Heterologous transfection with bacteriophage ΦX174 DNA An improved system

450

Biochimica et Biophysica Acta, 5 1 8 ( 1 9 7 8 ) 4 5 0 - - 4 5 6 © Elsevier/North-Holland

Biomedical Press

BBA 99157

HETEROLOGOUS TRANSFECTION WITH BACTERIOPHAGE ¢X174 DNA: AN IMPROVED SYSTEM

MASANORI

SUZUKI * and MASAO AZEGAMI

Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo (Japan) (Received October 12th 1977)

Summary A highly efficient and much more reproducible system for the heterologous transfection of several kinds of Gram-negative bacterial spheroplasts with bacteriophage ¢X174 DNA was established. By mild washing of the spheroplasts, the efficiency of transfection of all non-host heterologous bacterial species tested increased one or more orders of magnitude in producing the progeny phages and/or the infectious intermediates. Using the improved heterologous transfection systems, it has become clearer that a strong suppression system operates on the processes of ~bX174 progeny phage production and not on those of ¢X174 double-stranded replicative form DNA synthesis in the heterologous bacterial cells. Similar stimulatory effects of this washing procedure were observed in the homologous transfection. With this improved assay system, even less than 100 molecules of phage ¢X174 DNA can be detected and the number of molecules can be determined with accuracy.

Heterologous transfection with ¢X174 DNA has been developed as a unique system of virus-bacterium interaction [1,2]. This system seems to be very useful for studying several interesting problems on biological and biochemical functions of bacteria and the virus, for example, restriction, virus-host specificity beyond the barrier of cell wall, evolutionary relationship between bacterium and bacterium or virus, and so on. In general, however, the efficiency of heterologous transfection was quite low and comparative analyses of the products synthesized in the recipient cells appeared to be rather difficult in most cases. * To w h o m correspondence s h o u l d b e a d d r e s s e d . P r e s e n t address: S e c t i o n of Biochemistry, Molecular and Cell Biology, Comell University, Ithaca, N.Y. 14853, U.S.A. A b b r e v i a t i o n s : HARIM, hydroxylamine-resistant infectious materials; ~bX-SS-DNA, single-stranded circular DNA of bacteriophage ~bX174; ~bX-RF-DNA, double-stranded circular replicative form DNA o f p h a g e ~X174.

451 We tried to circumvent these difficulties in the course of studies on CX-DNA heterologous transfection using various kinds of bacteria belonging to the Enterobacteriaceae as well as to the other families (e.g. Pseudornonas, Bacillus, Haemophilus). In the earlier studies on them, we had observed that the recovery of the input infectivity of CX DNA greatly decreased as the time of incubation with some kinds of bacterial spheroplasts was increased [2]. On the basis of this, the low transfection efficiency (at least some part of it) was thought to be due to the action of extracellular nucleases. In addition, other reports have indicated the release of nucleases during the preparation of Escherichia coli spheroplasts [3--5] and that the competence of E. coli spheroplasts for phage RNA transfection was affected by centrifugation and resuspension of the spheroplasts [6,7]. Moreover, we recently found several nucleolytic enzyme activities different in their specificity from those which were released from E. coli cells in the supernatants after centrifugation of the heterologous bacterial spheroplasts. Some of them were apparently endodeoxyribonucleolytic enzymes (unpublished data). In the light of these observations, the effect of washing spheroplasts (both E. coli and the heterologous bacterial species) was extensively examined. The materials used and the general methods have been previously described [1,2]. It was observed that metal ions such as Cu 2÷ critically inhibited transfection of the heterologous bacterial spheroplasts. To obtain highly reproducible and efficient results, we used deionized water which was redistilled b y means of a glass apparatus in almost all the steps involved in transfection. The improved m e t h o d is as follows: Soon after the addition of MgSO4 to stop lysozyme action, 5 ml of the spheroplast suspension (2 • 109 spheroplasts per ml) in PAM medium (pH 7.3) was overlayered on a cushion of 2.5 ml of PAM containing 2.5 M sucrose and 0.45% bovine serum albumin (Armour, 22% sterilized solution) and then centrifuged in a swinging bucket rotor at 1900 X g for 25 min at 4 ° C. The resultant interband of the spheroplasts was carefully removed from the upper and lower layers with a large bore pipette and resuspended in 5 ml of fresh PAM containing 0.45% albumin with gentle shaking. The suspension was again overlayered on the same cushion and recentrifuged as described above. The spheroplasts removed from the second centrifuge tube were resuspended in 10 ml of PAM containing 0.45% albumin and stored on ice or immediately used for transfection. Table I shows the stimulatory effect of the washing procedure on the progeny phage production in the homologous and heterologous transfection with CX DNA. This m e t h o d was remarkable in that n o t only double-stranded replicative form (RF) DNA b u t also single-stranded (SS) DNA transfection could be equally improved in contrast with protam.ine treatment which stimulates only double-stranded DNA transfection [ 8]. Improvements must have been made in t w o areas for our heterologous transfection studies: one appeared to augment the efficiency of heterologous transfection itself and the other appeared to increase the sensitivity in assaying the infectious materials synthesized in the recipient heterologous bacterial spheroplasts using the spheroplasts of the most susceptible species of E. coli. The great increment in the efficiency of homologous transfection would be very important especially to the latter purpose. With this improved system, we could usually raise the specific biological activ-

