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Studies on the association of E. coli phage λ DNA and the host chromosome: Lack of a role of membranes
VIROLOGY
119,159-168 (1982)
Studies on the Association of E. co/i Phage X DNA and the Host Chromosome: Lack of a Role of Membranes MARC BETTER AND DAVID FREIFELDER Department of Biochemistry, Brandois University, Waltham, Massachusetts 0225.4 Received November 2, 1981;accepted February 5, 1982 The majority of parentally radiolabeled Escherichia co.3 phage X DNA becomes associated with E. coli DNA after infection of sensitive bacteria; this association is resistant to several detergents, RNase, and proteases. Removal of bacterial cell membranes does not greatly affect the sedimentation properties of the (host DNA)-(X DNA) complex or cause release of X DNA. Our findings suggest that host cell membranes are not involved in the rapid sedimentation of X DNA after infection of E. coli, contrary to a widely held view. INTRODUCTION
is enriched for the molecules undergoing A variety of experiments have suggested DNA replication (Valenzuela, 1975); this that E. coli phage X DNA becomes asso- has been taken as evidence for the view that replicating DNA is membrane-associated with bacterial components several minutes after phage DNA is injected into ciated. Close examination of most sedimentaa sensitive cell (Segal and Schaechter, 1973) or following induction of a supertion experiments taken to support the noinfected lysogenic host cell (Hallick et a/+, tion that h DNA and E. coli membranes 1969; Sakakibara and Tomizawa, 1971). are directly associated indicates that there The usual observation is that most of the is a fallacy in the logic used. The presence of two components on a high-density shelf phage DNA sediments rapidly and is found on a high density shelf along with fragmay mean that the substances are associated or alternately that sedimentation ments of the cell membrane and cell wall and most of the bacterial DNA. The cause has proceeded so long that one component has been deposited on top of another. of the rapid sedimentation of phage DNA Clearly cosedimentation and not “copelis unknown but it is commonly believed leting” is needed to establish that comthat phage X DNA becomes part of a rapidly sedimenting complex (RX) by spe- ponents are associated. Recently Pato and cific association with bacterial cell mem- Waggoner (1981), in a study of Mu phage, branes (Salivar and Gardinier, 1970; Segal reported a few experiments with X which indicate that an appreciable amount of the and Schaechter, 1973, for review). This view of DNA-membrane association has intracellular X DNA (a mixture of an unbeen strengthened by the association of a known ratio of parental and daughter large fraction of the X DNA with Mg sarmolecules) cosediments with the E. coli cosinate crystals in M bands (Firshein and nucleoid even when the latter has been Kourilsky, 1976); these crystals bind cell subjected to treatments that disrupt chromembranes but not free DNA. Also a com- mosome structure. Unfortunately, there plex containing X DNA and E. coli cell was no direct assay for membranous commembranes has been described from CsCl ponents in these experiments. density gradients (Witkiewicz and Taylor, In this paper we have used cosedimen1978). Other experiments indicate that the tation, under a variety of conditions, to fraction of X DNA that is found in a RSC reinvestigate the points just raised. The 159
0042-6822/82/070159-10$02.00/O Copyright 0 1982 by Academic Press, Inc. All right8 of reproduction in any form reserved.
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results of these experiments have led us to question previous conclusions and have provided new information about the fate of phage h DNA following infection. We have found that a significant fraction of parentally radiolabeled X DNA does cosediment with the bacterial DNA, but that h DNA remains associated with bacterial DNA under conditions where host cell membranes have been completely solubilized by detergents. MATERIALS
AND METHODS
Bacteria and phages. The bacterial strains C600 and C6OO(XcIs5’7)were used throughout, except for the experiments described in part E of the Results section. For part E, the strain DF4 was used. DF4 is CGOOthrZeu met thi str thgA. Bacteriophage XcI857 was used in all experiments. Chemicals and radiolabels. Octyl glucoside was purchased from Boehringer Mannheim. Sodium cholate (Sigma Chemical Co.) was recrystallized as described by Tzagoloff and Penefsky (1971). Growth of bacteria and preparation of radiolabeled phage. Bacteria were grown in media containing 50 m2MNazHP04, 20 mM KH2P04, 18 mlM NH&l, 8 mlK NaCl, 5 mlKMgS04, 0.1 mlMCaClz, 0.35% glucose, 0.4% maltose, and 1% casamino acids (Difco). Thymine at 10 pg/ml was added for growth of DF4. For radiolabeling of bacteria, cells were grown for three generations in the above media supplemented with the appropriate radiochemical at the concentrations specified in the figure legends. 32P-labeled phage were prepared by thermoinduction of the temperature-sensitive X lysogen C600@&5’7) in media containing 5-10 &i/ml [32P]phosphate. After bacterial lysis, cell debris was removed from lysates by low-speed centrifugation and then phage were purified by two highspeed centrifugations, 30,000 rpm for 90 min in a Beckman type 40 rotor. Infection of cells with radiolabeled X Bacteria were grown to a final cell density of 2-4 x lO*/ml and collected by centrifugation at 5000 rpm for 5 min. The pellet was washed once with 10 mM Tris, pH 8,
10 mlM MgSO, (TM), and then suspended in TM at 10’ cells/ml. Phage were added at a multiplicity of infection (m.0.i.) of 35 and adsorption was carried out at 34” for 10 min. After adsorption, bacteria were collected by centrifugation and suspended in prewarmed growth media. Preparation of bact&al &sates. Method I: This is a modification of the procedure described by Freifelder (1976) for the preparation of phage X covalent circles. Bacteria were collected by centrifugation at 5000 rpm for 5 min at 0”. The pellet was gently suspended with a glass rod in lysis buffer at 0” containing 50 mM Tris, pH 8.0, 20 mM EDTA, and 1 ti spermidine to produce a final cell density of 5 X log/ ml. Lysozyme was added to make 0.1 mg/ ml and bacteria were incubated for 5 min at 0”. Lysates were then subjected to two rapid freeze-thaw cycles using a liquid Nz or dry ice-acetone bath. These lysates were usually clear and nonviscous. Method II: The procedure used was the same as described by Worcel and Burgi (1974). This method involves the addition of Brij-58 and sodium deoxycholate to lysozyme treated bacteria in the presence of 1 M NaCl. For experiments in which digestion with trypsin was used, 15 mM sodium citrate was substituted for EDTA. For equilibrium centrifugation, lysates were diluted 1:l with 10 mMTris, pH 8.0, after cell lysis. Cent~ugatim in neutral sucrase and CsC1.In most experiments 0.05-0.1 ml of a cell lysate was layered onto a 4.5-ml preformed gradient of 5-20% sucrose in 750 mMNaC1, 10 mMTris, pH 8.0,l mlMEDTA formed on a 0.5 ml shelf of 1.5 g/ml CsCl in 40% sucrose. When lysates were prepared at higher salt concentrations, the NaCl concentration of the sucrose solution was increased to 1 or 2 1M.Centrifugation time and speed are as stated in the figure legends. For equilibrium centrifugation, 1.528 g of saturated CsCl in 10 mM Tris, pH 8.0, 1 mM EDTA was added to 0.229 g of a bacterial lysate yielding a final density of 1.73 g/ml. The solution was centrifuged for 20 hr at 36,000 rpm at 22” in a Beckman SW50.1 rotor.
X DNA ASSOCIATION WITH THE HOST CHROMOSOME 20
I
a.
