Biochimica et Biopl~vsica Acta 923 (1987) 29-34 29 Elsevier

BBA 22444

The inhibitory e f fec t of dithiothreitol on the assembly

of the f i lamentous phage fd

M a r i l y n V a c c a r o , B e v e r l y B o e h l e r - K o h l e r , W o l f r a m Mi i l l e r a n d I h a b R a s c h e d

Fakulth't j'~r Biologie der Unwersitiit Konstan~, D-7750 Constance (F. R. G.)

(Received 16 June 1986)

Key words: Dithiothreitol; Gene 5 protein; ssDNA-protein complex; (Filamentous phage)

Assembly of the filamentous phage fd is preceded by the formation of a complex between the viral single-stranded (ss) DNA and the virally coded gene 5 protein (gene 5 protein-ssDNA complex). The presence of 5 mM dithiothreitol in the growth medium prevents phage production; however, phage infection, phage DNA replication and phage genome expression are still observed. In contrast, the gene 5 protein- ssDNA complex is not formed in the presence of dithiothreitoi in vivo, although the complex is not affected by the disulfide reducing agent in vitro. Furthermore, host lipid composition is altered by growth in the presence of dithiothreitol. The zwitterionic lipid, phosphatidylethanolamine, increases while the cationic phospholipid content, cardiolipin and phosphatidyiglycerol, decreases. This suggests a role for iipids or membranous structures in the process of gene 5 protein-ssDNA complex formation.

Introduction

Bacteriophage fd is a male-specific, single- stranded DNA phage closely related to phages M13 and fl. Their common hosts are E. coli strains harboring an F-factor. These phages are neither lytic nor lysogenic; infected cells multiply while continuing to release progeny phage through their membranes. The phage genome codes for ten genes [1]. The products of gene 2, gene X and gene 5 are involved in phage DNA replication [2]. After formation of a double-stranded circular superheli- cal DNA (RF I) by host enzymes, viral plus strand synthesis is initiated by gene 2 protein which nicks the RF I. Elongation at the 3'-end occurs via the rolling circle mechanism [3]. Gene 5 protein, a phage encoded DNA-binding protein, cooperatively associates with the nascent viral DNA-strand [4], thus initiating the first step in

Correspondence: Dr. I. Rasched, Fakultgt ft~r Biologie der Universit~it Konstanz, D-7750 Constance, F.R.G.

phage assembly. This nucleoprotein complex may be isolated from lysates of fd-infected cells [5] and appears to be associated with the cellular mem- brane [6]. During the final stages of assembly, gene 5 protein is displaced into the cytoplasm where it is recycled [5], and the DNA is covered with phage coat proteins 8, 3, 6, 7 and 9 before emerging outside the cell as a mature phage par- ticle. In addition to the coat proteins, morphogen- esis also requires the products of genes 1 and 4 and the presence of the host membrane protein thioredoxin [7].

Here we show that the disulfide reducing agent dithiothreitol inhibits phage morphogenesis at the level of DNA-protein complex formation. Our studies indicate that the target of dithiothreitol inhibition is not a phage component. Rather, growth of E. coli in the presence of dithiothreitol has an effect on the lipid composition of the host cell, suggesting that a particular membrane struc- ture or component plays a role in the formation of the viral nucleoprotein complex in vivo.

0304-4165/87/$03.50 ~,c,, 1987 Elsevier Science Publishers B.V. (Biomedical Division)

30

Materials and Methods

Preparation of the gene 5 protein-DNA complex Bacteriophage fd and E. coli strain K37 (supD)

