DNA synthesis in toluene-treated bacteriophage-infected minicells of Bacillus subtilis

82

Biochimica et Biophysica Acta, 520 (1978) 82--87 © Elsevier/North-Holland Biomedical Press

BBA 99213

DNA SYNTHESIS IN TOLUENE-TREATED BACTERIOPHAGE-INFECTED MINICELLS OF B A C I L L US S U B T I L I S

EGON AMANN and JOHN N. REEVE Max-Pianck-Institut fiir molekulare Genetik, Abteilung Trautner, Ihnestrasse 63/73, D-IO00 Berlin 33 (Germany)

(Received October 12th, 1977) (Revised manuscript received January 23rd, 1978)

Summary Bateriophage (¢29, SPP1, or SPO1)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthe-

sis as measured by incorporation of [3H]dTTP into a trichloroacetic acid-precipitable form. The [3H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2 • 10s). Short exposure of the DNA molecules containing the incorporated [3H]dTMP to Escherichia coli exonuclease III results in over 90% of the [3H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, naiidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p-hydroxyphenylazo)-uracil.

Introduction Minicells are anucleate, quasi-spherical cells produced by aberrant bacterial cell division [1--3]. Although chromosomeless, it is possible to obtain minicells which contain a plasmid molecule by segragation into the minicell or to introduce a bacteriophage genome into the minicells by the normal bacteriophage infection process [3,4]. It has been consistently observed that although both plasmid DNA and phage DNA can be transcribed and translated in minicells there is little, if any, DNA replication in minicells [3,4,5]. The lack of DNA synthesis in minicells would suggest that either a precursor(s) of DNA synthesis or one or more enzymes responsible for DNA synthesis are absent from minicells. The inability to initiate DNA synthesis is observed in minicells infected with phages capable of synthesizing their own DNA-replicating enzymes (e.g.

83 SPO1 [4]) and with phages which are dependent on host-cell DNA-replicating enzymes (e.g. SPP1 [6], unpublished results) suggesting that the inhibition of DNA synthesis occurs at a stage c o m m o n to both types of phage infection, e.g. supply of precursors. We have therefore investigated the possibility that by supplying the direct precursors of DNA synthesis using toluene-permeabilized minicells we could produce a DNA-replicating system. The results presented here indicate that although DNA synthesis occurs under these conditions, it is not replicative DNA synthesis. Materials and Methods Bacteria; minicell purification. Bacillus subtilis CU403 thyA- thyB- metBdivIVB1 was grown in minimal medium at 30°C as previously described [7]. Minicells were separated from nucleated cells by sucrose gradient centrifugation [7] and stored at --70°C suspended (2 • 101° minicells/ml) in minimal medium containing 10% (w/v) glycerol. Minicell preparations contained less that one colony-forming unit per 10 s miniceUs. Bacteriophage. SPO1, SPP1 and ~b29 were grown as previously described [4]. 14C-labelled SPP1 were obtained by infection (input multiplicity = 3) of B. subtilis VUB12 t h y A - thyB- trpC2 novA1 (from N. Hartford) at an Aeo0n m = 0.5 in MIII minimal medium [6] containing 0.02% casamino acids plus 3 #g thymine/ml (0.5/~Ci [2-14C]thymine/ml, Amersham). 14C-labelled SPP1 phage lysates were concentrated by centrifugation and purified by CsCl density gradient centrifugation as previously described [4]. 14C-labelled SPO1 phage lysates were obtained by incorporation of [2-14C]uridine as previously described [4]. Toluene treatment. The toluene permeabilization procedure [8,9] was modified to optimize DNA synthesis in infected minicells. Minicells ( 2 - 1 0 1 ° 5" 101°/ml) were suspended in phosphate buffer (70 mM KH2PO4/K2HPO4, pH 7.4) and exposed to 1% (v/v) toluene for 10 min at 25°C with gentle shaking. Permeabilized miniceUs were washed twice with 10 volumes of phosphate buffer (70 mM KH2PO4/K2HPO4, pH 7.4) in a Sorvall RC2-B refrigerated centrifuge (4°C; 1 2 0 0 0 × g ; 1 0 m i n ) and resuspended in minimal medium ( 2 . 1011 minicells/ml) at 0°C. Bacteriophage adsorption was allowed for 5 min at 0°C (SPO1 input multiplicity = 2; SPP1 input multiplicity = 10) and phage DNA injection into the minecells was facilitated by placing the infected minicells at 37°C for 5 min. (Controls showed no differences in subsequent DNA synthesis experiments when phage infection was permitted before the toluene treatment). Toluene-treated, phage-infected minicells were diluted 10-fold with phosphate buffer, pelleted by centrifugation (12 000 X g; 10 min) and resuspended in phosphate buffer at a concentration of 2 • 1011 miniceUs/ml. DNA synthesis and sucrose gradient analysis. DNA synthesis was measured by incorporation of [Me-3H]thymidine 5'-triphosphate ([3H]dTTP) (30 Ci/ mmol, Amersham) into a trichloroacetic acid-precipitable form. The reaction mixture was as described by Billen and Hellermann [10]. A reaction volume of 0.5 ml contained 2 . 1 0 1 ° minicells and 5 pCi [3H]dTTP (final specific activity = 0.5 pCi/nmol). Upon completion of DNA synthesis the reaction mixture was placed at 0°C and ~ volume of lysozyme solution (20 mg lysozyme in TES buffer (0.05 M Tris • HC1/0.05 M EDTA/0.1 M NaC1 (pH 7.4)) was added.

