The synthesis of coliphage T1 DNA: Degradation of the host chromosome

VIROLOGY

108,

373-380

The Synthesis

(1981)

of Coliphage

J. R. CHRISTENSEN,*,’ *Department Ro’chester,

Tl DNA: Degradation D. H. FIGURSKI,*,2

AND W. H. SCHREILt

of Microbiology, University of Rochester School New York 1.4662, and tlnstituto Znternazionale Napoli, Ztalia Accepted

July

of the Host Chromosome

of Medicine di Genetica

and Dentistry, e Biojisica,

10, 1980

Tl derives most of the thymine for synthesis of its DNA from the host DNA. Nevertheless, degradation of host DNA to acid-soluble products cannot be observed even when synthesis of phage DNA is inhibited. The methods of inhibition were infection of a nonpermissive host with amber mutants with a DO (no DNA synthesis) phenotype, infection of a nonpermissive host with an amber mutant with a DA (DNA synthesis arrest) phenotype, shift to a nonpermissive temperature after infection with a ts mutant with a DO phenot.ype, and addition of nalidixic acid. We conclude that degradation of the host chromosome is linked to ongoing synthesis of phage DNA. During Tl’s development cycle, the bacterial nuclear region maintains a normal appearance by electron microscopy. Filling and filled phage heads are observed chiefly near the boundary of the nuclear region. INTRODUCTION

MATERIALS

For a virulent phage, the host chromosome is a potential source of precursors for the synthesis of viral DNA. Using one example of each of the four major groups among the T phages, Labaw (1953) demonstrated that a substantial fraction of the phosphorous atoms of each of these phages was derived from the material of the host cell. We report here the results of a study of the host cell as a source of thymine for Tl DNA synthesis, from which we conclude that, like phosphorous atoms, most thymine residues in Tl DNA are derived from host DNA. We also report on a study of the degradation of the host’s chromosome by phage Tl. The results imply an unusual linkage between the degradation and ongoing synthesis of phage DNA. Finally, we report electron microscopic studies of the appearance of the host’s nuclear body during Tl’s development.

AND METHODS

Strains. Tl+ and the amber mutants used here have been described (Figurski and Christensen, 1974). Tlts 120 was isolated in this laboratory after hydroxylamine mutagenesis (Freese et al., 1961). T4amN55 and T7 were provided by John Wiberg. Escherichia coli B was the host bacterium throughout. Media. NB is nutrient broth. M9 was made according to the formula given by Studier (1969). Supplements were added as indicated for individual experiments. Labeling procedures. Pulse-labeling and assay of acid-insoluble radioactivity were as described previously (Figurski and Christensen, 1974). Except in the experiment reported in Table 1, prelabeling of bacteria was carried out by growing the bacteria to a density of 1 x lO*/ml in NB supplemented with adenosine (1.25 mmol) and [3H]thymidine (5.5 @.X/ml; 6.7 Ci/mmol). The cells were twice sedimented by centrifugation and resuspended in NB, and then incubated for an additional 15-20 min for the depletion of internal [3H]thymidine pools before infection.

’ To whom requests for reprints should be addressed. 2 Present address: Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10027. 373

0042~6822/81/020373-08$02.00/O Copyright All rights

B 1981 by Academic Prela. Inc. of reproduction in any form reserved

374

CHRISTENSEN, TABLE SOURCE OFTHYMIDINE SYNTHESISOFT~

FIGURSKI,

1 FORTHE

DNA”

Analysis of peak fractions from C&l gradient [3H]TdR was present Before and during infection Before infection only After infection only

TCA-insol. ‘H cpmml (x10-5)

PFUiml (x lo-“‘)

CPm lo5 PFU

5.8

1.97

2.96

10.3

4.24

2.43

4.29

0.95

4.06

a Bacteria were grown in M9 medium supplemented with adenosine (0.3 mgiml) and with thymidine (0.45 pg/ml), either unlabeled or 3H labeled (6.7 Ci/ mmol), as indicated. Log-phase cells were twice pelleted and resuspended in 5 x 1O-4 M MgCI, before Tl infection (m.o.i. = 14). After infection, the suspensions were diluted in M9, supplemented as above, but with the addition of bovine serum albumin (0.5 mgiml) to protect the viability of Tl. After 30 min at 37”, the lysates were clarified by low-speed centrifugation and treated with DNAse (final concentration, 0.1 mgiml; MgS& added to give 5 x 10m3M). The phages were pelleted by ultracentrifugation, resuspended in buffered CsCl (Brady et al., 19&I), and centrifuged to equilibrium. Fractions were collected and titrated for PFU. Fractions containing the highest titer were assayed for cpm. RESULTS