are from our collection. Fd-tet constructed by Zacher et al. [8] is a gift of these authors. L-[U- 14C]Arginine (> 300 mCi /mmol ) and [methyl- 3H]thymidine (> 40 mCi /mmol ) were purchased from Amersham Buchler, F.R.G. The isolation of the gene 5 protein-ssDNA complex from fd-in- fected E. coli cells proceeded according to Ref. 9, with the following modifications. Cells to be grown in the presence of dithiothreitol (Sigma) contained 5 mmol/1 of the freshly dissolved powder in all cultivating media. The 10 ml culture containing infected cells in minimal medium [13] was doubly labeled with 100 /iCi [14C]arginine and 150 /~Ci [3H]thymidine. Following an incubation of 90 min these cells were mixed with 200 ml of unlabeled infected bacteria, collected by centrifugation and washed twice to remove unincorporated label and phage. The cells were gently lysed with lysozyme, cleared, and the gene 5 protein-ssDNA complex was pelleted as described [9]. The resuspended sample was subsequently layered onto a linear 10-40% sucrose gradient in a 10 mM Tris-HC1 buffer (pH 7.6) (1 mM in EDTA and 10 mM in NaCI; TEN buffer) and centrifuged at 34000 rpm in the Beckman SW40 rotor for 10 h. Fractions were collected from the bottom of the gradient and the location of the complex was inferred from the radioactivity profile. Fractions to be analysed were pooled accordingly, dialysed against TEN buffer followed by distilled water and lyophilized. In cases where the radioactivity did not show well separated peaks, the resuspended fractions were pooled and purified by sedimentation through a second 10-40% sucrose gradient.

Phospholipid analyses Phospholipids from E. coli cells were extracted

according to Ref. 10 and separated by ascending thin layer chromatography on acetone-activated silica gel G plates (Merck) in a ch loroform/ methanol /water (65 : 25 : 4, v /v ) developing sys- tem. Phospholipids were visualized and identified with iodine vapor, phosphate spray reagent [11] and ninhydrin spray. Further identification was by chromatography with commercial standards

and by mild alkaline hydrolysis [12]. The fractionated lipids were then extracted from the silica gel plate with chloroform/methanol (1:2). This extraction procedure was repeated two times. The relative quantities of each phospholipid were determined by measuring their inorganic phos- phate content [13].

Results and Discussion

Growth of E. coli" strain K37 is slightly affected by the presence of dithiothreitol up to a con- centration of 5 mM. The generation time due to dithiothreitol was increased by approx. 10 min and the final concentration of cells after growth was reduced by one third. In contrast, the effect of dithiothreitol on the production of fd phage in the host is drastic. At a concentration of 5 raM, dithiothreitol completely inhibited the production of progeny phage as measured by the standard plaque assay in which case efficiency of plaque formation was less than 1 0 - 6 . In addition, no phage were precipitated from the culture medium with poly(ethylene glycol), ruling out the possibil- ity that uninfectious particles are produced.

To investigate the possibility that dithiothreitol might interfere with phage infection, an fd-tet phage carrying the genes coding for tetracycline resistance [8] was used. E. coli cells grown in the presence of 5 mM dithiothreitol and infected with fd-tet were screened for their ability to grow on LB plates containing 10/~g/ml tetracycline and 5 mM dithiothreitol. Following a 10 min incubation, aliquots from the culture were washed twice and diluted with ice cold medium containing 5 mM dithiothreitol before spreading. The number of transduced colonies formed was the same as the number formed from parallel control cultures grown and infected with fd-tet in the absence of dithiothreitol. Thus, added dithiothreitol does not affect phage infection. Furthermore, the fact that colonies (containing approximately 20 generations of cells) grew on plates containing tetracycline and dithiothreitol indicates that the fd-genome must have been replicated.

Since dithiothreitol was shown to prevent phage production but not phage infection, we set out to determine at what point it interferes in the life cycle of fd. E. coli cells were cultured and infected

with fd in the presence and absence of 5 m M di th io thre i to l . Sucrose grad ien ts were pe r fo rmed f rom lysates of b o t h cul tures in o rde r to s tudy the fo rma t ion of the gene 5 p r o t e i n - s s D N A complex. The sed imen ta t ion prof i les shown in Fig. l a clear ly show the presence of gene 5 p r o t e i n - s s D N A com- plex ( 1 4 C / 3 H doub le peak in f ract ions 6 -10 ) in

cells grown in the absence of d i th iothre i to l . How- ever, this doub le peak is not observed in the prof i le ob ta ined f rom cells grown in d i th io thre i to l (Fig. lb ) .