I
I
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I
Sedimentation
15
E 8 7 5 5
10
E 8 i5 a 5
0 25
Fraction number FIG. 1. Sedimentation profiles of bacterial lysates prepared by method I (a) and method II (b). JZ.coli C606 was labeled with 0.5 &i/ml [‘HImethyl thymidine and infected with =P-labeled Xcl357 at a m.o.i. = 3. After adsorption for 10 min at 34’ and subsequent growth at 3’7” for 10 min, cells were collected and lysed by method I or II. Samples (0.1 ml) were layered on top of a 4.5 ml 520% sucrose gradient containing a 0.5 ml shelf of 1.5 g/ml CsCl in 40% sucrose. Centrifugation was for 20 min at 15,006 rpm in an SW 50.1 rotor at 22”. Closed circles, ‘H-labeled bacterial DNA; open circles, q-labeled X DNA. The horizontal arrow shows the direction of sedimentation. The vertical arrow shows the top of the CsCl shelf.
Fractions were collected by puncturing the bottom of the tube and slowly (0.1 ml/ min) collecting five-drop fractions. Very slow collection was necessary to eliminate turbulence while the fractions containing bacterial DNA were collected. For counting radioactivity, fractions were acid-precipitated.
In this experiment, 50-60s of the parentally 32P-labeled phage DNA cosediments with the bulk of the chromosomal DNA while the remainder sediments freely (Fig. la). A second aliquot of the cells was lysed by method II under conditions where the “membrane-dissociated” form of the folded bacterial chromosome predominates (Worcel and Burgi, 1974); the RESULTS sedimentation profile is shown in Fig. lb. The sedimentation profiles of the bacterial A. Sedimentation Behavior of X DNA from DNA prepared by either method I or Phage-Iqfected Cells method II are very similar. However, by Sensitive bacteria, E. coli C600, labeled method I we reproducibly found about with [3H]thymidine, were collected and re- 60% of the X DNA in association with host suspended at 0” in nonradioactive phage- DNA, while a larger fraction (30%) of the adsorption buffer and infected with 32P- parental X DNA remained associated after labeled phage. After growth at 37” for 10 lysis by method II. These experiments inmin, one aliquot of the cells was lysed as dicate that a significant fraction of the described in method I and sedimented phage DNA is associated in some way with through a 520% linear sucrose gradient. host DNA. Attempts to release the phage
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a.
I
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Sedimentation
I
I
I
b.
Fraction number FIG. 2. Sedimentation profiles of bacterial lysates prepared by method I after treatment with the detergents sodium cholate (a) and octyl glucoside (b). Sample preparation and lysis is as described in Fig. 1. (a) Sodium cholate was added to a final concentration of 1.5% and NaCl was added to a final concentration of 300 mM; centrifugation was for 20 min at 15,000 rpm. (b) Octyl glucoside was added to a final concentration of 100 mM; centrifugation was for 30 min at 15,000 rpm. Closed circles, 3H-labeled bacterial DNA, open circles, “P-labeled X DNA. The horizontal arrow shows the direction of sedimentation. The vertical arrow shows the top of the CsCl shelf.
DNA are described in the following sections. B. Effect of Mild Detergents on the Association of X DNA and E. coli DNA Since detergents often break down the association of macromolecules, several mild detergents were tested for their ability to release h DNA from host DNA. Figures 2a and 2b show the sucrose gradient profiles of lysates (prepared by method I) of rH]thymidine-labeled bacteria after infection with =P-labeled X and following addition of either the nonionic detergent, octyl glucoside, or the bile salt, sodium cholate. These detergents do not greatly affect the sedimentation of the chromosomal DNA (compare Fig. la with Figs. 2a and b), indicating that chromosome structure probably remains unaltered and only at most 20% of the associated X DNA is released even when the detergent concentration is increased (data not shown). The nonionic detergent Triton X-100 and the bile salt sodium deoxycholate had sim-
ilar effects. Table 1 summarizes these data. There are small quantitative differences in the effect of different detergents, but these are probably not significant. The effect of some of these detergents on the cell membrane will be described shortly. C. Effect of Other Agents and Conditions on (X DNA)-(Host DNA) Association Other agents that are known to disrupt chromosomal structure or that have been reported to reduce the amount of rapidly sedimenting parentally radiolabeled X DNA (Hallick and Echols, 1973) were tested to see if additional X DNA could be released from the complex with host DNA in lysates prepared by method I. These included treatment with proteases, pancreatic RNase, incubation with ionic detergents, changes in ionic strength and pH, and incubation at elevated temperatures. Table 2 summarizes the results of these experiments. We found that treatment with proteases, pancreatic RNase, or ionic detergents cause unfolding of the
163
h DNA ASSOCIATION WITH THE HOST CHROMOSOME
host chromosome to various degrees but release X DNA from host components only marginally, if at all. Pronase or trypsin (which had been shown to lack nuclease activity) disrupted chromosome structure more than did treatment with nonionic detergents and bile salts, as judged by the reduced sedimentation of the bacterial DNA, but released approximately the same amount of associated X DNA. Other workers (Stonington and Pettijohn, 1971; Worccl and Burgi, 1974) have found that Pronase does not disrupt the structure of the membrane-dissociated nucleoid prepared in high salt (method II), indicating that the bacterial DNA isolated in lysates prepared by method I at lower salt concentrations differs from the membrane-dissociated nucleoid in that it is more sensitive to proteases. After incubation with the ionic detergent SDS, the bacterial DNA likewise sediments much more slowly than in untreated samples, consistent with the observations (Stonington and Pettijohn, 1971) that SDS disrupts nucleoid structure, but SDS was not sufficient to release associated X DNA. In lysates prepared at salt concentrations up to 2 M NaCl and varying from pH TABLE 1 PERCENTOF PARENTALLY LABELED X DNA AssoCIATED WITH BACTERIAL DNA AFTER TREATMENT WITH MILD DETERGENTS”
method
Detergent
Percentage associated
I I I I I II
100 mM octyl glucoside 1.5% sodium choiate 1% Triton X-100 0.5% sodium deoxycholate Untreated Untreated
45 40 36 41 55 82
Lysis
a E. coli C600 labeled with [‘Hlthymidine were infected with 32P-labeied phage and grown as outlined in the legend of Fig. 1. Bacterial lysates were prepared by method I or method II. Detergents were then added to iysates prepared by method I. Centrifugation of bacterial lysates was for 20 min at 15,000 rpm in a Beckman SW 50.1 rotor at 22”. Each “percentage associated” represents the average of at least two sucrose gradients.
TABLE 2 EFFECTS OF PROTEASES, IONIC DETERGENTS, CHAOTROPIC SALTS, AND OTHER DISRUPTANTS ON THE AsSOCIATION OF X DNA WITH HOST DNA”
Disruptant RNase Trypsin Pronase 1% SDS 1 M NaC104 0.5 M Na trichloroacetate Incubation at 50”, 10 min pH 6.0 pH 9.0 20,000 rads (y-rays)
Percentage associated 60 49 47
100 36
61 35
69 58 42
a E. wli C600 labeled with rH]thymidine were infected with 3sP-labeled phage and grown as outlined in the legend to Fig. 1. Bacterial lysates were prepared by method I. Aliquots of bacterial lysates were subjected to digestion with pancreatic RNase, trypsin, or Pronase at 100 pg/ml at 37” for 10 min. SDS, NaCIO,, or Na trichloroacetate was added to lysates to the concentrations listed. The dose of y-irradiation was measured by FeSO* dosimetry. Each “percentage associated” represents the average of at least two sucrose gradients.
