Bacteriophage N4-induced transcribing activities in Escherichia coli II. Association of the N4 transcriptional apparatus with the cytoplasmic membrane

VIROLOGY

95,466-475

Bacteriophage

(1979)

N4-Induced

Transcribing

II. Association of the N4 Transcriptional

S. C. FALCO’ Department

of Biophysics

Activities

in Escheric.l?ia

Apparatus with the Cytoplasmic

co/i

Membrane

AND L. B. ROTHMAN-DENES2

and Theoretical

Biology,

Accepted

The University

February

of Chicago, Chicago,

Illinois

60~~

27, 1979

Several lines of evidence indicate that the two rifampicin-resistant transcribing activities induced by Escherichia coli phage N4 are associated with the cell membrane. First, under lysis conditions designed to avoid nonspecific DNA-membrane association, the N4 RNAsynthesizing activities (as well as the endogenous N4 DNA) cosediment with the E. coli DNA membrane complex in sucrose gradients. Second, it has not been possible to dissociate the activities from the DNA-membrane complex by any treatment which has been tested; for example, after high salt treatments (up to 4 M NaCl), which do not irreversibly inactivate the activities, the activities remain stably associated with the complex. However, low detergent concentrations inhibit the activities, and irreversible inactivation results from treatment with the anionic detergent, sodium deoxycholate. Third, upon separation of the cell envelope of N4-infected E. coli into inner (cytoplasmic) and outer membrane fractions, the NQ-induced RNA-synthesizing activity is found associated with the cytoplasmic membrane. Based on electrophoretic migration in sodium dodecyl sulfate-polyacrylamide gels, the NCinduced polypeptides found associated with the DNA-membrane complex are also found selectively associated with the cytoplasmic membrane. INTRODUCTION

The binding of DNA to the cell membrane was tist proposed by Jacob et al. (1963) as a mechanism for chromosome segregation during bacterial division as part of their replicon model. Since then a substantial amount of evidence has accumulated indicating the association of DNA with the bacterial cell membrane (Ganesan and Lederberg, 1965; Smith and Hanawalt, 196’7, Ryter, 1968; Sueoka and Quinn, 1968; Tremblay et al., 1968). More recently the DNA of E. coli has been isolated in a folded, supercoiled form attached to cell membrane fragments (Stonington and Pettijohn, 1971; Worcel and Burgi, 1972, 1974; Pettijohn et al., 1973); and specific cytoplasmic membrane proteins have been implicated in this attachment (Portalier and Worcel, 1976). 1 Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge Mass. 02139. 2 To whom correspondence should be addressed. 004%6822/79/080466-10$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

466

The attachment of the DNA of several bacteriophages to the host cell membrane, at some stage of phage development, has also been observed. This has been shown for Escherichiu coli phages 4X174, A, and T4 (Knippers and Sinsheimer, 1968; Hallick et al., 1969; Earhart, 1970) and for Salmonella typhimurium phage P22 (Botstein and Levine, 1968) among others. The function of the phage DNA-membrane association is not yet known. Roles in DNA replication and/or maturation of phage heads have been suggested (Frankel et aZ., 1968; Botstein and Levine, 1968; Hallick et al., 1969). Another possibility is that attachment of phage DNA to the cell membrane might be required for, or be the consequence of, phage transcription. During lytic growth of phage lambda, DNA-membrane association requires the N gene product (Hallick et al., 1969) and may be the result of active early mRNA transcription (Kolber and Sly, 1971). Transcription of the late genes of phage T4 in vitro has only been possible using T4

BACTERIOPHAGE

NCINDUCED

DNA-membrane complexes (Snyder and Geiduschek, 1968; Rabussay and Geiduschek, 1973; Maor and Shalitin, 1974). We have shown that bacteriophage N4induced rifampicin-resistant transcribing activities are found in DNA-membrane complexes obtained from gently lysed cells (see preceding paper). In this report we present additional evidence indicating that the N4 transcriptional apparatus is associated with the cytoplasmic membrane of E. coli. MATERIALS

