging reaction. A surprising result is that the requirement for gp18 was not stringent (Table 1, row 4). It is possible that some in vitro condition partly compensates for the role(s) played by gp18. ATP, Mg2+, and a high molecular weight hydrophilic polymer, polyvinyl alcohol or polyethyelene glycol were absolutely required. The packaging reaction as a function of ATP concentration showed a sigmoidal dependence (Fig. l), consistent with a model involving an allosteric mechanism with ATP as effector. An ATP analog ATP-7-S inhibited DNA packaging. Therefore, it is concluded that DNA packaging requires the cleavage of ATP. The energy released by hydrolysis of ATP would be utilized for the movement of DNA into the head (Wood and King, 1979; Earnshaw and Casjens, 1980). The stimulatory effect of the high molecular weight polyols decreased with the decrease in molecular

ATP

concentration

,@4

FIG. 1. Phage production as a function of the concentration of ATP. In vitro DNA packaging reaction was run with the complete reaction mixture as described under Materials and Methods except that concentration of ATP was varied as shown.

HAMADA,

122 TABLE

FUJISAWA.

2

EFFECT OF MOLECULAR WEIGHTS OF HYDROPHILIC POLYMERS ON DNA PACKAGING

Polymers

Mol. wt. (Da)

Polyvinyl alcohol Polyethylene glycol Polyethylene glycol Polyethylene glycol

22,000 8,000 4,000 1,000

Phage production (PFU/ml) 426 53 116 202

X x X x

lo6 lo6 10’ 10’

Note. The packaging reactions were carried out as described under Materials and Methods except that 10% polymers was added in place of polyvinyl alcohol.

weight (Table 2) and sucrose and sorbitol were inactive. Serwer et al. (1983) have reported that polyols such as dextran 10, sucrose, or sorbitol are stimulatory for DNA packaging in a crude extract. The absolute requirement for a hydrophilic polymer with a high molecular weight has been reported for enzymatic replication of the origin of the E. coli chromosome (Fuller et aL, 1981) and for in vitro transposition of bacteriophage Mu (Mizuuchi, 1983). These polyols may increase the effective concentrations of macromolecular reactants by an “excluded volume” effect (Fuller et al, 1981; Mizuuchi, 1983). Although crude in vitro DNA packaging systems all require polyamine (Earnshaw and Casjens, 1980), the present system did not show a strong requirement for spermidine (Table 1, row 9). Hafner et al. (1979) have reported that T7 and T4 can grow on host strains which lack spermidine and putrescine entirely. Mature T3 DNA was not converted to concatemeric forms in the present in vitro system (data not shown). Therefore, mature DNA must be packaged directly without proceeding by way of concatemeric intermediates as has been observed in a crude packaging system (Fujisawa et aZ., 1978). Previously, we showed that T3 and T7 DNAs can be discriminated during packaging by a crude in vitro system (Fujisawa and Yamagishi, 1981), but in the present

AND

MINAGAWA

system, mature T7 DNA was packaged as well as T3 DNA (Table 1, row 16). The packaging specificity observed in the crude extract might reside in the mechanism of cutting of monomeric DNA from concatemerit DNA. The defined in vitro system described here is the first that we know of that can package phage DNA using purified components. Use of this system should work to elucidate the molecular mechanism involved in DNA packaging. ACKNOWLEDGMENTS We are grateful to Dr. F. K. Fujimura, La Jolla Cancer Research Foundation, for invaluable help with the manuscript. We thank Dr. Y. Ryo for his helpful discussions. This study was supported by the Research Fund of Education of Japan.

REFERENCES EARNSHAW, W. C., and CASJENS, S. R. (1980). DNA Packaging by the double-stranded DNA bacteriophages. CeU 21,319-331. FEISS, M., and BECKER, A. (1983). DNA packaging and cutting. In ‘Lambda II”(R. W. Hendrix, J. W. Roberts, F. W. Stahl, and R. A. Weisberg, eds.), pp. 305330. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. FUJISAWA, H., MIYAZAKI, J., and MINAGAWA, T. (1978). In Vitro packaging of phage T3 DNA. Virology 87, 394-400. FUJISAWA, H., and YAMAGISHI, M. (1981). Studies on factors involved in in vitro packaging of phage T3 DNA. Prog. CEin. Bid Rex 64.239-252. FUJISAWA, H., YAMAGISHI, M., MATSUO-KATO, H., and MINAGAWA, T. (1980). Purification of DNA-binding proteins of bacteriophage T3 and their role in in vitro packaging of T3 DNA. virology 105,480-489. FULLER, R. S., KAGUNI, J. M., and KORNBERG, A. (1981). Enzymatic replication of the origin of the Escherichia coli chromosome. Proc. Natl Acad. Sci. USA 78.7370-7374. GEORGOPOULOS,C., and TILLY, K. (1981). Bacteriophage-host interactions in assembly. Prog. Cl&. Biol. Res. 64,21-34. HAFNER, E. W., TABOR, C. W., and TABOR, H. (1979). Mutants of Eschevichia coli that do not contain 1,4diaminobutane (putrescine) or spermidine. J. Biol Chem

254,12419-12426.

HAMADA, K., FUJISAWA, H., and MINAGAWA, T. (1984).

DEFINED

IN

VITRO

SYSTEM FOR PACKAGING

Purification and properties of gene 18 product of bacteriophage T3. firology 139,251~259. HAMADA, K., FUJISAWA,H., and MINAGAWA,T. (1986). Overproduction and purification of the products of bacteriophage T3 genes 18 and 19, two genes involved in DNA packaging. Virology 151,110-118. MATSUO-KATO,H., FUJISAWA,H., and MINAGAWA,T. (1981). Structure and assembly of bacteriophage T3 tails. virology 109,157-164. MIYAZAKI, J., RYO,Y., FUJISAWA,H., and MINAGAWA, T. (1978). Mutation in bacteriophage T3 affecting host ceil lysis. virology 89,327-329. MIZUUCHI,K. (1983). In Vitro transposition of bacteriophage Mu: A biochemical approach to a novel reprication reaction. Cell 35, 785-794. NAKASU, S., FUJISAWA,H., and MINAGAWA,T. (1985).

T3 DNA

123

Purification and characterization of gene 8 product of bacteriophage T3. Virology 143,422-434. SERWER,P., MASKER,W. E., and ALLEN, J. L. (1983). Stability and in vitro DNA packaging of bacteriophages: Effects of dextrans, sugars, and polyols. J. Viral. 45,665-671.

YAMAGISHI, M., FUJISAWA, H., and MINAGAWA, T. (1981). Bacteriophage T3 DNA binding protein interaction with DNA. virology 109, 148-156. YAMAGISHI, M., FUJISAWA,H., YAMAGISHI, H., and MINAGAWA,T. (1980). Purification of gene 6 product of bacteriophage T3 and its role in in vitro DNA packaging. Virology 100,382-389. WOOD,W., and KING, J. (1979). In “Comprehensive Virology” (H. Fraenkel-Conrat and R. Wagner, eds.), Vol. 13, pp. 581-624. Plenum, New York.