6 to pH 9, the amount of X DNA found to associate with bacterial DNA was constant. Chaotropic salts such as sodium perchlorate or sodium trichloroacetate, which denature and often precipitate proteins and which disrupt phages and viruses, also did not release additional X DNA from its chromosomal constraints even though these agents greatly reduced the sedimentation of E. coli DNA and presumably disrupt chromosome structure greatly. The lack of an effect of RNase, chaotropic salts, or SDS indicates that unfolding of the nucleoid is insufficient to release X. Since lysates were prepared at a high cell density (5 X 10’ cells/ml), it was thought possible that the observed association is an artifact due to nonspecific trapping. If so, the fraction of X associated ought to vary with DNA concentration. However in lysates prepared by method I at cell densities from 5 X lO’/ml-5 X log/
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ml, X DNA remained associated with host DNA. Furthermore, lysates from cultures at various cell densities were incubated for up to 6 hr to allow time for the X DNA to diffuse out of the bacterial chromosomes. This also did not affect the association of X DNA with the host chromosome. We examined the possibility that breakage of the chromosome could release h DNA. Sensitive cells were y-irradiated prior to infection with 32P-phage with a dose sufficient to produce up to 15 doublestrand DNA breaks per cell, 20,000 rads (Freifelder, 1966a; Sinden and Pettijohn, 1981). As summarized in Table 2, preirradiation of the cells did not significantly improve the separation of bacterial and phage DNA. D. Localization Fraction
of the E. coli Membrane
The experiments described so far indicate that X DNA cosediments with host DNA after treatment with agents that alter the degree of condensation of the host chromosome, confirming earlier observations many of which have been interpreted in terms of DNA-membrane binding. If in fact membranes are responsible for the association, it would be expected that solubilization of membranes would release the X DNA. Experiments to investigate this are described in this section. We have examined the association of phage X DNA with the membrane fraction of E. co2i in lysates of cells labeled with [3Hp-glycerol, a specific marker for bacterial cell membranes (Miller, 1972). E. coli C600, grown in media supplemented with [3H]2-glycerol and resuspended in nonradioactive medium, were infected with ?Plabeled phage. At 12 min after infection, cells were lysed by method I and sedimented. Labeled bacterial membranes separated into three fractions after sedimentation (Fig. 3a). Approximately 35% of the 3H-labeled material sediments onto a shelf underlying the gradient, 60% sediments to a position where the majority of the bacterial DNA is known to sediment (see Fig. la), and 5% did not sediment
appreciably. Figures 3b and c show the sedimentation profiles of lysates treated with the detergents sodium cholate or octyl glucoside, respectively. Sodium cholate removes at least 99% of the chromosomeassociated [3Hlglycerol-labeled material while after incubation with octyl glucoside, no mlglycerol-labeled material could be detected eosedimenting with the bacterial DNA. However, phage X DNA still sediments with the bacterial DNA after treatment with these detergents (compare Figs. 2a and b with Figs. 3b and c). To be sure that the material labeled with [3Hlglycerol is indeed a membrane component, its lipid-like character was tested by extraction with chloroform-methanol (Ames, 1963); more than 95% of the material found to be 3H-labeled in the lysates was soluble in chloroform-methanol. These experiments indicate that most of the parental X DNA cosediments with host DNA even when host cell membranes are absent. E. Separation of Bacterial DNA and Phage X DNA 2ndEquilibrium CentrZfugation in a Density Gradient In this section we investigate the possibility that phage and host DNA are associated through covalent interactions, by subjecting lysates of infected cells to isopycnic centrifugation in CsCl using phage and bacterial DNA that differ in buoyant density. The density label bromouracil deoxyriboside (BUDR) was used to label bacterial DNA by growing the thyminerequiring strain DF4 for three generations in media supplemented with BUDR (10 pg/ml), thymine (3 pg/ml), and rH]BUDR. With the thymine supplement, cells do not show the many deleterious effects of labeling with BUDR, as has been reported earlier (Freifelder, 1966b), and growth of X is normal (Better and Freifelder, submitted for publication). Cells were infected with q-labeled X and grown in media supplemented with 50 &ml thymine for 12 min at 37”, after which the cells were harvested and lysed by method I. Complete separation of host and phage DNA does not occur when either untreated
165
X DNA ASSOCIATION WITH THE HOST CHROMOSOME
1
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Fraction number FIG. 3. Sedimentation profile of bacterial lysates prepared by method I from [BHj2-glycerol-labeled bacteria. E. coZiC600 was grown in the presence of 10 &i/ml [8Hj2-glycerol and infected with qlabeled Xc1857at a m.o.i. = 3. After adsorption for 10 min and subsequent growth at 37” for 10 min, cells were collected and lysed by method I. The lysate (0.1 ml) was layered onto a 5-2096 sucrose gradient. (a) Untreated lysate, centrifugation for 20 min at 15,000rpm. (b) Sodium cholatetreated lysate. Sodium cholate was added to make 1.5% and NaCl was added to make 300 mM; centrifugation was for 30 minutes at 15,000 rpm. (c) Octyl glucoside-treated lysate. Octyl glucoside was added to make 100 mM; centrifugation was at 15,000rpm for 30 min. Closed circles, 3H-glycerollabeled membranes; open circles, mP-labeled X DNA. The horizontal arrow shows the direction of sedimentation. The vertical arrow shows the top of the CsCl shelf.
lysates (data not shown) or lysates treated with either a detergent or a proteolytic enzyme were centrifuged to equilibrium in CsCl. In these cases, although some of the host and phage DNA separated in the gradient, a substantial fraction of the total (host + X) DNA floated on top of the density gradient (Figs. 4b and c), the position expected for a complex containing an excess of protein. Proteolytic digestion of the lysate with trypsin or Pronase was not sufficient to allow the release of the DNA from the floating fraction (Fig. 4b), probably because all of the bound protein is not accessible to the enzyme. However host and phage X DNA are both released by treatment with a protease and an ionic
detergent such as SDS (Fig. 4a), and E. coli and X DNA become separable by this treatment. This indicates that the association between host and X DNA is not a covalent one. DISCUSSION
The experiments just described show that parentally radiolabeled X DNA is associated with the bacterial chromosome in lysates of infected cells. The nature of the association is completely unclear but the association is independent of the presence of bacterial cell membranes. This conclusion is based on our experiment (section D) with radiolabeled bacterial membranes
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a. 25 -
FIG. 4. CsCl equilibrium gradient profiles of bacterial lysates prepared by method I. E. wZi DF4 was grown for three generations in media supplemented with BUDR (10 ag/ml), thymine (3 a/ ml), and [SI]BUDR (0.5 &i/ml) to a final cell density of 3 X 108/ml. Bacteria were collected by centrifugation, washed, and infected with =P-labeled Xc1857at a m.o.i. = 3. After adsorption for 10 min and subsequent growth at 37’ for 10 min, cells were collected and lysed by method I. CsCl gradients were prepared, centrifuged, and collected as described under Materials and Methods. (a) Treatment was with trypsin (100 rg/ml for 30 min at 37”) followed by SDS to make 0.1%. (b) Incubation of the bacterial lysate with trypsin alone (100 pg/ml for 30 min at 3’7”). (c) Incubation of the bacterial lysate at 37” for 30 min followed by addition of SDS to make 0.1%. Closed circles, 8H-labeled bacterial DNA, open circles, =P-labeled X DNA. The heavily and lightly hatched vertical bars represent the 3H and BP radioactivity, respectively, that floated on the gradient. This material was recovered by adding 1 ml of 1 M NaOH to the tube after the contents had been recovered by drop collecting, mixing, neutralizing with HCl, and precipitating.