The Host as a Source of DNA Precursors

Although Tl derives a majority of its phosphorous atoms from host material (Labaw, 1953), this does not necessarily imply that the purines and pyrimidines of the host are similarly used. Phage T5 degrades the host DNA (for review see McCorquodale, 1975) and derives much of its phosphorous from the host (Labaw, 1953), but excretes bases and nucleosides into the medium rather than using them as precursors (Warner et al., 1975). We have determined the relative contributions of host DNA and of the medium as sources of thymidine for the synthesis of Tl DNA by comparing the radioactivity of Tl particles produced under three differ-

AND SCHREIL

ent labeling regimens: prelabeling of the host by growth in [3H]thymidine, adding label to the medium at the time of infection, and both. The results are shown in Table 1 and indicate that a substantial majority of the thymidine found in Tl particles is derived from host material. Quite similar results were obtained in experiments carried out in broth medium and utilizing the thymine-requiring strain E. coli B3 (data not shown). In addition, one-step growth curves were obtained for Tl growing in the Thy- strainE. coli B3, in synthetic medium, with and without the addition of thymine. In the absence of thymine, the yield of Tl is about two-thirds of that obtained in the presence of excess thymine (data not shown). These results suggest that, like phosphorous atoms, thymine residues from the host are used for the synthesis of Tl DNA, and that this is true whether or not the host has the capacity for thymine synthesis. The possibility remained, however, that phage-coded enzymes might provide for the synthesis of thymine, and that contributions from the host and from the medium would both be minor thymine sources. We therefore measured the specific activity of the DNA of bacteria labeled with [3H]thymidine and of phages propagated on them. Results are shown in Table 2, and it can be seen that the DNA of the phage had about twothirds to three-fourths the specific activity of the host DNA before infection. We conclude then, that the host DNA is the source for the thymine residues utilized for Tl DNA synthesis. Presumably this applies to other DNA precursors as well. Tl DNA has a molecular weight of 31-32 x lo6 (Bresler et al., 1967) and a base composition of 25 mol% thymine (Creaser and Taussig, 1957). Therefore, a typical burst of 100 Tl particles contains 2.4 x106 thymine residues. Using the estimate that two-thirds of these come from the host, about 1.6 x lo6 residues are derived from that source. Our measurements (Table 2) give about 1.3 x lop8 ,ug DNA per bacterium. Harold and Ziporin (1958) report 1.7 x 1O-8 pg, with 23 mol% thymine, which corresponds to 7.1 X lo6 thymine residues per cell.

SYNTHESIS

OF COLIPHAGE

Tl DNA

375

TABLE 2 SPECIFIC ACTIVITY OF HOST AND PHAGE DNA Host”

Phage*

Expt No.

DNA’ (&lo8 cells)

3H (cpm/lO* cells)

DNA’ (pg/lO’” PFU)

3Hd (&lO’o PFU)

Ratio of specific activities, phage/host

1 2

1.32 1.15

1.43 x 104 5.15 x 103

1.86 1.49

1.30 x 104 5.03 x 103

0.65 0.75

-

(2Cultures of 100 ml were prelabeled as described in Materials and Methods, except that in Expt 2 [3H]labeled thymidine was added to a final concentration of 2.4 &i/ml rather than 5.5 &i/ml. * After lysis, phage was pelleted by ultracentrifugation, banded in CsCl, collected by syringe, and dialyzed overnight against a large excess of buffer (10 m&f Tris, 1 mM EDTA, pH 8.1). c A slightly scaled-up version of the method of Friesen (1968) was used. Each value is based on three or four samples analyzed. d Samples of 75 to 150 ~1 were diluted into 5 ml cold 5% trichloroacetic acid and then processed according to the procedure of Figurski and Christensen (1974). Each value is baaed on three samples counted.

