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The initial steps in infection with coliphage M13
VIROLOGY
24, 372380
(19G4)
The Initial
Steps in Infection
HELEP\’ TZAGOLOFF Department
of Bacteriology,
with
AND
Coliphage
DAVID
of Wisconsin,
University Accepted July
M13’
PRATT Madison,
Wisconsin
15, 1964
Infection of Escherichia coli with the rod-shaped DNA bacteriophage Ml3 begins with an irreversible attachment of the phage to one of a very limited number of sites on the cell surface. The attachment rate is maximal in media of molar ionic strength of about 0.1. Temperature influences the rate only to the extent that it affects the rate of phage diffusion. Following attachment, the phage DNA penetrates into the cell, leaving behind over 90% of the protein. The penetration process is extremely temperature sensitive, with an energy of activation of about 25,000 Cal, can occur in less than 20 seconds at 37”, and depends on the host cell metabolism. Thus, the initial steps of infection with Ml3 fit into the general pattern known for other bacteriophages. The penetration is not analogous to the release of Ml3 phage from infected cells, where intact virus particles pass through the cell wall. INTRODUCTION
The rod-shaped male-specific phages of Escherichia coli can escape from the intact host cell by passage through the cell wall (Hoffmann-Berling et al., 1963; Hofschneider and Preuss, 1963). To determine whether the phage invades the host by an analogous mechanism, and to establish the optimum conditions for adsorption, we have examined the nature of the initial steps of WI13 infection. The term adsorption will be used here in its broadest sense, to mean fixation of the phage to the bacterium so that the two will sediment together (Adams, 1959). The terms attachment and penetration will be given more detailed operational definitions later in the paper. MATERIALS
AND
METHODS
The accompanying paper by Salivar et al. (1964) describes the growth, purification, and assay of MI3 phage stocks, the preparation of anti-Ml3 rabbit sera, and most of the media used in experiments with M13. 1 Supported by grants 5Tl CM 686 and AI 05627 from the IJnited States Public Health Service and an institutional grant. from t,he American Cancer Society.
Bacteria. E. coli strain 112-12 (Ah) (Edgar, 1958) was used in all experiments. This is a K12 F+ strain which will hereafter be designated simply as K. Unless otherwise specified, K bacteria were prepared for adsorption experiments as follows. An overnight culture in H-broth was diluted 5O@fold into fresh H-broth and grown with aeration (on a shaker) at 37” to 5 X 108 cells/ml. The cells were centrifuged, resuspended in an equal volume of 0.08 M NaCl, and starved with aeration at 37” for 45 minutes. The starved culture was centrifuged and the pellet resuspended at 5 X 109 cells/ml in 0.08 M NaCl for phage attachment, or in 0.08 64 NaCl + 0.05 M ammonium phosphate buffer, pH 6.8, for attachment and penetration. Preparation of SWabeled phage. Carrierfree radioactive sulfur, as HzSO+ was obtained from Oak Ridge National Laboratories. The labeled phage stock was prepared in essentially the same manner as standard Ml3 stocks except that a modified M9 medium, low in sulfur (see Media), was substituted for the 3XD medium described by Salivar et al. (1964). S35was added to the 372
INITIAL
STEPS
IN
medium to a specific activity of 8 mc/mg at the onset of bacterial growth. The radioactive phage were purified by 3 cycles of differential centrifugation in distilled water. In the resulting stock, 90 % of the S35was precipitable by anti-Ml3 serum, and there were approximately 1 X 10-s radioactive disintegrations per minute per infective phage particle. Enzymes. Crystalline bacterial proteinase, Nagarse, was obtained from the Enzyme Development Corporation (64 Wall Street, New York City). Treatment of free, attached, and penetrated phage was usually carried out at 37” in phosphate buffer, pH 7. The enzyme was used at the very high concentration of 10 mg/ml to ensure rapid digestion of the phage protein. Such treatment did not affect the host cell viability. Crystalline deoxyribonuclease I, DNase from beef pancreas (Worthington Biochemical Corporation Freehold, New Jersey) was used at 30” at a concentration of 50 pg/ml in 0.05 M RIgS04. Media. The following media were used. Modified 119 medium: 7 g Na2HP04, 3 g KH2P04, 1 g SH,Cl, 0..5 g NaCl, 1 liter distilled water, 1 ml 0.1 M CaCl?, 0.25 ml 1 M MgSO4, 0.3 ml 0.01 M FeC13, 10 ml 20% glucose, plus 10 rg/ml each of the following nonsulfur-containing amino acids : threonine, leucine, arginine, glutamic acid, aspartic acid, phenylalanine, serine, isoleucine, valine, alanine, and proline. 