452 TABLE I EFFICIENCY OF TRANSFECTION

OF WASHED AND UNWASHED SPHEROPLASTS

( ~ X - D N A ( 1 0 1 0 m o l e c u l e s / m l , SS- o r R F - D N A ) w a s i n c u b a t e d w i t h a n e q u a l v o l u m e o f s p h e r o p l a s t susp e n s i o n ( 1 0 9 s p h e r o p l a s t s / m l ) . I n c o m p a r a t i v e e x p e r i m e n t s to e s t i m a t e t h e e x t e n t o f f a c i l i t a t i o n o f t r a n s fection by the washing procedure, the spheroplasts which were prepared according to the routine proced u r e [ 1 ] w e r e d i v i d e d in t w o p o r t i o n s . T h e o n e w a s s t o r e d o n ice, w h i l e t h e o t h e r w a s w a s h e d as d e s c r i b e d i n t h e t e x t a n d r e s u s p e n d e d in f r e s h P A M m e d i u m ( p H 7 . 3 ) in t h e p r e s e n c e o f 0 . 4 5 % a l b u m i n a n d s t o r e d o n i c e u n t i l u s e d . E f f i c i e n c y o f t r a n s f e c t i o n is d e f i n e d b y t h e r a t i o o f p l a q u e - f o r m i n g u n i t s o b t a i n e d p e r D N A m o l e c u l e . T h e n u m b e r s in p a r e n t h e s e s i n d i c a t e t h e n u m b e r o f e x p e r i m e n t s p e r f o r m e d . DNA

Bacterium

Efficiency Unwashed spheroplasts

Washed spheroplasts

Min

Min

Max (51)

SS

E s c h e r i c h i a coli K 1 2 W 6

7.6 • 10 -3

E s c h e r i c h i a coil Q 1 3

2.5 • 10 -2

9 . 2 " 10 -1 (4) 1.3 • 10 -1

Salmonella typhimurium

4 . 6 - 1 0 -6

Aerobacter aerogenes

7.8 • 1 0 -8

E s c h e r i c h i a coli K 1 2 W 6

1.1 • 10 --4

8.9 • 10 -1

(24) 144 (3) 27

9.3

(3) 5.3 • 1 0 - 4

9.9 • 1 0 - 3

5.1 • 1 0 - 2

(3) 730

1 . 4 • I 0 -S

(3) 2.2 • 1 0 - 3

8.1 • 10 - 3

(3) 891

2.6 • 10 -2

(7) 4 . 5 • 10 -2

4.1 • 10 -1

(7) 156

(12)

(16) RF

Max

(24) 5.2 • 10 -1 (3) 6.6 • 10 -1

(12)

Average facilitation over control

ity of ~bX DNA (plaque-forming units produced per DNA molecule) to more than one. On the other hand, any other methods would give far lower specific activity (and thus far lower sensitivity in assaying) of CX DNA. For example, the specific radioactivity attained by usual methods is at the most in the order of 10 -s to 10 -4 cpm per CX DNA molecule. In this connection, the range of linearity of the number of plaque-forming units produced with that of the transfecting DNA molecules was shown to be extended to a low concentration of DNA as compared with the conventional system with unwashed spheroplasts as shown in Fig. 1. The lower limit of detection and quantification of the DNA would fall below 100 molecules under our condition. This is clearly one of the remarkable merits of this washing system.