AND METHODS

Bacteria and phage. Escherkhiu coli K-12 strain W3350 was used in all experiments. N4 and N4 amber mutant am15 were obtained from G. C. Schito. Reagents. rH]Thymidine 60 Cilmmol and [14C]GTP 50 mCi/mmol were purchased from New England Nuclear; ““SO:- was purchased from ICN; rifampicin and chloramphenicol was obtained from Sigma Chemicals; ATP, CTP, GTP, and UTP were purchased from P-L Biochemicals; streptolydigin was a gift of the Upjohn Chemical Company; T4 lysozyme was a gift of M. Inouye. Growth of cells and infection by N4. This procedure was as described in the preceding paper (Falco and Rothman-Denes, 1979). Preparation of the DNA -membrane complex. Sodium Azide (NaN,) was added to a concentration of 0.01 M 7 min after infection. The cells were collected by centrifugation, washed in media containing 10 mM NaN,, plus rifampicin (200 pg/ml) and chloramphenicol (100 pg/ml) if applicable, and again collected by centrifugation. All operations were at 4”. Cells were lysed as described by Silberstein and Inouye (1974). A washed cell pellet obtained from 10 ml of culture was resuspended in 0.2 ml of 20% sucrose 0.1 M NaCl, 0.01 M Tris-HCl, pH 80.01 M NaN,. T4 lysozyme, 0.05 ml, at a concentration of 0.05 @g/ml in 0.12 M Tris-HCl pH 8, 0.05 M EDTA pH 8 was added. The mixture was kept at 30” for 5 min. Then 0.25 ml of 0.1% Triton X-100, 0.01 M EDTA pH 8, 0.01 M NaN, was added. Lysis was complete after 20 min at 30”. Using a wide-bore pipet the lysate was carefully layered onto a lo-ml, lo-30% linear sucrose gradient which con-

TRANSCRIBING

ACTIVITIES

467

tained 0.01 M Tris-HCl pH 8, 0.001 M EDTA, and 0.001 M 2-mercaptoethanol. The sucrose gradient had been layered onto a l-ml shelf of 62% sucrose containing CsCl, p = 1.5. The gradients were centrifuged at 23,000 rpm in a Beckman SW 41 rotor for 10 min at 5”. Alternatively, for large scale preparations of the DNA-membrane complex lysis procedure 1 described in the preceding paper was employed. Isolation and separation of the cell membrane fractions. Isolation of the cell membrane and its separation into cytoplasmic and outer membrane fractions was as described by Osborn et al. (1972). Assay for N.4 transcribing activities. The standard assay employed was as described in the preceding paper. Polyacrylamide gel electrophoresis of proteins. The procedure was as described in the preceding paper. The composition of the polyacrylamide gels was as described by Blattleret al. (1972). Fluorography was performed according to the method of Bonner and Laskey (1974). RESULTS

The association of the two distinct bacteriophage N4-induced transcribing activities with a DNA-membrane complex obtained from gently lysed E. coli has been demonstrated (see previous paper). The nature of this association is not clear. However, Silberstein and Inouye (1974) have shown that basic proteins, such as lysozyme, other polycations, such as spermidine, or the divalent cation Mg2+ can result in the formation of a rapidly sedimenting “secondary DNA-membrane complex.” The lysis procedures which yielded DNA-membrane complexes containing the N4 transcribing activities made use of fairly high concentrations of lysozyme as well as spermidine. It was therefore important to determine whether the association of the transcribing activities with these complexes resulted from the conditions of lysis employed. The experiments shown in Figs, 1 and 2 utilize the method developed by Silberstein and Inouye (1974), which is a modification of the procedure of Stonington and Pettijohn (1971). The procedure makes use of very low

FALCO AND ROTHMAN-DENES

468

concentrations of T4 lysozyme (0.01 pg/ml) and nonionic detergents and eliminates nonspecific aggregation of DNA and membrane into a rapidly sedimenting form. In Fig. 1A lysates of cells infected by N4, labeled with [3H]thymidine, have been sedimented through a lo-30% sucrose gradient. Sixty percent of the N4 DNA is found in a rapidly sedimenting form at the bottom of the gradient, cosedimenting with the E. coli DNA-membrane complex (Fig. 1A). The reason for the partitioning of the N4 DNA into rapidly and slowly sedimenting forms is not known (see Discussion). I