Using their values, at least 22% of the host DNA would have to be degraded to provide thymine residues for Tl production. Since measurements of our strain of E. coli B, grown under our conditions, give a lower quantity of DNA per cell, and since presumably some of the Tl DNA containing host-derived thymidine would not be matured into particles, this is a conservative estimate. Linkage

between Degradation and Ongoing

Synthesis

One does not expect to observe significant concentrations of low molecular weight DNA degradation products during ordinary infection, since their reincorporation into Tl DNA is likely to be rapid. By blocking DNA synthesis, however, one might expect to be able to follow degradation by monitoring the phage-induced release of acidsoluble label from prelabeled host cells. Following the precedent of experiments carried out with other phages, we infected [3H]thymidine-labeled B cells with Tl+ and with amber mutants in genes 1 and 2, both of which display a DO phenotype (no DNA synthesis) and quickly turn off the synthesis of host DNA, (Figurski and Christensen, 1974). A DO mutant of T4 (Kutter and Wiberg, 1968) was included as a control. As can be seen in the results presented in Fig. 1, no degradation is evident in the

case of either Tl DO mutant, in marked contrast to the results with T4umN55. We conclude that host DNA is not degraded, at least to acid-soluble products, after infection of a nonpermissive host by Tl DO mutants. This result suggests two possible explanations: The synthesis of hypothetical Tl gene products involved in degradation could depend on prior phage DNA synthesis. Alternatively, the degradation process itself may require ongoing phage DNA synthesis, just as synthesis of messenger for late proteins of T4 depends on concomitant DNA synthesis (Riva et al., 1970). One should be able to distinguish between these two possibilities by experiments in which one allows phage DNA synthesis to commence and then imposes some block to further DNA synthesis. If degradation has begun, and can continue without ongoing synthesis, any DNA that has not been already incorporated into phage sequences should be broken down. The results of one experiment of this kind are also presented in Fig. 1. Tlam23 has a DA (DNA arrest) phenotype: DNA synthesis commences normally, but rather abruptly halts about midway through the latent period (Figurski and Christensen, 1974). As can be seen in Fig. 1, no degradation of host label is observed after infection by Tlarn23. Thus, although the mutation blocks the continuation of DNA

CHRISTENSEN,

376

IO

20 TIME

30

40

FIGURSKI,

I

bin)

FIG. 1. Lack of solubilization of host DNA after infection by phage Tl with mutations effecting synthesis of phage DNA. Tlaml6 (gene l), Tlam5 (gene 2), and T4umN55 all have a DO phenotype. T4amN55 has its mutation in gene 42 (Chiu and Greenberg, 1973), the structural gene for deoxycytidine hydroxymethylase (Wiberg and Buchanan, 1964). Tlam23 (gene 4) has a DA phenotype. All phage mutants inhibit bacterial DNA synthesis. Bacteria were labeled by growth in NB supplemented with adenosine (1.25 mmol) and t3H]TdR (5.5 @Z/ml; 6.7 Ci/mmol). Early log-phase cells were thrice pelleted and resuspended in chilled NB, then warmed to 37” and grown for 15-20 min to deplete any pools of acid-soluble label. Aliquots of the labeled culture were infected with the indicated phages (m.o.i. from 6 to 14 in the various cases) and monitored for TCA-insoluble label at times shown. The radioactivity of an uninfected control (about 80,000 cpm per sample) was taken as 100%.

synthesis after 6-7 min, it does not provide conditions under which degradation of host DNA can be observed. It might be argued that, in the Tlam23 experiment, host material had already been degraded and reincorporated into phage DNA before the mutation could impose a block to further synthesis. To answer this, one needs to be able to inhibit phage DNA synthesis at any desired time after infection. One way of achieving this is first to infeet with a ts mutant in one of the DO genes, then allow a period of normal development at a permissive temperature, and finally shift to a nonpermissive temperature to block reincorporation. Such an experiment was performed with Tlts120, a mutant that falls in gene 2 by complementation tests (unpublished results). At 40”, Tlts120 has a DO phenotype; furthermore, in experi-