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k O G DNA MOk~CUkES/ML Fig. 1. L i n e a r i t y o f e x t e n t o f t r a n s f e c t i o n w i t h ~bX174 D N A (SS) c o n c e n t r a t i o n . T h e c u r v e s r e p r e s e n t t h e t i t e r o f p r o d u c e d p r o g e n y p h a g e . T h e w a s h e d ( A ) a n d t h e u n w a s h e d (B) E. coli K 1 2 W 6 s p h e r o p l a s t s (1 • 109 s p h e r o p l a s t s / m l ) w e r e p r e p a r e d f r o m t h e s a m e b a t c h o f s p h e r o p l a s t s in t h e p r e s e n c e o f a l b u m i n as d e s c r i b e d in t h e l e g e n d o f T a b l e I. T h e r e s u l t s o f t w o s e t s o f e x p e r i m e n t s w h i c h w e r e p e r f o r m e d w i t h t h e d i f f e r e n t s p h e r o p l a s t p r e p a r a t i o n s are s h o w n , o, E x p t . 1; e, E x p t . 2.

453 TABLE II STIMULATORY EFFECT OF WASHING PROCEDURE RESISTANT INFECTIOUS MATERIALS (HARIM)

ON THE

YIELD OF HYDROXYLAMINE-

E x p e r i m e n t a l details have b e e n previously described [ 1 , 2 ] . The i n f e c t i o u s materials e x t r a c t e d f r o m the transfected spheroplasts ( w a s h e d or u n w a s h e d ) b y S a r k o s y l - p h e n o l t r e a t m e n t at 3 0 m i n after t r a n s f e c t i o n w i t h ~ X - S S o D N A (multiplicity o f t r a n s f e c t i o n w a s 1 0 0 ) w e r e treated with 0 . 2 M h y d r o x y l a m i n e - H C 1 ( p H 6 . 0 ) a t 3 7 ° C f o r 6 h to inactivate c o m p l e t e l y i n f e c t i o u s i n p u t S S - D N A and t h e n titrated o n u n w a s h e d E. c o l i K 1 2 W 6 spheroplasts. T h e n u m b e r o f H A R I M as ~ X - R F - D N A equivalents w a s calculated f r o m the standard curve ( n o t s h o w n ) p l o t t e d w i t h purified ~ X - R F I - D N A , assuming that the remaining infectivities after 6 h h y d r o x y l a m i n e t r e a t m e n t , ( b y that t i m e t h e y had already reached plateau) w e r e due to R F - D N A . Each value is the average o f three e x p e r i m e n t s w i t h each bacterial spheroplast. Bacterium

R F equivalent per spheroplast Unwashed spheroplast

Esc herichia c o l i K 1 2 W 6 Escherichia coli Q13 Salmonella typhirnurium A e r o b a c ter a e r o g e n e s Klebsiella pneumoniae P r o t e u s vulgaris Serratia m a r c e s e e n s Pseudomonas aeruginosa

4.7 9.6 2.8 2.2 2.0 6.4 9.9 2.1

Washed spheroplast

10 -2 10-3 10 -3 10 -3

1.3 • 10 1.8 4 . 4 " 10 -1 5.9 • 10 -1

10 -3

2.1 • 10 -1

10 -3

2.5 • 10 -I

10-3

1.7 • 1 0 -I

10-2

8.8 • 1 0 -2 *

* I n c o m p l e t e l y w a s h e d (see t e x t ) .