300

v

A

-

j

r 7

g 200 x

70 L

f 0 z r2

100

E 4 %

FIG. 1. Association of N4 DNA with the E. coli DNA-membrane complex. Lysis and sucrose gradient centrifugation as described under Materials and Methods; snow indicates direction of sedimentation; a 0.1~ml aliquot of each fraction was assayed for acid-insoluble radioactivity ae described (Rothman-Denes et al., 1973). (A) Cells were labeled with rH]thymidine (40 ~CiIml) for the final generation of growth. Rikmpicin (200 &ml) was added 15 min prior to infection by unlabeled N4 (O), or unlabeled cells were infected with [3H]thymidinelabeled N4 (30,000 cpm per 1 x 10”’ PFU) in the absence (A) or presence (A) of pretreatment with rifampicin. (B) Purified rH]thymidme-labeled N4 DNA (0.8Fg of 15,000 cpm/pg N4DNA) was added to unlabeled cells immediately preceding lysis; (0) uninfected, rifampicin-treated cells, (A) NCinfected, rifampicin-treated cells.

10 Fraction

20 Number

FIG. 2. Association of N4-induced transcribing activities with the E. coli DNA-membrane complex. Lysis and sucrose gradient centrifugation as described under Materials and Methods except sucrose-CsCl shelf was omitted from these gradients; arrow indicates direction of sedimentation. Fractions were collected from the top. Pellet (fraction 1) was resuspended in 0.7 ml of 30% sucrose, 0.01 &f Tris-HCl pH 8,O.OOl M EDTA, and 0.001 M 2-mercaptoethanol. (A) Cells were treated with rifampicin (200 pg/ml) 15 min prior to infection by [3H]thymidine-labeled N4 (30,000 epm per 1 x 10”’ PFU); a O.l-ml aliquot of each fraction was assayed for acid-insoluble radioactivity as described (Rothman-Denes et al., 1973). (B) Cells were treated with rifampicin (200 pg/ml) 15 min prior to infection by N4 (M) or rifampicin at 15 min and chloramphenicol at 10 min prior to infection (0). Aliquots of fractions (0.05 ml, (0); 0.02 ml, (m)) were assayed for RNA polymerase activity in the presence of 200 pg/ml of streptolydigin and 20 pg/ml of N4 DNA.

When purified 3H-labeled N4 DNA was added to E. coli (either uninfected or infected by unlabeled N4) before lysis, the labeled DNA did not sediment rapidly with the DNA-membrane complex after lysis, as shown in Fig. 1B. This result argues against the possibility that the cosedimentation of N4 DNA from infecting phage with the complex is due entirely to nonspecific aggregation. In the experiment shown in Fig. lA, the

BACTERIOPHAGE

NCINDUCED

TRANSCRIBING

469

ACTIVITIES

RNA-synthesizing activity in soluble fractions of cells lysed by a variety of techniques have been unsuccessful. As a result, considerable effort has been aimed at the dissociation of the transcribing activities from the DNA-membrane complex, so that purification and further characterization of the activities might be possible. For these studies DNA-membrane complexes which contained both N4 transcribing activities were used. However, it should be noted that activity II is present in great excess over activity I in these extracts (see Table 1 and preceding paper). The transcribing activities are quite stable in the complex and remain associated with it after rather harsh treatments. Inhibition of the activities by the presence of low salt concentrations (Falco and Rothman-Denes, 1979) is completely reversible. In fact, treatment of the complex with a variety of different salts [KCl, NaCl, NH&l, (NH,),SO,J over a wide range of concentrations (from 0.1 to 4 M) has no irreversible effect on the activities and does not release activity from the complex. Treatment at low pH (down to pH 3), even at high salt concentrations, neither inactivates the RNA polymerase activities, nor dissociates activity from the complex. Treatment at increasing pH does not release activity from the complex, but eventually (at pH 10) results in irreversible inactivation. Several methods designed to separate proteins from nucleic acid were employed

DNA-membrane complex sediments to the interface of the lo-30% sucrose gradient and a sucrose-CsCl shelf. This shelf has been eliminated in the experiments of Fig. 2 so that the N4 transcribing activities, which are sensitive to high salt concentrations, could be assayed. Under these conditions 60% of the N4 DNA is found in a pellet at the bottom of the gradient (Fig. 2), along with the E. coli DNA-membrane complex (not shown). All of the rifampicinand streptolydigin-resistant N4-induced RNAsynthesizing activity is found in the pellet of parallel gradients (Fig. 2B). This activity does not depend on the addition of exogenous DNA (Table 1). No drug-resistant RNAsynthesizing activity is found in the pellet fraction (Table 1) or any other fraction from gradients on which lysates of uninfected cells were run (not shown). These experiments show that the N4 transcribing activities are found associated with the cellular DNA-membrane complex under conditions designed to eliminate nonspecific rapidly sedimenting DNAmembrane aggregates. Furthermore, the observation that all of the N6induced RNApolymerizing activity sediments with the complex (which contains about 60% of the N4 DNA) and that no activity is found with the 40% of the N4 DNA which sediments slowly suggests that association of the N4 transcriptional apparatus with the cell membrane is essential for activity. All attempts to measure N4-induced TABLE