The componen t s of the sucrose grad ien ts shown in Fig. 1 were fur ther ana lysed by e lec t rophores is as descr ibed in the legends to Figs. 2 and 3. The doub le peak in rad ioac t iv i ty in the cont ro l sucrose g rad ien t (Fig. l a f ract ions 6 -10 ) was shown to con ta in the gene 5 p r o t e i n - s s D N A complex: anal- ysis of the D N A conten t of these f ract ions by agarose gel e lec t rophores is showed that viral s s D N A was the ma jo r D N A species (Fig. 2, lane 4); and analysis of the p ro te in con ten t by SDS- p o l y a c r y l a m i d e gel e lec t rophores is fol lowed by i m m u n o p r e c i p i t a t i o n showed that gene 5 p ro te in was the ma jo r p ro te in species presen t (Fig. 3, lane

2).

2~ i i i i i i ! 1 ,

{a) (b) 20 / \ f,.~.

~ II _m l ~ ~ _n , mr .

" ,i /L /1'l j" ":::--....../ "i:.'-<:.'...."-;./ I

0 r J: ~ , , , , [ L , . . . . ~' , A J 5 ,O 15 20 25 5 10 15 20 25

FRACTION Fig. 1. Sucrose gradient centrifugation of the gene 5 protein- ssDNA complex. [14C]Arginine and [3H]thymidine labelled complex was isolated and purified from equal amounts of cells grown and infected in the absence (a) and presence (b) of 5 mM dithiothreitol as described in Materials and Methods. Fractions of 0.5 ml were collected from the bottom of the 10-40% (w/v) sucrose gradients. 20 ~1 aliquots of each frac- tion were analysed in a scintillation counter. (O) I'*C activity; (A) 3H activity. Roman numerals denote the pooling of frac- tions for further analysis in Figs. 2 and 3.

31

1 2 3 4 5 6 7 8 9

RF/ I

R F I ~

SSL ~ , ~

SSC -

Fig. 2. Agarose gel electrophoresis of samples taken from sucrose gradients (Fig. 1). Fractions were pooled as indicated by roman numerals in Fig. 1, dialysed and concentrated as described in Materials and Methods and quantitatively resus- pended in buffer containing 10 mM Tris-HCl (pH gA) and 1 mM EDTA. Aliquots to be analysed were made up to 1% SDS and incubated for 10 min at 60°C. After addition of 1/4 vol. 0.125% bromphenol blue and 20% glycerol, DNA samples were subjected to electrophoresis through 1% agarose gels in buffer containing 40 mM Tris-acetate (pH 8.2) 20 mM sodium acetate and 1 mM EDTA. DNA staining was done with 1 ~g/ml ethidium bromide and visualized by ultraviolet light. Lanes: 1, fd RF I and II standards; 2, fd ss standard; 3 6, zones l-IV from the sucrose gradient in Fig. la; 7 9, peaks I-III from the sucrose gradient in Fig. lb. RF I, supercoiled rcplicative form; RF II, nicked replicative form: SSC, single-stranded closed: SSL, single-stranded linear. Linearization was at least partly caused by lyophilization and storage.

Similar analysis of the cy top la sm from cells infected in the presence of d i th io thre i to l revealed tha t nei ther gene 5 p ro te in nor fd s s D N A were con ta ined in the equivalent f ract ions of the sucrose gradient . G e n e 5 prote in , however, was present in the uppe rmos t f ract ions of the grad ien t (Fig. 3, lane 8) as well as in whole cell lysates of cells grown in d i th iothre i to l . This indicates that gene 5 p ro te in is synthesized, however fo rmat ion of the gene 5 p r o t e i n - s s D N A complex is inhibi ted by the presence of d i th iothre i to l . Whi le the smal ler a moun t s of gene 5 p ro te in observed in di thio- thre i to l - t rea ted cul tures could poss ib ly account for the lack of complex format ion , a subs tant ia l re- duc t ion in the total a m o u n t of gene 5 pro te in in