AND SCHREIL

ments in which infection by Tlts120 is initiated at 30”, a shift to 40” at any time after infection results in a rapid, drastic drop in the rate of [“Hlthymidine incorporation by the infected culture (data not shown, but a study of this phenomenon will be reported). The results of a temperature-shift experiment, done with prelabeled host cells, are shown in Fig. 2. Whether shifted to 40 after a 5-, lo-, or 15-min sojourn at 30”, the results are similar: No degradation is evident. In passing, it may be noted that the Tl+ controls included in this experiment confirm the prediction made above that no acid-soluble products would be seen after an ordinary Tl infection. As a further means of inhibiting Tl DNA synthesis during infection, we used nalidixic acid. That nalidixic acid promptly stops Tl DNA synthesis if added 5 min after infection

A 100 13001 F 3 120

90

TI+

IO

TIME

20

30

(min.)

FIG. 2. Temperature-shift experiments with Tl ts 120, a gene 2 mutant with a DO phenotype, fail to reveal degradation of host DNA. Cells were labeled and infected (m.o.i. = 6) as explained for Fig. 1. For the results shown in C, D, and E, lo-ml portions of the appropriate culture incubating at 30” were transferred, at the time shown by the arrows, to a prewarmed flask in a shaking water bath maintained at 40”. The radioactivity of an uninfected control culture (about 88,000 cpm per sample) was taken as 100%.

SYNTHESIS

OF COLIPHAGE

NAL

TIME (min.) FIG. 3. Nalidixic acid inhibits the synthesis of Tl DNA. Cells were grown to log phase in NB and infected with Tl+ (m.o.i. = 8). Half of the culture was transferred to a flask containing nalidixic acid such that the final concentration was 20 pg/ml. Points represent the amount of [3H]TdR incorporated during a 30-set pulse. Temperature throughout was 37”.

is indicated by the results presented in Fig. 3. To follow the degradation reaction, prelabeled cells were infected with Tl, and nalidixic acid was added to samples of the infected culture at various times thereafter. In Fig. 4A, the results of a control

Tl DNA

377

experiment with T7 are presented. Degradation is observed in all cases, although the extent of solubilization decreases as the addition of nalidixic acid is delayed beyond 5 min. No doubt this is due to the fact that, with time, increasing amounts of label released from the host chromosome have been reincorporated into T7 DNA. We note that the synthesis of T’7 DNA would require very nearly the same amount of host material as would Tl: T7 has slightly less DNA per particle, but it obtains slightly more of its phosphorous, at least, from the host (Labaw, 1953). In the T7 experiment, with reincorporation inhibited, about half of the host DNA is degraded to acid-soluble products if the nalidixic acid is added early. With Tl, the results (Fig. 4B) are very different: No degradation is seen, whatever the time of addition of nalidixic acid. In summary, whether ongoing Tl DNA synthesis is blocked by amber mutations with DO or with DA phenotypes, by shifting to a nonpermissive temperature at various times after infection by a ts mutant with a DO phenotype, or by adding nalidixic acid at various times after infection by Tl+, no degradation of the host chromosome to acid-soluble products is observed. The various experimental procedures presumably block ongoing synthesis by very different mechanisms, and it seems unlikely that each of them prevents degradation by

FIG. 4. Blocking DNA synthesis by nalidixic acid allows the demonstration of the solubilization of host chromosome after T7 infection, but not after Tl infection. Labeling of cells and infection with T? (m.o.i. = 8) or Tl+ (m.o.i. = 5) were as explained for Fig. 1. At times indicated, samples were transferred to flasks containing nalidixic acid to give a final concentration of 20 pg/ml. Acid-insoluble radioactivity was measured at times shown, with the radioactivity of an uninfected control (about 82,000 cpm per sample) taken as 100%. Temperature throughout was 37”.