84 The mixture was placed at 37°C for 2 min, returned to 0°C for 10 min and finally incubated at 37°C for a further 2 min before ~ volume of 10% (w/v) SDS solution (in TES buffer) was added and the mixture placed at 40°C for 10 rain. The resulting clear lysate was analyzed by centrifugation through 5--20% (w/v) sucrose gradients in TES buffer (4.4 ml over a 0.5 ml 60% sucrose cushion). Enzymatic digestion of DNA product. Upon completion of DNA synthesis minicell lysates were subjected to sucrose gradient centrifugation. Fractions from the sucrose gradient containing newly synthesized DNA were dialyzed against 0.06 M Tris • HC1/0.66 M MgC12/1.0 mM 2-mercaptoethanol. Escherichia coli exonuclease III was purchased from Miles Lab. Ltd., Slough England, and used under the standard reaction conditions [ 11]. Enzyme activity was terminated by the addition of 100 pg calf-thymus DNA and 20 vols. of 5% (w/v) ice-cold trichloroacetic acid. Results Uninfected, toluene-treated minicells do not incorporate [3H]dTTP whereas infected minicells (SPO1, SPP1, ¢29) convert [3H]dTTP to a trichloroacetic acid-precipitable form. This synthesis is dependent on the presence of all deoxyribonucleosides triphosphates (dNTP) (Table I). The synthesis results solely from the activity of enzymes present within the minicells at the time of infection as addition of chloramphenicol or rifampicin prior to infection does not decrease the amount of DNA synthesis observed. The inhibitory effect of

TABLE I INCORPORATION

OF

[3H]dTTP

BY

SPOI-INFECTED

MINICELLS

IN

REACTION

MIXTURES

LACKING DEFINED COMPONENTS 2 " 109 S P O l - i n f e c t e d , t o l u e n e - t r e a t e d m i n i e e l i s w e r e a l l o w e d t o i n c o r p o r a t e [ 3 H ] d T T P i n t o c o l d tric h l o r o a c e t i c a e i d - p r e c i p i t a b l e m a t e r i a l for I h at 3 7 ° C . 1 0 0 % w a s e q u i v a l e n t t o t h e i n c o r p o r a t i o n o f 8 2 5 0 c p m in t h e e x p e r i m e n t in w h i c h v a r i o u s c o m p o n e n t s w e r e o m i t t e d a n d e q u i v a l e n t t o t h e i n c o r p o r a t i o n o f 5 7 6 0 c p m in t h e e x p e r i m e n t in w h i c h t h e e f f e c t o f i n h i b i t o r s w a s t e s t e d . M e t a b o l i c i n h i b i t o r s w e r e u s e d at t h e f o l l o w i n g final c o n c e n t r a t i o n s : c h l o r a m p h e n i c o l ( S i g m a , St. L o u i s , M o . , U . S . A . ) 1 5 0 ~ g / m l ; r i f a m p i c i n ( C a l b i o c h e m , S a n D i e g o , C a l i f . U . S . A . ) 1 0 0 ~ g / m l ; n a l i d i x i c acid ( S i g m a , St. L o u i s , M o . , U , S . A . ) 1 0 0 /~g/ml; m i t o m y c i n C ( C a l b i o c h e m , S a n D i e g o , Calif., U . S , A . ) 1 0 0 ~zg/mL Reaction mixture

Residual activity

Complete -- dATP -- dCTP -- dGTP -- dATP, dCTP -- dATP, dGTP -- dCTP, dGTP -- 3-NTP -- ATP - - Mg 2+ complete + chloramphenicol -- complete + rifampicin - - c o m p l e t e r + n a l i d i x i c acid -- complete + mitomycin C