1

NI-INDUCED RNA POLYMERASE ACTIVITY IN THE DNA-MEMBRANE Incorporation

COMPLEX”

of GMP (pmoU15 min)

Assay conditions

Uninfected

Uninfected + l-if

N4 infected + rif

N4 infected + i-if, CAM

No additions +Streptolydigin (200 pg/ml) +Streptolydigin (200 pg/ml and N4 DNA 20 rglml

298 28

7 0

2680 2780

73 64

-

-

2160

60

a Preparation of DNA-membrane complex was as described under Materials and Methods and in legend to Fig. 2B. Pellet fractions were resuspended in 1150 vol of the original cell culture in 0.01 1M Tris-HCl pH 7.9, 0.01 M MgCI,, 10m4M EDTA, lo-‘M dithiothreitol, 20% glycerol. Assay conditions as in Materials and Methods. Twenty-microliter aliquots were assayed.

470

FALCO AND ROTHMAN-DENES

to try to dissociate the RNA-synthesizing activity from the complex and/or to make the activity dependent on the addition of exogenous DNA. These included precipitation of the DNA at low salt concentration using streptomycin sulfate or at high salt concentration (1 M NaCl) using polyethylene glycol (Molineaux et al., 1974), and phase partitioning of nucleic acid and protein at high and low salt concentrations (Okazaki and Kornberg, 1964; Babinet, 1967). In all cases the RNA-synthesizing activity remained associated with the DNA. Further attempts to dissociate activity from the DNA in the complex made use of both pancreatic and micrococcal nuclease. Treatments which resulted in up to 80% reduction of transcribing activity in the complex along with solubilization of most of the DNA were attained, but no RNA polymerase activity could be detected in the soluble fraction. The N4 transcribing activities in the DNA-membrane complex are inhibited by low concentrations of several detergents (Fig. 3). For the detergent deoxycholate (DOC) the reversibility of this inhibition has

CONCENTRATION

( % )

FIG. 3. Detergent sensitivity of NCinduced transcribing activities. Detergents were present during the assay at the indicated concentrations. Protein concentration 0.6 mg/ml. One hundred percent = 620 pmol GMP incorporated.

I

10 INCUBATION

I 20 TIME

30 (min)

FIG. 4. Irreversible inactivation of N4 transcribing activities by deoxycholate. N4 DNA-membrane complex was incubated in the absence of detergent for the times indicated and assayed as under Materials and Methods (0). N4 DNA-membrane complex was incubated in the absence of detergent for the times indicated and assayed in the presence of 0.025% deoxycholate as under Materials and Methods (0). N4 DNAmembrane complex was incubated in the presence of 0.1% deoxycholate and assayed at a fmal concentration of 0.026% deoxycholate as under Materials and Methods (X). Protein concentration in the assay: 0.6 mglml. One hundred percent = 850 pmol GMP incorporated.

been investigated. The experiment presented in Fig. 4 shows that the RNAsynthesizing activities are stable when incubated at 37” in the absence of detergent. Addition of DOC, for different periods of incubation, to a concentration of 0.025% in the RNA polymerase assay mixture results in a constant level of inhibition of activity throughout the incubation period. In contrast, upon incubation of the activities in the presence of 0.1% DOC, followed by dilution to a concentration of 0.025% in the assay mixture, a decrease in RNA-synthesizing activity is observed. Thus treatment with DOC irreversibly inactivates the N4 transcribing activities. Our inability to detect NQ-induced RNA polymerase activity in soluble fractions as well as our inability to release activity from

BACTERIOPHAGE

N4-INDUCED

the DNA-membrane complex again suggests the essential nature of the association of the N4 transcriptional apparatus with the cell membrane. The inactivation of the activities by mild detergent treatments lends further support to this hypothesis. Therefore, a more direct method for investigating the association of the N4 transcribing activities with the cell membrane was employed. The cell envelope of E. coli is composed of an inner and outer cell membrane (Schnaitman, 1971; Osborn et al., 1972). A technique for the isolation of these membranes and their separation by isopycnic sucrose gradient centrifugation has been developed (Osborn et al., 1972). This tech-