in which the total fraction of chromosomeassociated membranes was removed with detergents yet only a fraction of the chromosome-associated X DNA was released by this treatment. This conclusion contrasts with the commonly held view that the rapid sedimentation of X DNA results from membrane attachment. It is important to realize that our data have no bearing in the question of whether X DNA is membrane-associated at some time in its life cycle. We have only shown that the association between X DNA and E. coli DNA does not require membrane binding. The association between X DNA and the bacterial DNA is not easily broken down
by agents which are known to disrupt chromosome structure greatly and could only be disrupted by a combined treatment with detergents and proteases followed by equilibrium centrifugation in CsCl. Furthermore, there are either three kinds of association-(i) a protein-dependent one, (ii) a detergent-sensitive one, and possibly (iii) a CsCl-sensitive one-or a single type of association that can be broken only by the combined action of protease digestion, detergent treatment, and the high salt environment of a CsCl step. Our experiments do not distinguish these possibilities. Incubation of lysates prepared by method I with mild detergents such as so-
X DNA ASSOCIATION
WITH
dium cholate or octyl glucoside releases 20% of the chromosome-associated X DNA. We cannot directly rule out the possibility that this represents a fraction of the X DNA that is bound to cell membranes, although this seems unlikely to us for the following reasons. First, we have examined by electron microscopy the population of molecules freed from the bacterial chromosome after incubation with these detergents and we find that only nonreplicating circular molecules are present (Better and Freifelder, to be published). Second, in lysates prepared by method II, which yields the membrane-dissociated, folded, bacterial chromosome, 80% of the parentally radiolabeled X DNA is associated with the host DNA. We feel that the release of this small fraction of bound X DNA in treated samples prepared by method I represents the fraction of associated molecules which are not tightly bound to bacterial DNA and are freed as the chromosome structure is disrupted. The finding described here that phage X DNA is associated with bacterial DNA following infection of sensitive bacteria is reminiscent of previously described observations concerning autonomously replicating bacterial plasmids. The sex plasmid F is found to associate with the E. coli chromosome in bacterial lysates prepared essentially as described here for method II (Kline and Miller, 1975; Miller and Kline, 1979). Many other E. coli plasmids have been found to cosediment with folded bacterial DNA as well (Kline et al., 1976; Archibald et al., 1978). Kline et al. (1976) suggest that complexing of a plasmid to folded chromosomes is a universal condition of plasmids in E. coli strains. Furthermore, association of plasmids with the folded chromosome of E. coli is independent of the presence of bacterial cell membranes, just as we have shown here to be the case for phage X DNA. We have no data to indicate the factors that are involved in maintaining the association between X DNA and host DNA. Two points which may be important are the limited association known to occur between X DNA and host components when X is repressed (Sakakibara and Tom-
THE HOST CHROMOSOME
167
izawa, 1971; Hallick and Echols, 1973), for example, when a lysogen is superinfected, and the role of both the X N gene product and transcription in RSC formation (Hallick et al., 1969; Hallick and Echols, 1973). One possibility is that RNA polymerase, which is the major protein found to remain with isolated bacterial chromosomes (Pettijohn et al., 1973; Worcel and Burgi, 1974) by itself, or in conjunction with X-specified proteins, is reponsible for maintaining the (X DNA)-(host DNA) association. Alternatively, X DNA may be associated with host DNA through interaction with both proteins and nascent RNA chains. This would be consistent with observations that the association is RNaseor protease-insensitive and with the idea that RNA-DNA interactions stabilized by proteins maintain bacterial DNA in a condensed form in vitro (Hecht et al., 1977). Possibly there would be no association of X DNA with host components in a repressed superinfected lysogen if the few promoters that are actively being transcribed in repressed phage were lacking. ACKNOWLEDGMENTS This work was supported by Grant GM-14358 from the National Institutes of Health. We acknowledge the many suggestions given to us by Chris Miller and Mike White. This is publication No. 1398 of the Department of Biochemistry, Brandeis University. REFERENCES AMES, G. F. (1968). Lipids of Salmonella typhimurium and Escherichia coli: Structure and metabolism. J. BacterioL 95,8X3-843. ARCHIBALD, E. R., CLARK, C. W., and SHEEHY, R. J. (19’78). Relationship of R6K replicating forms to the folded chromosome of Escherichia coli. J. Bacteriol 135,476-482. FIRSHEIN, W., and KOURILSKY, P. (1976). Attempts to purify a membrane attached cromoid of bacteriophage lambda. Biochimie 58,417-425. FREIFELDER, D. (1966). Lethal changes in bacteriophage DNA produced by X-rays. Radiat. Res. Suppl 6, 80-96. FREIFELDER, D. (1966). Replication of DNA during F’Lac transfer. B&hem. Biophys. Res. Cammun 23,576-582. FREIFELDER, D. (1976). Preparation of covalently closed and open circular DNA molecules of. phage X. Biochint. Biophys. Acta 432,113-117.
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HALLICK, L., BOYCE, R. P., and ECHOLS, H. (1969). Membrane association of bacteriophage X DNA: Possible direct role of regulator gene N. Nature (London) 223, 1239-1242. HALLICK, L. M., and ECHOLS,H. (1973). Genetic and biochemical properties of an association complex between host components and lambda DNA. Virdogy 52, 105-119. HECHT, R. M., STIMPSON,D., and PETTIJOHN, D. (197’7). Sedimentation properties of the bacterial chromosome as an isolated nucleoid and as an unfolded DNA fiber: Chromosomal DNA folding measured by rotor speed effects. J. Mol. Biol 111,257277.
KLINE, B. C., and MILLER, J. R. (1975). Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosomes of Escherichiu col? Physicochemical approach to studying the unit of segregation. J. Bacterid 121.165-172. KLINE, B. C., MILLER, J. R., CRESS,D. E., WLODARCZYK, M., MANIS, J. J., and OTTEN, M. R. (1976). Nonintegrated plasmid-chromosome complexes in Escherichia co& J. Bacterial 127.881439. MILLER, J. R., and KLINE, B. C. (1979). Biochemical characterization of nonintegrated plasmid-folded chromosome complexes: Sex factor F and the Escherichia cdi nucleoid. J. Bacterid 137, 885-890. MIUER, R. C. (1972). Association of replicative T4 deoxyribonucleic acid and bacterial membranes. J. Viral
10, 920-924.
PATO, M. L., and WAGGONER,B. J. (1981). Cellular location of Mu DNA replicas. J. Viral 38,249-255. PE’ITIJOHN,D. E., HECHT, R. M., STONINGTON,0. G., and STAMATO, T. D. (1973). Factors stabilizing DNA folding in bacterial chromosomes. In “DNA Synthesis in Vitro” (R. D. Wells and R. B. Inman,
eds.), pp. 145-162. University Park Press, Baltimore. SAKAKIBARA, Y., and TOMIZAWA,J. (1971). Gene N and membrane association of lambda DNA. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 691-760. Cold Spring Harbor Laboratory, New York. SALIVAR,W. O., and GARDINIER,J. (1970). Replication of bacteriophage lambda DNA associated with the host cell membrane. Virology 41, 38-51. SEGAL, P. J., and SCHAECHTER,M. (1973). The role of the host cell membrane in the replication and morphogenesis of bacteriophage. Annu. Rev. Microbid 27.261-282. SINDEN, R. R., and PETTLJOHN,D. E. (1981). Chro-
mosomes in living Escherichia c&i cells are segregated into domains of supercoiling. Proc Nat. Ad Sci USA 78, 224-228. STONINGTON,0. G., and PETTLJOHN,D. E. (1971). The folded genome of Escherichia coli isolated in a protein-DNA-RNA complex. Proc. Nat. Ad Sci USA 68, 6-9.
TZAGOLOFF,A., and PENEFSKY,H. S. (1971). Extraction and purification of lipoprotein complexes from membranes. In “Methods in Enzymology” Vol. XXII, (W. B. Jakohy, ed.), Vol. XXII, pp. 219-230. Academic Press, New York. VALENZUELA, M. (1975). Intermediates of the first round of X DNA replication are preferentially found in a rapidly sedimenting complex. Biochwn. Biophys. Res. Commun 65,1221-1228. WITKIEWICZ,H., and TAYLOR,K. (1978). X DNA-membrane complex isolated in the CsCl density gradient. FEBS L&t. SO,313-317. WORCEL,A., and BURGI, E. (1974). Properties of a membrane-attached form of the folded chromosome of Escherichia coli J. Mol. Biol 82,91-105.