378

CHRISTENSEN,

FIGURSKI,

some mechanism that is independent of their synthesis-inhibiting activity. Rather, we interpret the results to mean that ongoing DNA synthesis is required for degradation of the host DNA to occur after Tl infection, so that any agent that blocks that synthesis will also prevent the degradation. Electron Microscopic Observation of the Cell Nucleoplasm after Tl Infection All of the results presented above point to a linkage between degradation of host

FIG. 5. Thin sections of Tl-infected bacteria at 3, 6, 9, and 12 min after infection (a, b, c, and d, respectively). At the times indicated 0~0, was added to samples of the infected culture to give a final concentration of O.l%, and the cells were immediately pelleted by centrifugation. Pellets of the “prefixed” cells were mixed with liquid agar held at 45”, and samples of the mixture were taken up into Pasteur pipets. After solidification, the agar was blown out onto a glass slide to be cut into small blocks, which were then fixed overnight in 1% 0~0, with exact Ryter-Kellenberger conditions (Ryter et al., 1958; Schreil, 1964). Fixed blocks were treated with uranyl acetate (Schreil, 1964), dehydrated in acetone, and embedded in Vestopal W. Thin sections were cut and stained with lead by the method of Karnovsky (1961). Size marker is 1 pm.

AND SCHREIL

DNA and ongoing Tl DNA synthesis. In this regard, it is of interest to examine the state of the DNA in the Tl-infected cell. Observations by light miscroscopy long ago revealed that Tl-infected cells do not undergo the nuclear disintegration characteristic of cells infected by the T-even phages or T5 (Luria and Human, 1950). We have studied the state of the nuclear region of Tl-infected cells by electron microscopy, observing thin sections of cells fixed by the standard Ryter-Kellenberger procedure (Ryter et al., 1958; Schreil, 1964). Figure 5 shows typical results. Figure 5a shows cells 3 min after infection: The nuclear region appears in no way different from that of an uninfected bacterium. Six minutes after infection (Fig. 5b) one observes a few dark-staining areas, suggestive of partially filled phage heads. These are characteristically seen at the margin of the DNA-containing region. The appearance of the nuclear region itself is essentially unchanged. After 9 min (Fig. 5c), the nuclear region still appears normal; darkstaining condensations suggestive of full phage heads are found not only near the margins of the DNA region but also in small clusters, with no obvious contact with the nuclear region. On observations of 200 cells, about twice as many were found to contain clusters of phages in the nuclear region as contained phages closely surrounded by the granular cytoplasmic material. By 12 min (Fig. 5d), near the end of the latent period, the DNA-containing region of many of the cells seemed to have expanded to fill a larger portion of the cell, but the fibrillar appearance of the regions was still very similar to that of uninfected bacteria. The great majority of the dark-staining condensates was again found to be associated with these fibrillar DNA regions. In summary, by electron microscopy there was no obvious disruption of the organization of the nuclear regions despite the fact that the DNA comprising them must have been undergoing a conversion from bacterial to phage DNA during the period covered by the observations. Phage heads, apparently full or in the process of being filled by DNA, were found predominantly in close physical proximity to the nuclear regions, from their first appearance until just before lysis.

SYNTHESIS

OF COLIPHAGE

DISCUSSION

After infection by the other virulent, double-stranded DNA phages of E. coli, degradation of host cell DNA occurs even when synthesis of phage DNA is inhibited. For instance, with T4, degradation can be observed after infection by a DO mutant (Kutter and Wiberg, 1968); with T5, after nalidixic acid or hydroxyurea treatment or after infection by a DO mutant (Zweig et al. , 1972); and with T7, after DO infection (Sadowski and Kerr, 19’70) or after nalidixic acid treatment (Fig. 4). In the case of T5, the two processes are temporally separated during a normal, uninhibited infection: Degradation is essentially complete before significant phage DNA synthesis occurs, whereas with the other two phages, with their shorter latent periods, the time periods for degradation and synthesis overlap, and it is only by inhibition of DNA synthesis that the degradation reaction can be conveniently studied. For all three phages, it is clear that degradation is a multistage process and that several phagecoded functions are involved. With Tl, we have an apparent paradox. On the one hand, most of the thymine for the synthesifs of Tl DNA comes from the host, and the host DNA seems to be the only plausible source for this thymine. On the other hand, attempts to observe degradation of the host DNA by blocking synthesis of Tl DNA by a variety of different procedures have failed even though these procedures work for other phages. The paradox is resolved, however, if we assume that, during Tl infection, there is a close coupling of degradation of the host DNA with ongoing synthesis of phage DNA. For instance, if a key enzyme involved in degradation were associated with the Tl DNA replication complex, and if the process of active replication were to impart a conformational change to that enzyme which served to activate degradation, such a coupling would be found: Degradation would depend on active synthesis, though the converse would not necessarily be true. Other mechanisms can be imagined. Whatever the mechanism may be, we propose that such a coupling exists. While we know of no precedent for it, it does not