The washing procedure proved very highly efficient in increasing the efficiency of heterologous transfection itself. The spheroplasts of Salmonella typhimurium and Aerobacter aerogenes became two orders of magnitude more competent both in producing CX progeny phages and the hydroxylamine-resistant infectious materials (HARIM [1] ) after being washed (Tables I and II). On the other hand, Klebsiella pneurnoniae, Proteus vulgaris and Serratia marcescens became one to two orders of magnitude more competent in producing the HARIM, but without any detectable increment in the yield of the progeny phage (Table II). Pseudornonas aeruginosa spheroplasts could not be effectively washed because of their increased viscosity after EDTA-lysozyme treatment; however, some improvement on the HARIM yield was observed after washing. These facts indicate that the washing procedure facilitates the incorporation of the transfecting DNA molecules into those spheroplasts of heterologous as well as homologous bacterial species and the synthesis of the infectious intermediates such as HARIM through the input ~bX-SS-DNA. On the other hand, it has become clearer that a strong suppression system operates on the processes of @X phage development in the heterologous bacterial cells. This is shown by noting the differences in the extent of increases in the yield of CX mature progeny phages between the bacterial species relatively close to ~bX natural host bacterium E. coli and distantly related ones. For the former, S. typhimurium and A. aerogenes produced ~bX phage at a rate proportional to that in HARIM synthesis by the washing procedure. The latter species contrast this by showing no detectable stimulatory effect on the progeny phage yield in such bacteria as K. pneumoniae and P. aeruginosa. This suggests that the critical difference in the fate of the transfecting DNA between the homologous or close heterologous and distant-heterologous transfection is not at the stage of its incorporation

454

into the spheroplasts and the synthesis of the RF-DNA or the infectious intermediates such as HARIM containing RF-DNA, b u t at the subsequent stage(s) leading to the mature progeny phage formation. Concerning homologous transfection, it should be especially noted that HARIM are synthesized in great quantities in E. coli K12W6 spheroplasts upon transfection with ¢X-SS-DNA (13 molecules per spheroplast as CX-RF-DNA equivalent, Table II). We demonstrated that almost all of such HARIM synthesized in the spheroplasts of E. coli species are ¢X-RF-DNA [1]. Therefore, we can compare directly the transfection efficiency in our improved system and the efficiency of normal infection with the intact phage and cell, which had been already extensively studied by others. Apparently the number of CX-RFDNA molecules synthesized in both systems is almost equal (15--20 RF-DNA molecules are synthesized in normal infection system [9]). This fact implies that the CX DNA transfection system can be used as efficiently as (or even instead of) the normal phage infection system. This is very important to various studies on the interaction of the DNA with several macro- and small molecules; e.g., examination of the effects of drugs such as carcinogens on the DNA. In preliminary studies, MS2 phage RNA transfection to the spheroplasts of several kinds of bacterial species was found to have remarkable stimulation by this washing method. The procedure of washing of E. coli spheroplasts through sucrose layers to measure the extent of CX DNA adsorption to the spheroplasts has been reported b y Guthrie and Sinsheimer [10]. But no mention was made of the effect of it on transfection efficiency. The procedure of washing can also be found in the report of T4 phage transformation of penicillin-induced spheroplasts o f E . coli [11]. To examine the nature of the washed spheroplasts more precisely and in detail, E. coli K12W6 was used for further experiments, because we could easily see the course of transfection and the o u t c o m e of it. However, the heterologous bacterial spheroplasts showed features very similar to those of E. coli species in almost all aspects. Fig. 2 shows the stability of competence. The washed spheroplasts were unstable and lost their competence rapidly if bovine serum albumin was omitted from the suspending medium. The presence of albumin is essential to keep the competence stable and it seemed best to use the spheroplast preparations immediately after washing. Gelatin could n o t be substituted for albumin. Protamine sulfate [8,12--14] was found to be effective on the washed spheroplasts as well as the unwashed ones (Fig. 3). By using the washed, protaminetreated E. coli K12W6 spheroplasts, the efficiency of transfection with CX-RFDNA could be raised up to approximately 10 plaque-forming units per input RF-DNA molecule, i.e. one infective center per 10 input RF-DNA (one infective center produces a b o u t 100 plaque-forming units (unpublished data; ref. 10)). Nevertheless, the protamine treatment alone (without a washing procedure) caused little stimulatory effect in titrating the HARIM which were synthesized in the heterologous bacterial spheroplasts. This p h e n o m e n o n may be due to the fact that most of such HARIM have single-stranded segments of R N A which are sensitive to ribonuclease I [1]. It should be noted that protamine sulfate raised equally the transfection efficiency of the washed and unwashed E. coli spheroplasts. A similar effect was