FRACTION

TRANSCRIBING

ACTIVITIES

471

nique has been used for the study of membranes from N4 infected E. coli. In the experiment shown in Fig. 5, cells were labeled for 1 hr with a 3H-amino acid mix. Rifampicin was then added, followed 15 min later by N4 infection. At 7 min after N4 infection chloramphenicol was added, the cells were collected and the membranes were isolated and fractionated as described (Osborn et al., 1972). Simultaneous experiments were performed with unlabeled E. coli, either infected or uninfected by N4. The separation of the outer (H) and inner membranes and the fractionation of the inner membrane into three components (M, 4, L,) shown in Fig. 5 is as described by Osborn indicating that the drug treat-

NUMBER

FIG. 5. Association of N4 transcribing activites with the E. coli inner cell membrane. Fractionation of E. coli cell membranes was as described in Osborn et al. (1972). Marker membranes (A) were from cells labeled with 3H-amino acids (10 $3/ml) for 1 hr before infection by N4. Rifampicin (206 wg/ml) was added 15 min before infection. H, outer membranes; M, unseparated envelope tiagments; L, and L,, inner membranes. Unlabeled, parallel membrane preparations were assayed for streptolydigin-resistant RNA polymerase activity in the presence of 60 pg/ml N4 DNA. (0) Uninfected cell membranes; (0) N4-infected cell membranes. Assay conditions as described under Materials and Methods. (x) Buoyant density.

472

FALCO AND ROTHMAN-DENES

ments and infection by N4 do not interfere with the isolation procedure. Superimposed on this profile, on the basis of the density of the fractions, are the results of the measurements of streptolydigin-resistant RNAsynthesizing activity across parallel gradients of the unlabeled membrane preparations. Little RNA polymerase activity is found in the membranes from uninfected cells. In the membranes of cells infected by N4, however, streptolydigin-resistant RNA-synthesizing activity comigrates with the inner membrane. This inner membrane associated activity is independent of, and not stimulated by the addition of exogenous N4 DNA (not shown). The recovery of RNAsynthesizing activity by this technique is low; less than 10% of the activity per infected cell, obtained by lysis procedures which yield the DNA-membrane complex, is present in the inner cell membrane. However, the membrane isolation and fractionation technique is a much longer and less gentle procedure. Furthermore, very little of the cellular DNA remains associated with the isolated membranes. Since the NCinduced activity found associated with the inner membrane depends on endogenous N4 DNA, the low recovery of activity may be due to the lack of endogenous template (analogous to the result obtained by treating the DNAmembrane complex activity with DNase). An investigation of the NCinduced proteins associated with the isolated membranes has been performed. For this analysis cells were labeled for 7 hr with a 3H-amino acid mixture. Rifampicin was then added, followed 15 min later by addition of 35S to an uninfected sample, 35S and N4 to a second sample, and 35S and an N4 amber mutant in cistron 4(am15) (a pleiotropic mutant which yields only transcribing activity I in viva) to a third sample. Seven minutes later chloramphenicol was added, the cells were collected in the cold, and the membranes were isolated. Figure 6 shows a fluorogram of SDS-polyacrylamide gel on which fractions from these samples were electrophoresed. The fractions shown for each sample are the total lysate (a), the supernatant from a high-speed centrifugation (b), and the total membranes (c). The N4- and amE-induced proteins found associ-

abcabcabc

FIG. 6. N4- and aml5-induced proteins associated with isolated E. coli membranes. At times t = 0 3H-amino acids (10 &i/ml) were added to growing E. coli cells (1.5 x lo* cells/ml); at t = 45 min, rifampitin (200 kg/ml) was added; at t = 60 min, phage and 35S (10 &i/ml) were added. Cells were collected at t = 6’7 min and unfractionated membranes were prepared as described in Osborn et al. (1972). Electrophoresis and fluorography as described under Materials and Methods. The two panels represent different exposures of the same experiment. (a) Total lysate; (b) lysate super&ant; (c) total membranes. A 15% polyaqlamide gel was employed.