Tl DNA

379

seem in any way unreasonable. On the contrary, release of potential precursors of DNA synthesis in the immediate vicinity of a synthetic complex would seem to be an efficient procedure. Also, Tl DNA synthesis is different from other virulent coliphages in another way: It requires the activity of several host gene products that are involved in the elongation stage of DNA thesis (Bourque and Christensen, 1980). With degradation and synthesis so closely coupled, it is not suprising that the DNAcontaining region of the bacterium maintains a normal appearance, or that the headfilling process, as it occurs, seems to do so in close proximity to the “nuclear” region. In cases in which clusters of filled heads are seen to be closely surrounded by cytoplasm, it may be that one is seeing the end result of the sequence: degradation of E. coli DNA, synthesis of Tl DNA, and head filling, with complete exhaustion of the local supply of host DNA. However, late in the infection, substantial pools of unpackaged DNA are present in the cells, and it is clear that the rate of synthesis of Tl DNA must be sufficient to maintain the pools despite their depletion through packaging. ACKNOWLEDGMENTS We thank John Wiberg for T4 and T7 phages. Tom Kustra found that Tltsl20 is a gene 2 mutant. Support was provided by Training Grant 5-701-GM00592 from the U. S. Public Health Service. REFERENCES L. W., and CHRISTENSEN, J. R. (1980). The synthesis of coliphage Tl DNA: Requirement for host dna genes involved in elongation. Virology 102, 310-316. BRODY, E., COLEMAN, L., MACKAL, R. P., WERNINGHAUS, B., and EVANS, E. A., JR. (1964). Properties of infectious deoxyribonucleic acid from Tl and A bacteriophage. J. Biol. Chem. 239, 285-289. BRESLER, S. E., KISELEV, N. A., MANJAKOV, V. F., MOSEVITSKY, M. I., and TIMKOVSKY, A. L. (1967). Isolation and physicochemical investigation of Tl bacteriophage DNA. Virology 33, l-9. CHIU, C., and GREENBERG, G. (1973). Intragenic complementation between temperature-sensitive mutants of gene 42 (dCMP hydroxymethylase) of bacteriophage T4. J. Viral. 12, 415-416. CREASER, E. H., and TAUSSIG, A. (195’7). The purifica-

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LURIA, S. E., and HUMAN, M. L. (1950). Chromatin

AND SCHREIL

staining of bacteria during bacteriophage infection. 59, 551-560. MCCORQUODALE, D. J. (1975). The T-odd bacteriophages. CRC Cd. Rev. Microbial. 4, 101-159. RIVA, S., CASCINO, A., and GERDUSCHEK, E. P. (1970). Coupling of late transcription to viral replication in bacteriophage T4 development. J. Mol. Biol. 54, 85- 102. RYTER, A., KELLENBERGER, E., BIRCH-ANDERSON, A., and MAALOE, 0. (1958). Etude au microscope electronique de plasmas coytenant de I’acide desoxyribonucleique. Z. Natur$orsch. 136, 597-605. SADOWSKI, P. D., and KERR, C. (1970). Degradation of Escherichia coli B deoxyribonucleic acid after infection with deoxyribonucleic acid-defective amber mutants of bacteriophage T7. J. Viral. 6, 149-155. SCHREIL, W. (1964). Studies on the fixation of artificial and bacterial plasms for the electron microscopy of thin sections. J. Cell. Biol. 22, I-20. STUDIER, F. W. (1969). The genetics and physiology of bacteriophage T7. Virology 39, 562-574. WARNER, H. R., DRONG, R. F., and BERGET, S. M. (1975) Early events after infection of Escherichia coli by bacteriophage T5. I. Induction of a 5’nucleosidase activity and excretion of free bases. J. Viral. 15, 273-280. ZWEIG, M., ROSENKRANZ, H. S., and MORGAN, C. (1972). Development of coliphage T5: Ultrastructural and biochemical studies. J. Viral. 9, 526-543. J. Bacterial.