100 18 26 26 15 9 8 7 52 50 106 101 104 93

85 6-(p-hydroxyphenylazo)-uracil (HPUra) [12] on this system has been assayed many times and in the majority of experiments approx. 20% inhibition of synthesis (range 0--40%) was observed. To determine the size of the molecules labelled by incorporation of [3H]d T T P and the fate of the infecting phage genome, minicells were infected with SPP1 phage containing 14C-labelled D N A and, following toluene treatment, were allowed to incorporate [3H]dTTP. Sucrose gradient analyses (Fig. 1) indicated that the majority of input molecules are found at the position at which intact genome-length molecules are located whereas the product D N A is located near the top of the sucrose gradient indicating that it is composed of small molecules. There is, however, a small percentage of input 14C-labelled phage D N A which has been degraded to small molecules of the same size as the 3H-labelled product material. The molecular weight of the material labelled by incorporation of [3H]dTTP (2.10s--4 • 10 s) was estimated by comparison with the restriction fragments obtained by BsuI [13] digestion of 3H-labelled C O L E 1 D N A subjected to identicalsucrose gradient centrifugation.Analysis of of the newly synthesized material by isopycnic CsCI gradient centrifugation following incorporation of density labels ([3H]dTTP for SPO1 [14]; Br[3H]UTP for SPP1) demonstrated that the newly synthesized material was covalently bound to the small molecular weight template D N A (resultsnot shown). The products of d T T P incorporation were further characterized by exposure

1

i ~,

,

,

, ~

100

•-

3H ..o...........i

~

so

5

10 15 20 Froction No.

25

I

I

15

30 rn'm

F i g . I . S u c r o s e g r a d i e n t a n a l y s i s o f t h e i n f e c t i n g a n d n e w l y s y n t h e s i z e d D N A in S S P l - i n f e c t e d , t o l u e n e t r e a t e d m i n i c e U s . T o l u e n e - t r e a t e d m i n i c e l l s w e r e i n f e c t e d b y SSP1 c o n t a i n i n g 1 4 C - l a b e l l e d D N A (e o ) a n d a l l o w e d t o i n c o r p o r a t e [ 3 H ] d T T P f o r 1 h a t 8 7 ° C (e -'). T h e m i n i e e l l s w e r e l y s e d and the lysate placed on top of a 5--20% (w/v) neutral sucrose gradient. Centrifugation was for 2.5 h at 1 5 ° C i n a SW 5 0 . 1 r o t o r a t 4 5 0 0 0 r e v . / m i n . F o l l o w i n g c e n t r i f u g a t i o n t h e g r a d i e n t w a s f r a c t i o n a t e d a n d t h e r a d i o a c t i v i t y c o n t a i n e d in t r i c h l o r o a c e t i c a c i d - p r e c i p l t a b l e m a t e r i a l w a s d e t e r m i n e d . F r a c t i o n 1 is t h e b o t t o m o f t h e g r a d i e n t a n d t h e a r r o w i n d i c a t e s t h e p o s i t i o n o f i n t a c t , g e n o m e - l e n g t h SPP1 m o l e c u l e s . Fig. 2. E. coU e x o n u c l e a s e III d i g e s t i o n o f p r o d u c t D N A . T o l u e n e - t r e a t e d m i n i c e l l s w e r e i n f e c t e d b y S P O 1 containing 14C.labelied DNA and allowed to incorporate [3H]dTTP for 1 h at 37°C. The minicells were l y s e d a n d t h e l y s a t e w a s s e d i m e n t c d t h r o u g h a s u c r o s e g r a d i e n t as d e s c r i b e d i n Fig. 1. F r a c t i o n s 1 2 - - 2 4 f r o m t h e s u c r o s e g r a d i e n t w e r e p o o l e d a n d d i a l y z e d a g a i n s t 0 . 0 6 M Tris • H C I / 0 . 6 6 M MgCI 2 / 1 . 0 m M 2-mercaptoethanol. The solution was concentrated 10-fold by exposure to polyethylene-glycol. Exonuclease III [ 1 1 ] w a s a d d e d t o t h e c o n c e n t r a t e d s o l u t i o n a n d t h e r a d i o a c t i v i t y r e t a i n e d i n a t r i c h l o r o a c e t i e a c i d - p r e c i p l t a b l e f o r m w a s d e t e r m i n e d w i t h i n c r e a s i n ~ l e n g t h o f i n c u b a t i o n a t 8 7 ° C.