ated with the total isolated membranes are the same ones previously shown to be associated with the DNA-membrane complex (Table 2; see also the preceding paper). The isolated membranes from each of these samples were then fractionated on sucrose gradients (Figs. 7A-C). In all these cases the separation of the inner and outer

BACTERIOPHAGE TABLE

N4INDUCED

2

N4-INDUCED PROTEINS ASSOCIATED wrru THEE. Coli CELL MEMBRANE

Protein designation Pl P2 (PC2) P3 p4 (PC41 P5 ~6 P7 P3 P9 P12 (PC3) P13 ~16 PI7 ~13 P20 P21

Proteins associated with the DNAmembrane complex +a +I-+I+ + +/+ + + + +/-

Proteins associated with the cytoplasmic membrane nd +I+I+I+/+ + nd + + nd

a + indicates membrane associated protein, - indicates supernatant protein; nd indicates not determined.

membranes is indicated by the 3H radioactivity profile. As expected, in the uninfected sample little 35S radioactivity is

FAACTION

NUMBER

FRACTION

TRANSCRIBING

473

ACTIVITIES

detectable, since rifampicin was added 15 min before the labeling period (Fig. 7A). In contrast considerable Y3 radioactivity is present in N4- and aml&infected cell inner membranes, due to the incorporation of label into phage-induced proteins (Fig. 7B and C). Aliquots from all fractions of these three gradients were electrophoresed on SDSpolyacrylamide gels (not shown). The fluorograms of the gels show that all of the N4and aml5-induced proteins observed in the total isolated membrane fraction (Fig. 6 and Table 2) are selectively associated with the inner cell membrane, as the sucrose gradients (Fig. ‘7) suggest. While the inner membrane isolated from NCinfected cells contain as many as 10 NCinduced proteins, the inner membrane from aml5-infected cells contains low molecular weight proteins, designated ~17 and ~20, both of which are overproduced. DISCUSSION

The data presented here and in the preceding paper indicate that the bacteriophage NCinduced transcriptional machinery is associated with the cytoplasmic membrane of the host cell. Under conditions which minimize the possibility of nonspecific aggregation of DNA and membrane, the N4 transcribing activities cosediment with the

NUWER

FRACTION

NUYOER

FIG. ‘7. Membrane-associated proteins from uninfected cells (A), N4-infected cells (B), and am15infected cells (C). The cells were treated with rifampicin (2OOpg/ml) 15 min before infection. Preparation and labeling ofE. coli membranes and N4 proteins was as described in the legend to Fig. 6. Fractionation of membranes was as described in Osborn et al. (1972). (O), 3H; (O), [Y?,]; (X), buoyant density.

474

FALCO AND ROTHMAN-DENES

cellular DNA-membrane complex. Sixty percent of the infecting N4 DNA also sediments with this complex and acti as template for N4 RNA synthesis. The remaining forty percent of the infecting N4 DNA sediments slowly. This DNA (which is not due to unadsorbed virions) may represent DNA which has not been bound to the membrane or which has been released from the membrane as a result of the lysis procedure. The second possibility is probably correct since the lysis procedure employs the detergent Triton X-100 which is known to disrupt components of the inner membrane (Schnaitman, 1971). In any case RNA-synthesizing activity is found only in the DNA-membrane complex. The inability to dissociate the N4 transcribing activities from the DNA-membrane complex by treatments at very high ionic strength suggests that hydrophobic rather than hydrophilic interactions are involved in the association. Inhibition and irreversible inactivation of the activity by mild detergent treatments is consistent with this hypothesis. Additional support for such an interaction comes from the observation that treatment of the DNA-membrane complex with phospholipase A, results in inactivation of the RNA-synthesizing activities (unpublished observations). These results suggest an interaction between the N4 transcribing activities and the cell membrane which is essential for function. By isolating the E. coli cell envelope and separating it into outer and inner (cytoplasmic) membrane fractions, it has been possible to show that the N4 transcribing activities axe associated with the cytoplasmic membrane. In addition, the same N4induced proteins which were shown to be associated with the DNA-membrane complex (Falco and Rothman-Denes, 1979) are also found associated with the cytoplasmic membrane (Table 1). However, certain proteins, for example protein p4, which are found almost exclusively associated with the DNA-membrane complex, are not selectively associated with the isolated cytoplasmic membrane. The protein p4 is of particular interest because it is the product of cistron 4, a gene whose function is required for the appearance of transcribing activity II in viva. Thus the low recovery