119,159-168 (1982)
Studies on the Association of E. co/i Phage X DNA and the Host Chromosome: Lack of a Role of Membranes MARC BETTER AND DAVID FREIFELDER Department of Biochemistry, Brandois University, Waltham, Massachusetts 0225.4 Received November 2, 1981;accepted February 5, 1982 The majority of parentally radiolabeled Escherichia co.3 phage X DNA becomes associated with E. coli DNA after infection of sensitive bacteria; this association is resistant to several detergents, RNase, and proteases. Removal of bacterial cell membranes does not greatly affect the sedimentation properties of the (host DNA)-(X DNA) complex or cause release of X DNA. Our findings suggest that host cell membranes are not involved in the rapid sedimentation of X DNA after infection of E. coli, contrary to a widely held view. INTRODUCTION
is enriched for the molecules undergoing A variety of experiments have suggested DNA replication (Valenzuela, 1975); this that E. coli phage X DNA becomes asso- has been taken as evidence for the view that replicating DNA is membrane-associated with bacterial components several minutes after phage DNA is injected into ciated. Close examination of most sedimentaa sensitive cell (Segal and Schaechter, 1973) or following induction of a supertion experiments taken to support the noinfected lysogenic host cell (Hallick et a/+, tion that h DNA and E. coli membranes 1969; Sakakibara and Tomizawa, 1971). are directly associated indicates that there The usual observation is that most of the is a fallacy in the logic used. The presence of two components on a high-density shelf phage DNA sediments rapidly and is found on a high density shelf along with fragmay mean that the substances are associated or alternately that sedimentation ments of the cell membrane and cell wall and most of the bacterial DNA. The cause has proceeded so long that one component has been deposited on top of another. of the rapid sedimentation of phage DNA Clearly cosedimentation and not “copelis unknown but it is commonly believed leting” is needed to establish that comthat phage X DNA becomes part of a rapidly sedimenting complex (RX) by spe- ponents are associated. Recently Pato and cific association with bacterial cell mem- Waggoner (1981), in a study of Mu phage, branes (Salivar and Gardinier, 1970; Segal reported a few experiments with X which indicate that an appreciable amount of the and Schaechter, 1973, for review). This view of DNA-membrane association has intracellular X DNA (a mixture of an unbeen strengthened by the association of a known ratio of parental and daughter large fraction of the X DNA with Mg sarmolecules) cosediments with the E. coli cosinate crystals in M bands (Firshein and nucleoid even when the latter has been Kourilsky, 1976); these crystals bind cell subjected to treatments that disrupt chromembranes but not free DNA. Also a com- mosome structure. Unfortunately, there plex containing X DNA and E. coli cell was no direct assay for membranous commembranes has been described from CsCl ponents in these experiments. density gradients (Witkiewicz and Taylor, In this paper we have used cosedimen1978). Other experiments indicate that the tation, under a variety of conditions, to fraction of X DNA that is found in a RSC reinvestigate the points just raised. The 159
0042-6822/82/070159-10$02.00/O Copyright 0 1982 by Academic Press, Inc. All right8 of reproduction in any form reserved.
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results of these experiments have led us to question previous conclusions and have provided new information about the fate of phage h DNA following infection. We have found that a significant fraction of parentally radiolabeled X DNA does cosediment with the bacterial DNA, but that h DNA remains associated with bacterial DNA under conditions where host cell membranes have been completely solubilized by detergents. MATERIALS
AND METHODS
Bacteria and phages. The bacterial strains C600 and C6OO(XcIs5’7)were used throughout, except for the experiments described in part E of the Results section. For part E, the strain DF4 was used. DF4 is CGOOthrZeu met thi str thgA. Bacteriophage XcI857 was used in all experiments. Chemicals and radiolabels. Octyl glucoside was purchased from Boehringer Mannheim. Sodium cholate (Sigma Chemical Co.) was recrystallized as described by Tzagoloff and Penefsky (1971). Growth of bacteria and preparation of radiolabeled phage. Bacteria were grown in media containing 50 m2MNazHP04, 20 mM KH2P04, 18 mlM NH&l, 8 mlK NaCl, 5 mlKMgS04, 0.1 mlMCaClz, 0.35% glucose, 0.4% maltose, and 1% casamino acids (Difco). Thymine at 10 pg/ml was added for growth of DF4. For radiolabeling of bacteria, cells were grown for three generations in the above media supplemented with the appropriate radiochemical at the concentrations specified in the figure legends. 32P-labeled phage were prepared by thermoinduction of the temperature-sensitive X lysogen C600@&5’7) in media containing 5-10 &i/ml [32P]phosphate. After bacterial lysis, cell debris was removed from lysates by low-speed centrifugation and then phage were purified by two highspeed centrifugations, 30,000 rpm for 90 min in a Beckman type 40 rotor. Infection of cells with radiolabeled X Bacteria were grown to a final cell density of 2-4 x lO*/ml and collected by centrifugation at 5000 rpm for 5 min. The pellet was washed once with 10 mM Tris, pH 8,
10 mlM MgSO, (TM), and then suspended in TM at 10’ cells/ml. Phage were added at a multiplicity of infection (m.0.i.) of 35 and adsorption was carried out at 34” for 10 min. After adsorption, bacteria were collected by centrifugation and suspended in prewarmed growth media. Preparation of bact&al &sates. Method I: This is a modification of the procedure described by Freifelder (1976) for the preparation of phage X covalent circles. Bacteria were collected by centrifugation at 5000 rpm for 5 min at 0”. The pellet was gently suspended with a glass rod in lysis buffer at 0” containing 50 mM Tris, pH 8.0, 20 mM EDTA, and 1 ti spermidine to produce a final cell density of 5 X log/ ml. Lysozyme was added to make 0.1 mg/ ml and bacteria were incubated for 5 min at 0”. Lysates were then subjected to two rapid freeze-thaw cycles using a liquid Nz or dry ice-acetone bath. These lysates were usually clear and nonviscous. Method II: The procedure used was the same as described by Worcel and Burgi (1974). This method involves the addition of Brij-58 and sodium deoxycholate to lysozyme treated bacteria in the presence of 1 M NaCl. For experiments in which digestion with trypsin was used, 15 mM sodium citrate was substituted for EDTA. For equilibrium centrifugation, lysates were diluted 1:l with 10 mMTris, pH 8.0, after cell lysis. Cent~ugatim in neutral sucrase and CsC1.In most experiments 0.05-0.1 ml of a cell lysate was layered onto a 4.5-ml preformed gradient of 5-20% sucrose in 750 mMNaC1, 10 mMTris, pH 8.0,l mlMEDTA formed on a 0.5 ml shelf of 1.5 g/ml CsCl in 40% sucrose. When lysates were prepared at higher salt concentrations, the NaCl concentration of the sucrose solution was increased to 1 or 2 1M.Centrifugation time and speed are as stated in the figure legends. For equilibrium centrifugation, 1.528 g of saturated CsCl in 10 mM Tris, pH 8.0, 1 mM EDTA was added to 0.229 g of a bacterial lysate yielding a final density of 1.73 g/ml. The solution was centrifuged for 20 hr at 36,000 rpm at 22” in a Beckman SW50.1 rotor.
X DNA ASSOCIATION WITH THE HOST CHROMOSOME 20
I
a.
I
I
161
I
Sedimentation
15
E 8 7 5 5
10
E 8 i5 a 5
0 25
Fraction number FIG. 1. Sedimentation profiles of bacterial lysates prepared by method I (a) and method II (b). JZ.coli C606 was labeled with 0.5 &i/ml [‘HImethyl thymidine and infected with =P-labeled Xcl357 at a m.o.i. = 3. After adsorption for 10 min at 34’ and subsequent growth at 3’7” for 10 min, cells were collected and lysed by method I or II. Samples (0.1 ml) were layered on top of a 4.5 ml 520% sucrose gradient containing a 0.5 ml shelf of 1.5 g/ml CsCl in 40% sucrose. Centrifugation was for 20 min at 15,006 rpm in an SW 50.1 rotor at 22”. Closed circles, ‘H-labeled bacterial DNA; open circles, q-labeled X DNA. The horizontal arrow shows the direction of sedimentation. The vertical arrow shows the top of the CsCl shelf.