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Fig. 2. S t a b i l i t y o f c o m p e t e n c e o f s p h e r o p l a s t s . P r e p a r a t i o n o f E. coli K 1 2 W 6 s p h e r o p l a s t s w a s p e r f o r m e d as d e s c r i b e d in t h e l e g e n d o f Fig. 1 e x c e p t t h a t t h e y w e r e r e s u s p e n d e d in PAM ( p H 7 . 3 ) e i t h e r in t h e prese n c e o r a b s e n c e o f 0 . 4 5 % a l b u m i n a n d s t o r e d o n ice u n t i l u s e d . T h e a l i q u o t s (0.4 m l , 109 s p h e r o p l a s t s / m l ) w e r e t r a n s f e c t e d w i t h ~ X - S S - D N A ( 0 . 4 m l , 107 m o l e c u l e s / m l ) a t t h e i n d i c a t e d t i m e . o o, w a s h e d s p h e r o p l a s t s in t h e p r e s e n c e of a l b u m i n ; ~ . . . . . . A, w a s h e d s p h e r o p l a s t s in t h e a b s e n c e o f a l b u m i n ; e--~e, u n w a s h e d s p h e r o p l a s t s in t h e p r e s e n c e o f a l b u m i n . Because t h e u n w a s h e d s p h e r o p l a s t s in t h e a b s e n c e o f a l b u m i n w e r e q u i t e u n s t a b l e a n d gave little r e p r o d u c i b l e results a n d l o w efficiencies, t h e y are n o t given in this figure. Fig. 3. S t i m u l a t o r y e f f e c t of w a s h i n g p r o c e d u r e in t h e p r e s e n c e o f p r o t a m i n e sulfate. T h e w a s h e d a n d unw a s h e d E. eoli K 1 2 W 6 s p h e r o p l a s t s w e r e p r e p a r e d as d e s c r i b e d in t h e l e g e n d o f T a b l e I. T h e y w e r e s t o r e d o n ice f o r 2 h a f t e r t h e a d d i t i o n o f 0 . 0 5 m l o f e a c h c o n c e n t r a t i o n o f s a l m i n e s u l f a t e , w h i c h w e r e p r e p a x e d b y t h e m e t h o d o f R a s m u s s e n [ 1 5 ] , i n t o 0 . 9 5 m l o f t h e s p h e r o p l a s t s u s p e n s i o n s (109 s p h e r o p l a s t s ] m l ) , a n d t h e n t r a n s f e c t e d w i t h ~ K - R F - D N A (1.0 ml, 107 m o l e c u l e s / m l ) . T h e s y m b o l s are t h e s a m e as in Fig. 2.

observed with the other bacterial species. Because inhibitory bacterial DNA [16] would be removed b y the repeated centrifugation, it would seem that protamine sulfate stimulates transfection through a direct interaction with spheroplasts. This is n o t consistent with the speculation that it makes a non-inhibitory complex with the bacterial DNA resulting in the stimulation of viral doublestranded DNA transfection [8]. In fact, protamine could bind to ~X-RF-DNA as well as bacterial DNA. We observed that the preincubation of the transfecting ~bX-RF-DNA with protamine sulfate and an excess a m o u n t (more than 200 pg per ml) of it inhibited the DNA's transfection as reported by others [8,14]. However, our results rather support those of Sabelnikov et al. [17]. As these workers supposed from their experimental results, protamine may increase the permeability of the cell wall and the attachment of viral DNA to the cell membrane along with the increase in the DNA adsorption to the spheroplast. The stimulatory effect of the washing procedure described in the present report is probably due to the removal of the inhibitory substances such as nucleases, bacterial nucleic acids which may be released from lysed cells, and others, if any. Because of its simplicity and applicability to the widely selected bacterial species, the washing of spheroplasts as described here should be very effective in obtaining high transfection efficiency and reproducible result for many bacterial species and viruses as compared with the other conventional transfection systems.

Acknowledgements We thank Dr. R. Benzinger for his suggestions on the use of protamine sulfate and albumin. The authors are grateful to Dr. G. Petersen for careful reading and correction of the manuscript.

456

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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