86 to nuclease digestion. Toluene-treated minicells were infected b y SPO1 containing '4C-labelled DNA and allowed to incorporate [3H]dTTP. The product was insensitive to the single-strand specific nuclease $1 [15] but was sensitive to digestion by E. coli exonuclease III [11]. In Fig. 2 the kinetics of conversion of '4C-labelled and 3H-labelled DNA to a trichloroacetic acid-soluble form are presented. It can be seen that whereas the 3H-labelled DNA was rapidly digested (75% within 5 min at 37°C) the conversion of 14C-labelled DNA to a soluble form occurs much more slowly. In excess of 90% of the 3H-labelled DNA was solubilized whereas only 45% o f the 14C-labelled DNA was converted to a trichloroacetic acid-soluble form by exonuclease III digestion. Discussion

Toluene-treated phage-infected minicells incorporated dTTP into DNA, however the total incorporation is very low (less than 1% increase in the a m o u n t of infecting phage DNA within 30 min) and depends entirely on host-cell enzymes which degrade a small percentage of the infecting DNA into molecules which act as substrate for the incorporation o f [3H]dTTP. B. subtilis is known to contain an endonuclease with precisely the activity we have observed in minicells, i.e. degradation o f double-stranded DNA into fragments with molecular weights of approx. 1 • 10 s [16]. The endonuclease is reported to produce molecules having free 5'-phosphate and 3'-hydroxyl ends. These molecules would serve as a substrate for DNA polymerase synthetic activity. We therefore propose that the synthetic activity we have observed in minicells results from a minority of infecting DNA molecules undergoing the following series of events: 5~/

input DNA "5' ] endonuclease

I

5/

/ 5 a

/ /

degraded DNA /

[ polymerase (I?) I

5/ ......

...... /5'

/ •....

/

/

7 product

DNA

This model explains w h y the newly synthesized DNA is totally sensitive to degradation by E. coli exonuclease III whereas the input DNA is m u c h less sensitive and degradation of the newly synthesized DNA occurs before degradation of input DNA (Fig. 2). The observed properties of the synthesis, i.e. partial ATP dependence and general insensitivity to HPUra make assignment o f a single known B. subtilis polymerase to the synthetic step difficult, however a similar t y p e of DNA synthesis, namely ATP-stimulated, HPUra-resistent incorporation of dTTP has been reported to occur in cold-shocked nucleated B. subtilis cells and to require the presence of DNA polymerase I [17].

87 References 1 Adler, H.I., Fisher, W.D., Cohen, A. and Hardigree (1967) Proc. Natl. Aead. Sei. U.S. 57,321--326 2 Reeve, J.N., Mendelson, N.H., Coyne, S.I., Halloek, L.L. and Cole, R.M. (1973) J. Bacteriol. 114, 660--873 3 Frazer, A.C. and Curtiss, llI, R. (1975) Curt. Top. Mierobiol. Immunol. 69, 1--84 4 Reeve, J.N. and Cornett, J.B. ( 1 9 7 5 ) J . Virol. 15, 1308--1316 5 Reeve, J.N. (1976) Microbiology 1976, ASM Publications, pp. 332---339 6 Esche, H., Schweiger, M. and Trautner, T.A. (1975) Mol. Gen. Genet. 124, 57--63 7 Mertens, G. and Reeve, J.N. (1977) J. Bacteriol. 129, 1198--1207 8 Moses, R.E. and Richardson, C.C. (1970) Proc. Natl. Acad. Sci. U.S. 67, 674--681 9 Matsushita, T., White, K.P. and Sueoka, N. (1971) Nat. N. Biol. 232, 111--114 10 Billen, D. and Hel]ermann, G. (1975) Biochim. Biophys. Acta 383,379--387 11 Richardson, C.C., Lehmann, I.R. and Kornberg, A. (1964) J. Biol. Chem. 239,251--268 12 Gems, K.B. and Cozzaretli, N.R. (1973) J. Biol. Chem. 248, 7688--/700 13 Bron, S. and Murray, K. (1975) Mol. Gen. Genet. 143, 25--33 14 Yehle, C.O. and Ganesan, A.T. (1972) J. Virol. 9,263--272 15 Ando, T. (1966) Bioehim. Biophys. Acta 114,158--168 16 Burke, Jr., W.F. and Spizizen, J. (1977) Biochemistry 16, 403---410 17 Hennebery, R.C. and Freese, E. (1973) Biochem. Biophys. Res. Commun. 55,788--797