Fractions were collected by puncturing the bottom of the tube and slowly (0.1 ml/ min) collecting five-drop fractions. Very slow collection was necessary to eliminate turbulence while the fractions containing bacterial DNA were collected. For counting radioactivity, fractions were acid-precipitated.
In this experiment, 50-60s of the parentally 32P-labeled phage DNA cosediments with the bulk of the chromosomal DNA while the remainder sediments freely (Fig. la). A second aliquot of the cells was lysed by method II under conditions where the “membrane-dissociated” form of the folded bacterial chromosome predominates (Worcel and Burgi, 1974); the RESULTS sedimentation profile is shown in Fig. lb. The sedimentation profiles of the bacterial A. Sedimentation Behavior of X DNA from DNA prepared by either method I or Phage-Iqfected Cells method II are very similar. However, by Sensitive bacteria, E. coli C600, labeled method I we reproducibly found about with [3H]thymidine, were collected and re- 60% of the X DNA in association with host suspended at 0” in nonradioactive phage- DNA, while a larger fraction (30%) of the adsorption buffer and infected with 32P- parental X DNA remained associated after labeled phage. After growth at 37” for 10 lysis by method II. These experiments inmin, one aliquot of the cells was lysed as dicate that a significant fraction of the described in method I and sedimented phage DNA is associated in some way with through a 520% linear sucrose gradient. host DNA. Attempts to release the phage
162
BETTER AND FREIFELDER I
a.
I
I
Sedimentation
I
I
I
b.
Fraction number FIG. 2. Sedimentation profiles of bacterial lysates prepared by method I after treatment with the detergents sodium cholate (a) and octyl glucoside (b). Sample preparation and lysis is as described in Fig. 1. (a) Sodium cholate was added to a final concentration of 1.5% and NaCl was added to a final concentration of 300 mM; centrifugation was for 20 min at 15,000 rpm. (b) Octyl glucoside was added to a final concentration of 100 mM; centrifugation was for 30 min at 15,000 rpm. Closed circles, 3H-labeled bacterial DNA, open circles, “P-labeled X DNA. The horizontal arrow shows the direction of sedimentation. The vertical arrow shows the top of the CsCl shelf.
DNA are described in the following sections. B. Effect of Mild Detergents on the Association of X DNA and E. coli DNA Since detergents often break down the association of macromolecules, several mild detergents were tested for their ability to release h DNA from host DNA. Figures 2a and 2b show the sucrose gradient profiles of lysates (prepared by method I) of rH]thymidine-labeled bacteria after infection with =P-labeled X and following addition of either the nonionic detergent, octyl glucoside, or the bile salt, sodium cholate. These detergents do not greatly affect the sedimentation of the chromosomal DNA (compare Fig. la with Figs. 2a and b), indicating that chromosome structure probably remains unaltered and only at most 20% of the associated X DNA is released even when the detergent concentration is increased (data not shown). The nonionic detergent Triton X-100 and the bile salt sodium deoxycholate had sim-
ilar effects. Table 1 summarizes these data. There are small quantitative differences in the effect of different detergents, but these are probably not significant. The effect of some of these detergents on the cell membrane will be described shortly. C. Effect of Other Agents and Conditions on (X DNA)-(Host DNA) Association Other agents that are known to disrupt chromosomal structure or that have been reported to reduce the amount of rapidly sedimenting parentally radiolabeled X DNA (Hallick and Echols, 1973) were tested to see if additional X DNA could be released from the complex with host DNA in lysates prepared by method I. These included treatment with proteases, pancreatic RNase, incubation with ionic detergents, changes in ionic strength and pH, and incubation at elevated temperatures. Table 2 summarizes the results of these experiments. We found that treatment with proteases, pancreatic RNase, or ionic detergents cause unfolding of the
163
h DNA ASSOCIATION WITH THE HOST CHROMOSOME
host chromosome to various degrees but release X DNA from host components only marginally, if at all. Pronase or trypsin (which had been shown to lack nuclease activity) disrupted chromosome structure more than did treatment with nonionic detergents and bile salts, as judged by the reduced sedimentation of the bacterial DNA, but released approximately the same amount of associated X DNA. Other workers (Stonington and Pettijohn, 1971; Worccl and Burgi, 1974) have found that Pronase does not disrupt the structure of the membrane-dissociated nucleoid prepared in high salt (method II), indicating that the bacterial DNA isolated in lysates prepared by method I at lower salt concentrations differs from the membrane-dissociated nucleoid in that it is more sensitive to proteases. After incubation with the ionic detergent SDS, the bacterial DNA likewise sediments much more slowly than in untreated samples, consistent with the observations (Stonington and Pettijohn, 1971) that SDS disrupts nucleoid structure, but SDS was not sufficient to release associated X DNA. In lysates prepared at salt concentrations up to 2 M NaCl and varying from pH TABLE 1 PERCENTOF PARENTALLY LABELED X DNA AssoCIATED WITH BACTERIAL DNA AFTER TREATMENT WITH MILD DETERGENTS”
method
Detergent
Percentage associated
I I I I I II
100 mM octyl glucoside 1.5% sodium choiate 1% Triton X-100 0.5% sodium deoxycholate Untreated Untreated
45 40 36 41 55 82
Lysis
a E. coli C600 labeled with [‘Hlthymidine were infected with 32P-labeied phage and grown as outlined in the legend of Fig. 1. Bacterial lysates were prepared by method I or method II. Detergents were then added to iysates prepared by method I. Centrifugation of bacterial lysates was for 20 min at 15,000 rpm in a Beckman SW 50.1 rotor at 22”. Each “percentage associated” represents the average of at least two sucrose gradients.
TABLE 2 EFFECTS OF PROTEASES, IONIC DETERGENTS, CHAOTROPIC SALTS, AND OTHER DISRUPTANTS ON THE AsSOCIATION OF X DNA WITH HOST DNA”
Disruptant RNase Trypsin Pronase 1% SDS 1 M NaC104 0.5 M Na trichloroacetate Incubation at 50”, 10 min pH 6.0 pH 9.0 20,000 rads (y-rays)
Percentage associated 60 49 47
100 36
61 35
69 58 42
a E. wli C600 labeled with rH]thymidine were infected with 3sP-labeled phage and grown as outlined in the legend to Fig. 1. Bacterial lysates were prepared by method I. Aliquots of bacterial lysates were subjected to digestion with pancreatic RNase, trypsin, or Pronase at 100 pg/ml at 37” for 10 min. SDS, NaCIO,, or Na trichloroacetate was added to lysates to the concentrations listed. The dose of y-irradiation was measured by FeSO* dosimetry. Each “percentage associated” represents the average of at least two sucrose gradients.