32

1 2 3 4 5 6 7 8 1 2 3

RFrr - -

gSp - - I RFI

g B p - - I

Fig. 3. SDS-polyacrylamide gel electrophoresis of samples from the sucrose gradients (Fig. 1). Fractions were pooled, dialysed, lyophilized and resuspended in 10 mM Tris-HCl (pH 8.5) to the volume of one fourth the original sucrose gradient fraction. One tenth of the resulting volume was applied and run on 12.5% acrylamide, 8 M urea gels as described in Ref. 22. The gel was subjected to western blotting according to Ref. 23 using antibodies directed against gene 8 and gene 5 proteins /gSp, g5p) before silver staining as in Ref. 24. Lanes 1-4, zones I - IV from the sucrose gradient in Fig. la; lane 5, isolated gene 5 and gene 8 proteins standards: lanes 6-8, zones I - I I I from Fig. lb. All bands which migrated to the marked gene 5 and gene 8 protein position were verified to be these proteins through western blotting (nitrocellulose sheets not shown).

dithiothreitol-treated cells is to be expected in light of the observation that no complex was formed in these cells. The pool of gene 5 protein is carefully regulated and only when it is bound to the viral D N A is it found in substantial amounts within the cell [14]. Furthermore, it should be emphasized that synthesis of single stranded DNA and therefore complex formation normally occurs in the cell shortly (4 min) after infection. At this time, the amount of accumulated gene 5 protein is also low [15]. Thus the cooperative nature of the bonding of ssDNA to gene 5 protein [4] enables complex formation even at low gene 5 protein concentrations. In addition to gene 5 protein, comparable amounts of gene 8 protein were ob- tained from both dithiothreitol-grown and control cultures, indicating that expression of phage pro- teins is not impaired by the reagent.

Analysis of the D N A content of whole cell lysates showed that the phage replicative form is indeed present within infected ceils grown in the presence of dithiothreitol, although to a lesser

Fig. 4. Electrophoretic analysis of the RF DNA from infected whole cell tysatcs grown in the presence and absence of 5 mM dithiothreitol. Log phase cells were infected with phage fd (muhiplicity of infection, 50) and grown (in the presence of 30 ~ g / m l chloramphenicol) under moderate aeration at 37°( ; for 90 rain. Equal amounts of cells were harvcsted and their phage replication form DNA extracted according to the rapid al kaline extraction procedure described in Ref. 25. Lane 1:10 ~tl from control lysates contaMing the DNA from approx. 107 cells: lane 2 : 1 . 2 ~g replicative form DNA purified lhrough CsCI gradient: lane 3: as in lane 1, from dithiothrcitol-lreated cells.

degree than that found in control cultures (Fig. 4). On the other hand, only trace amounts of viral ssDNA were seen in cells infected and grown in dithiothreitol. This latter observation is to be ex- pected since nascent viral strands are only stable when they are protected by the binding of gene 5 protein [2]. Likewise, the absence of complex for- mation can also explain the decreased amounts of replicative form DNA observed in dithiothreitol- treated cultures (which could be an additional cause for the decrease in viral strand synthesis). Gene 5 protein has been shown to repress the translation of the protein coded for by genes 2 and X [16]. In the case of dithiothreitol-grown cells, if the rapidly synthesized gene 5 protein is not bound to the emerging DNA, its accumulation would cause the repression of gene 2 (and gene X) proteins. This in turn would result in a decrease in phage DNA replication and hence fewer repli- cative form DNA copies per cell.

It is also improbable that gene 2 protein (con- taining five Cys residues) is the target of dithio- threitol's reducing activity, since experiments done in vitro demonstrating the endonuclease and ligat- ing activities of this gene product (both functions

being prerequisites for complex formation) have also been performed in the presence of mercapto- ethanol [17]. Similarly, the morphogenesis proteins coded for by genes 1 and 4 are not likely targets for dithiothreitol's action, since gene 5 protein- ssDNA complex has been detected in cells in- fected with phage particles carrying mutations in these genes [9].