of RNA-synthesizing activity in the isolated cytoplasmic membrane compared to the DNA-membrane complex may be related to the reduced proportion of p4 associated with the cytoplasmic membrane. It is also known that little endogenous DNA is found with the isolated membranes; this should certainly affect the level of RNA-synthesizing activity since it is required as template. Therefore, further investigation of the interaction between p4, N4 DNA, and the cytoplasmic membrane may lead to a better understanding of N4 transcription in vivo. The experiments presented suggest that the E. coli membrane may play a central role in phage N4 development. The details of the interaction between N4-induced RNA polymerase activities, N4 DNA, and the cytoplasmic membrane and the effect of the interaction on the regulation of N4 transcription remain to be worked out. Whether the cell membrane is also directly involved in late events of N4 development such as DNA replication and maturation of virions has not yet been investigated. In any case, N4 provides a useful system for the study of a number of roles of the host membrane in virus development. ACKNOWLEDGMENTS We gratefully acknowledge K. Vander Laan for technical assistance. This research was supported by Grant AI 12575 and Research Career Development Award 1K 04-A100078 from the National Institute of Allergy and Infectious Diseases to L.B.R.D. S.C.F. was a trainee on USPHS Grant GM 780. REFERENCES BABINET, C. (1967). A new method for the purification of RNA-polymerase. Biochem. Biophys. Res. Commun.

26, 639-644.

BLATPLER, D. P., GARNER, F., VAN SLYKE, K., and BRADLEY, A. (1972). Quantitative electrophoresis in polyacrylamide gels of 2-40%. J. Chromatogr. 64, 147-155. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for trltium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biohem.

46, 83-88.

BOTSTEIN, D., and LEVINE, M. (1968). Intermediates in the synthesis of phage P22 DNA. Cold Spring Harbor Symp. Qua&. Biol. 33, 659-667. EARHART, C. F. (1970). The association of host and

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phage DNA with the membrane of Escherichia coli. Virology 42, 429-436. FALCO, S. C., and ROTHMAN-DENES, L. B. (1979). Bacteriophage N4 induced transcribing activities in E. coli. I. Detection and characterization in cell extracts. Virology 95,454-465. FRANKEL, F. R., MAJUMDAR, C., WEINTRALJB, S., and FRANKEL, D. M. (1968). DNA polymerase and the cell membrane after T4 infection. Cold Sp&g Harbor Symp. Quant. Biol. 33, 495-500. GANEBAN, A. T., and LEDERBERG, J. (1965). A cellmembrane bound &action of bacterial DNA. B&hem. Biophys. Res. Commun. 18, 824-835. HALLICK, L., BOYCE, R. P., and ECHOLS, H. (1969). Membrane association by bacteriophage A DNA: Possible direct role of regulatory gene N. Nature (London) 223, 1239- 1242. JACOB, F., BRENNER, S., and CUZIN, F. (1963). On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28, 329-348. KNIPPERS, R., and SINSHEIMER, R. L. (1968). Process of infection with bacteriophage 4X174. XX. At&hment of the parental DNA of bacteriophage +X174 to a fast-sedimenting cell component. J. Mol. Biol. 34, 17-29. KOLBER, A. R., and SLY, W. S. (1971). Association of lambda bacteriophage DNA with a rapidly sedimenting Escherichia coli component. Virology 46, 638-654. MAOR, G., and SHALITIN, C. (1974). Competence of membrane-bound T4rII: DNA for in vitro “late” mRNA transcription. Viirology 62, 500-511. MOLINEAIJX, I. J., FRIEDMAN, S., and GE~ER, M. L. (1974). Purification and properties ofthe Eschwichia coli deoxyribonucleic acid-unwinding protein. J. Biol. Chem. 249, 6090-6098. OKAZAKI, T., and KORNBERG, A. (1964). Enzymatic synthesis of deoxyribonucleic acid XV. Purification and properties of a polymerase fi-om Bacillus subtilis. J. Biol. Chem. 229, 259-268. OSBORN, M. J., GANDER, J. E., PARISI, E., and CARSON, J. (1972). Mechanism of assembly of the outer membrane ofSalmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J. Biol. Chem. 247, 3962-3972. PE~JOHN, D. E., HECHT, R. M., STONINGTON, 0. G., and STAMATO, T. D. (1973). Factors stabilizing DNA folding in bacterial chromosomes. In “Steenback

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