6 to pH 9, the amount of X DNA found to associate with bacterial DNA was constant. Chaotropic salts such as sodium perchlorate or sodium trichloroacetate, which denature and often precipitate proteins and which disrupt phages and viruses, also did not release additional X DNA from its chromosomal constraints even though these agents greatly reduced the sedimentation of E. coli DNA and presumably disrupt chromosome structure greatly. The lack of an effect of RNase, chaotropic salts, or SDS indicates that unfolding of the nucleoid is insufficient to release X. Since lysates were prepared at a high cell density (5 X 10’ cells/ml), it was thought possible that the observed association is an artifact due to nonspecific trapping. If so, the fraction of X associated ought to vary with DNA concentration. However in lysates prepared by method I at cell densities from 5 X lO’/ml-5 X log/
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ml, X DNA remained associated with host DNA. Furthermore, lysates from cultures at various cell densities were incubated for up to 6 hr to allow time for the X DNA to diffuse out of the bacterial chromosomes. This also did not affect the association of X DNA with the host chromosome. We examined the possibility that breakage of the chromosome could release h DNA. Sensitive cells were y-irradiated prior to infection with 32P-phage with a dose sufficient to produce up to 15 doublestrand DNA breaks per cell, 20,000 rads (Freifelder, 1966a; Sinden and Pettijohn, 1981). As summarized in Table 2, preirradiation of the cells did not significantly improve the separation of bacterial and phage DNA. D. Localization Fraction
of the E. coli Membrane
The experiments described so far indicate that X DNA cosediments with host DNA after treatment with agents that alter the degree of condensation of the host chromosome, confirming earlier observations many of which have been interpreted in terms of DNA-membrane binding. If in fact membranes are responsible for the association, it would be expected that solubilization of membranes would release the X DNA. Experiments to investigate this are described in this section. We have examined the association of phage X DNA with the membrane fraction of E. co2i in lysates of cells labeled with [3Hp-glycerol, a specific marker for bacterial cell membranes (Miller, 1972). E. coli C600, grown in media supplemented with [3H]2-glycerol and resuspended in nonradioactive medium, were infected with ?Plabeled phage. At 12 min after infection, cells were lysed by method I and sedimented. Labeled bacterial membranes separated into three fractions after sedimentation (Fig. 3a). Approximately 35% of the 3H-labeled material sediments onto a shelf underlying the gradient, 60% sediments to a position where the majority of the bacterial DNA is known to sediment (see Fig. la), and 5% did not sediment
appreciably. Figures 3b and c show the sedimentation profiles of lysates treated with the detergents sodium cholate or octyl glucoside, respectively. Sodium cholate removes at least 99% of the chromosomeassociated [3Hlglycerol-labeled material while after incubation with octyl glucoside, no mlglycerol-labeled material could be detected eosedimenting with the bacterial DNA. However, phage X DNA still sediments with the bacterial DNA after treatment with these detergents (compare Figs. 2a and b with Figs. 3b and c). To be sure that the material labeled with [3Hlglycerol is indeed a membrane component, its lipid-like character was tested by extraction with chloroform-methanol (Ames, 1963); more than 95% of the material found to be 3H-labeled in the lysates was soluble in chloroform-methanol. These experiments indicate that most of the parental X DNA cosediments with host DNA even when host cell membranes are absent. E. Separation of Bacterial DNA and Phage X DNA 2ndEquilibrium CentrZfugation in a Density Gradient In this section we investigate the possibility that phage and host DNA are associated through covalent interactions, by subjecting lysates of infected cells to isopycnic centrifugation in CsCl using phage and bacterial DNA that differ in buoyant density. The density label bromouracil deoxyriboside (BUDR) was used to label bacterial DNA by growing the thyminerequiring strain DF4 for three generations in media supplemented with BUDR (10 pg/ml), thymine (3 pg/ml), and rH]BUDR. With the thymine supplement, cells do not show the many deleterious effects of labeling with BUDR, as has been reported earlier (Freifelder, 1966b), and growth of X is normal (Better and Freifelder, submitted for publication). Cells were infected with q-labeled X and grown in media supplemented with 50 &ml thymine for 12 min at 37”, after which the cells were harvested and lysed by method I. Complete separation of host and phage DNA does not occur when either untreated
165
X DNA ASSOCIATION WITH THE HOST CHROMOSOME
1
5
10
15
20
1
5
10
15
20
1
5
10
15
20
Fraction number FIG. 3. Sedimentation profile of bacterial lysates prepared by method I from [BHj2-glycerol-labeled bacteria. E. coZiC600 was grown in the presence of 10 &i/ml [8Hj2-glycerol and infected with qlabeled Xc1857at a m.o.i. = 3. After adsorption for 10 min and subsequent growth at 37” for 10 min, cells were collected and lysed by method I. The lysate (0.1 ml) was layered onto a 5-2096 sucrose gradient. (a) Untreated lysate, centrifugation for 20 min at 15,000rpm. (b) Sodium cholatetreated lysate. Sodium cholate was added to make 1.5% and NaCl was added to make 300 mM; centrifugation was for 30 minutes at 15,000 rpm. (c) Octyl glucoside-treated lysate. Octyl glucoside was added to make 100 mM; centrifugation was at 15,000rpm for 30 min. Closed circles, 3H-glycerollabeled membranes; open circles, mP-labeled X DNA. The horizontal arrow shows the direction of sedimentation. The vertical arrow shows the top of the CsCl shelf.
lysates (data not shown) or lysates treated with either a detergent or a proteolytic enzyme were centrifuged to equilibrium in CsCl. In these cases, although some of the host and phage DNA separated in the gradient, a substantial fraction of the total (host + X) DNA floated on top of the density gradient (Figs. 4b and c), the position expected for a complex containing an excess of protein. Proteolytic digestion of the lysate with trypsin or Pronase was not sufficient to allow the release of the DNA from the floating fraction (Fig. 4b), probably because all of the bound protein is not accessible to the enzyme. However host and phage X DNA are both released by treatment with a protease and an ionic
detergent such as SDS (Fig. 4a), and E. coli and X DNA become separable by this treatment. This indicates that the association between host and X DNA is not a covalent one. DISCUSSION
The experiments just described show that parentally radiolabeled X DNA is associated with the bacterial chromosome in lysates of infected cells. The nature of the association is completely unclear but the association is independent of the presence of bacterial cell membranes. This conclusion is based on our experiment (section D) with radiolabeled bacterial membranes
166
BETTER AND FREIFELDER
a. 25 -
FIG. 4. CsCl equilibrium gradient profiles of bacterial lysates prepared by method I. E. wZi DF4 was grown for three generations in media supplemented with BUDR (10 ag/ml), thymine (3 a/ ml), and [SI]BUDR (0.5 &i/ml) to a final cell density of 3 X 108/ml. Bacteria were collected by centrifugation, washed, and infected with =P-labeled Xc1857at a m.o.i. = 3. After adsorption for 10 min and subsequent growth at 37’ for 10 min, cells were collected and lysed by method I. CsCl gradients were prepared, centrifuged, and collected as described under Materials and Methods. (a) Treatment was with trypsin (100 rg/ml for 30 min at 37”) followed by SDS to make 0.1%. (b) Incubation of the bacterial lysate with trypsin alone (100 pg/ml for 30 min at 3’7”). (c) Incubation of the bacterial lysate at 37” for 30 min followed by addition of SDS to make 0.1%. Closed circles, 8H-labeled bacterial DNA, open circles, =P-labeled X DNA. The heavily and lightly hatched vertical bars represent the 3H and BP radioactivity, respectively, that floated on the gradient. This material was recovered by adding 1 ml of 1 M NaOH to the tube after the contents had been recovered by drop collecting, mixing, neutralizing with HCl, and precipitating.