To ensure that dithiothreitol does not affect the stability of the complex itself, the following ex- periment was performed. Isolated, radioactively labeled complex was incubated with 5 mM di- thiothreitol at 37°C for 1 h and subsequently sedimented through a sucrose gradient as de- scribed in the legend to Fig. 1. The radioactivity profile obtained was the same as seen in Fig. la.

The results presented thus far indicate that dithiothreitol does not prevent any essential pro- cess (i.e., phage DNA replication, genome expres- sion, protein synthesis) required to occur prior to complex formation. In the presence of dithioth- reitol the products of both genes 2 and 5 are present the only viral proteins necessary for complex formation [9]. No other host component is mandatory for complex formation as ascer- tained from in vitro complex formation experi- ments [4]. However, it should be mentioned that several structural distinctions exist between com- plex found in vitro and in vivo [5], suggesting that the location of complex formation is important. In this regard, it has already been established that the products of genes 2 and 5 as well as the phage RF

33

DNA and gene 5 protein-ssDNA complex itself have all been found associated with the cell en- velope [6]. Therefore, we decided to study the effect of dithiothreitol on E. coli membrane lipid composition.

Table I shows the results obtained from experi- ments measuring the phospholipid content of cells grown and infected in the presence and absence of dithiothreitol. Infection caused a relative decrease in phosphatidylethanolamine content as compared to uninfected cultures, an observation previously reported by other laboratories [18,19]. In contrast, E. coli grown in the presence of dithiothreitol had a significantly higher phosphatidylethanolamine content. Cells containing this dithiothreitol-altered lipid composition did not show the phage-media- ted decrease in phosphatidylethanolamine content but rather the higher dithiothreitol-related phos- phatidylethanolamine content. This dithiothreitol- related increase in phosphatidylethanolamine at the expense of the anionic lipids phosphatidyl- glycerol and cardiolipin in the E. coli membrane would lead to a difference in the ratio of zwit- terionic phosphatidylethanolamine to anionic phosphatidylglycerol and cardiolipin lipid content. The reason for putting the emphasis on anionic or zwitterionic content stems from a report [20] in which it was demonstrated that the surface and consequently binding properties of the E. co6 membrane are not dependent on the exact chem- ical structure of the phospholipid polar head group, but rather on its physical properties. More specifi-

TABLE I

PHOSPHOLIPID COMPOSITION OF E. COLI GROWN IN THE PRESENCE AND ABSENCE OF DITHIOTHREITOL AND PHAGE fd

Results are expressed as percentage of total phospholipids. Values represent an average of 12 individual inorganic phosphate determinations from three different preparations. PE = phosphatidylethanolamine: PG = phosphatidylglycerol: CL = cardiolipin.

Percent Percent Percent Zwitterionic/ zwitterionic anionic other anionic lipids (PE) (PG + CL)

Control uninfected 75.6 22.3 2.1 3.4 infected 71.8 26.8 1.4 2.7

+ dithiothreitol uninfected 80.6 17.9 1.5 4.5 infected 81.4 16.7 1.9 4.9

34

cally it shows that the ratio of anionic to zwit- terionic phospholipids is more relevant than the concentration of any particular phospholioid in the insertion of fd gene products mto the cellular membrane. Because the effect of dithiothreitol on host cell lipids is seen in both infected and unin- fected cells, the possibility that inhibition of phage assembly causes the observed membrane alter- ation can be ruled out.

In conclusion, the disulfide reducing agent has been shown to specifically inhibit fd phage assem- bly, probably by causing changes in the host mem- brane lipid composition. Whether the inhibitory effect of dithiothreitol is due exclusively to dis- turbances in the membrane, which in vivo prob- ably serves as an organizing surface on which the necessary phage and host components are brought together, or whether dithiothreitol exerts an ad- ditional effect on some other components, is cur- rently being studied in this laboratory. Other potential candidates are the protein necessary for producing the proton gradient across the cell membrane which has been shown to be necessary for fd phage assembly [21] or the redox enzyme thioredoxin which also plays a role in assembly [7]. In any case, dithiothreitol should prove to be helpful in elucidating the mechanism of fd phage assembly and perhaps also other membrane-in- volved assembly processes.

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