in which the total fraction of chromosomeassociated membranes was removed with detergents yet only a fraction of the chromosome-associated X DNA was released by this treatment. This conclusion contrasts with the commonly held view that the rapid sedimentation of X DNA results from membrane attachment. It is important to realize that our data have no bearing in the question of whether X DNA is membrane-associated at some time in its life cycle. We have only shown that the association between X DNA and E. coli DNA does not require membrane binding. The association between X DNA and the bacterial DNA is not easily broken down
by agents which are known to disrupt chromosome structure greatly and could only be disrupted by a combined treatment with detergents and proteases followed by equilibrium centrifugation in CsCl. Furthermore, there are either three kinds of association-(i) a protein-dependent one, (ii) a detergent-sensitive one, and possibly (iii) a CsCl-sensitive one-or a single type of association that can be broken only by the combined action of protease digestion, detergent treatment, and the high salt environment of a CsCl step. Our experiments do not distinguish these possibilities. Incubation of lysates prepared by method I with mild detergents such as so-
X DNA ASSOCIATION
WITH
dium cholate or octyl glucoside releases 20% of the chromosome-associated X DNA. We cannot directly rule out the possibility that this represents a fraction of the X DNA that is bound to cell membranes, although this seems unlikely to us for the following reasons. First, we have examined by electron microscopy the population of molecules freed from the bacterial chromosome after incubation with these detergents and we find that only nonreplicating circular molecules are present (Better and Freifelder, to be published). Second, in lysates prepared by method II, which yields the membrane-dissociated, folded, bacterial chromosome, 80% of the parentally radiolabeled X DNA is associated with the host DNA. We feel that the release of this small fraction of bound X DNA in treated samples prepared by method I represents the fraction of associated molecules which are not tightly bound to bacterial DNA and are freed as the chromosome structure is disrupted. The finding described here that phage X DNA is associated with bacterial DNA following infection of sensitive bacteria is reminiscent of previously described observations concerning autonomously replicating bacterial plasmids. The sex plasmid F is found to associate with the E. coli chromosome in bacterial lysates prepared essentially as described here for method II (Kline and Miller, 1975; Miller and Kline, 1979). Many other E. coli plasmids have been found to cosediment with folded bacterial DNA as well (Kline et al., 1976; Archibald et al., 1978). Kline et al. (1976) suggest that complexing of a plasmid to folded chromosomes is a universal condition of plasmids in E. coli strains. Furthermore, association of plasmids with the folded chromosome of E. coli is independent of the presence of bacterial cell membranes, just as we have shown here to be the case for phage X DNA. We have no data to indicate the factors that are involved in maintaining the association between X DNA and host DNA. Two points which may be important are the limited association known to occur between X DNA and host components when X is repressed (Sakakibara and Tom-
THE HOST CHROMOSOME
167
izawa, 1971; Hallick and Echols, 1973), for example, when a lysogen is superinfected, and the role of both the X N gene product and transcription in RSC formation (Hallick et al., 1969; Hallick and Echols, 1973). One possibility is that RNA polymerase, which is the major protein found to remain with isolated bacterial chromosomes (Pettijohn et al., 1973; Worcel and Burgi, 1974) by itself, or in conjunction with X-specified proteins, is reponsible for maintaining the (X DNA)-(host DNA) association. Alternatively, X DNA may be associated with host DNA through interaction with both proteins and nascent RNA chains. This would be consistent with observations that the association is RNaseor protease-insensitive and with the idea that RNA-DNA interactions stabilized by proteins maintain bacterial DNA in a condensed form in vitro (Hecht et al., 1977). Possibly there would be no association of X DNA with host components in a repressed superinfected lysogen if the few promoters that are actively being transcribed in repressed phage were lacking. ACKNOWLEDGMENTS This work was supported by Grant GM-14358 from the National Institutes of Health. We acknowledge the many suggestions given to us by Chris Miller and Mike White. This is publication No. 1398 of the Department of Biochemistry, Brandeis University. REFERENCES AMES, G. F. (1968). Lipids of Salmonella typhimurium and Escherichia coli: Structure and metabolism. J. BacterioL 95,8X3-843. ARCHIBALD, E. R., CLARK, C. W., and SHEEHY, R. J. (19’78). Relationship of R6K replicating forms to the folded chromosome of Escherichia coli. J. Bacteriol 135,476-482. FIRSHEIN, W., and KOURILSKY, P. (1976). Attempts to purify a membrane attached cromoid of bacteriophage lambda. Biochimie 58,417-425. FREIFELDER, D. (1966). Lethal changes in bacteriophage DNA produced by X-rays. Radiat. Res. Suppl 6, 80-96. FREIFELDER, D. (1966). Replication of DNA during F’Lac transfer. B&hem. Biophys. Res. Cammun 23,576-582. FREIFELDER, D. (1976). Preparation of covalently closed and open circular DNA molecules of. phage X. Biochint. Biophys. Acta 432,113-117.
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HALLICK, L., BOYCE, R. P., and ECHOLS, H. (1969). Membrane association of bacteriophage X DNA: Possible direct role of regulator gene N. Nature (London) 223, 1239-1242. HALLICK, L. M., and ECHOLS,H. (1973). Genetic and biochemical properties of an association complex between host components and lambda DNA. Virdogy 52, 105-119. HECHT, R. M., STIMPSON,D., and PETTIJOHN, D. (197’7). Sedimentation properties of the bacterial chromosome as an isolated nucleoid and as an unfolded DNA fiber: Chromosomal DNA folding measured by rotor speed effects. J. Mol. Biol 111,257277.
KLINE, B. C., and MILLER, J. R. (1975). Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosomes of Escherichiu col? Physicochemical approach to studying the unit of segregation. J. Bacterid 121.165-172. KLINE, B. C., MILLER, J. R., CRESS,D. E., WLODARCZYK, M., MANIS, J. J., and OTTEN, M. R. (1976). Nonintegrated plasmid-chromosome complexes in Escherichia co& J. Bacterial 127.881439. MILLER, J. R., and KLINE, B. C. (1979). Biochemical characterization of nonintegrated plasmid-folded chromosome complexes: Sex factor F and the Escherichia cdi nucleoid. J. Bacterid 137, 885-890. MIUER, R. C. (1972). Association of replicative T4 deoxyribonucleic acid and bacterial membranes. J. Viral
10, 920-924.
PATO, M. L., and WAGGONER,B. J. (1981). Cellular location of Mu DNA replicas. J. Viral 38,249-255. PE’ITIJOHN,D. E., HECHT, R. M., STONINGTON,0. G., and STAMATO, T. D. (1973). Factors stabilizing DNA folding in bacterial chromosomes. In “DNA Synthesis in Vitro” (R. D. Wells and R. B. Inman,
eds.), pp. 145-162. University Park Press, Baltimore. SAKAKIBARA, Y., and TOMIZAWA,J. (1971). Gene N and membrane association of lambda DNA. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 691-760. Cold Spring Harbor Laboratory, New York. SALIVAR,W. O., and GARDINIER,J. (1970). Replication of bacteriophage lambda DNA associated with the host cell membrane. Virology 41, 38-51. SEGAL, P. J., and SCHAECHTER,M. (1973). The role of the host cell membrane in the replication and morphogenesis of bacteriophage. Annu. Rev. Microbid 27.261-282. SINDEN, R. R., and PETTLJOHN,D. E. (1981). Chro-
mosomes in living Escherichia c&i cells are segregated into domains of supercoiling. Proc Nat. Ad Sci USA 78, 224-228. STONINGTON,0. G., and PETTLJOHN,D. E. (1971). The folded genome of Escherichia coli isolated in a protein-DNA-RNA complex. Proc. Nat. Ad Sci USA 68, 6-9.
TZAGOLOFF,A., and PENEFSKY,H. S. (1971). Extraction and purification of lipoprotein complexes from membranes. In “Methods in Enzymology” Vol. XXII, (W. B. Jakohy, ed.), Vol. XXII, pp. 219-230. Academic Press, New York. VALENZUELA, M. (1975). Intermediates of the first round of X DNA replication are preferentially found in a rapidly sedimenting complex. Biochwn. Biophys. Res. Commun 65,1221-1228. WITKIEWICZ,H., and TAYLOR,K. (1978). X DNA-membrane complex isolated in the CsCl density gradient. FEBS L&t. SO,313-317. WORCEL,A., and BURGI, E. (1974). Properties of a membrane-attached form of the folded chromosome of Escherichia coli J. Mol. Biol 82,91-105.