0.05 M ammonium phosphate buffer, pH 6.8: 4.0 g (IYH&HPO,, 2.3 g SH~HZPO~, 1 liter distilled water. Assaying for attached phage. Attachment was usually determined indirectly by measuring the proportion of the input phage still remaining unattached at any given time. An aliquot of the adsorption mixture was either shaken gently with a few drops of chloroform and then assayed for surviving phage, or centrifuged and the supernatant fluid assayed for phage. Attachment was measured directly in other experiments, either by centrifuging the adsorption mixture and assaying for plaque formers in the resulting pellet, or by allowing the attached phage to penetrate and then measuring penetrated phage as described below. The
INFECTION
WITH
Ml3
373
details of these methods are described in the (Adams, appendix to “Bacteriophages” 1959). Assaying for penetrated phage. The adsorption mixture was treated with anti-Ml3 serum at K = 20 for 5 minutes at 37” and then diluted and assayed for surviving plaque formers. The serum inactivates both free phage and those phage which have attached but not yet penetrated. RESULTS
Rate of Ml3 Adsorption a. Elffeet of host cell concentration. Adsorption of Ml3 phage to the host cells proceeds extremely slowly at the cell concentrations routinely used for measuring adsorption of other phages (Adams, 1950). The adsorption rate may be increased, however, by raising the host cell concentration, as shown in Fig. 1. The proportion of phage remaining unadsorbed after 9 minutes under optimum conditions is plotted as a function of cell concentration. It can be seen that the
O.Olb ! I I I I 1 I I I I I I , I
1x108lxd
IXIO’O CELL CONCENTRATION/ml
FIG. 1. Ml3 adsorption as a function of host cell concentration. K bacteria were grown to 5 X lo* cells/ml in H-broth, chilled, centrifuged, and resuspended in cold broth at a concentration of 1.4 X lOI cells/ml, as measured by assaying for colony formers and confirmed by microscopic count. Dilutions were made in cold broth to obtain a series of 9 concentrations from 3.5 X 10’ cells/ml to 1.4 X 10’0 cells/ml. The cell suspensions were warmed to 37”, infected with 3.5 X 107M13 phage/ ml, and aerated by vigorous shaking. After 9 minutes, unadsorbed phage were measured by the chloroform method and adsorbed phage by serum as described under Materials and treatment, Methods.
374
TZAGOLOFF
adsorption rate increases directly in proportion to cell concentration over the range tested, 3.5 X 1O’to 1.4 X lOlo cells/ml. This means that the frequency of collisions between phage and adsorption site limits the rate of Ml3 adsorption over this entire range. This is unlike the adsorption of T4 and +X174 phages, where collisions limit the adsorption rate only at low cell concentrations, and some second step becomes rate limiting at higher concentrations (Stent and Wollman, 1952; Fujimura and Kaesberg, 1962). Either there is no such second step for RI13, or else the first step proceeds so slowly that the second never becomes rate limiting. The adsorption rate constant, K, calculated from the experiment shown in Fig. 1 is approximately 3 X lo-l1 ml/min, which is lOO-fold lower than that for T-even phages under similar conditions (Adams, 1950) and also less than that for the small phages 4X174 (Fujimura and Kaesberg, 1962) and R17 (Paranchych and Graham, 1962). To compensate for the low adsorption rate, further adsorption experiments described
FIG. 2. Ml3 adsorption as a function of the number of input phage per cell. Aliquots of starved K cells at 5 X 109 cells/ml in 0.08 M N&l+ 0.05 M ammonium phosphate buffer, pH 6.8, were mixed with Ml3 at phage:cell ratios of 7, 3.5, 0.7, and 0.007. The mixtures were incubated at 30” for 3 hours, and unadsorbed phage were measured every 15 minutes by the chloroform method.
AND
PRATT
in this paper were carried out at a high cell concentration, 5 X lo9 cells/ml. The cells were always starved to increase their ability to survive for long periods at this high concentration under the different conditions of medium and temperature used. With these starved cells, Ml3 adsorption rates were somewhat lower than with growing cells, due at least in part to mechanical damage sustained by the adsorption sites during the washings carried out before and after starvation. b. E$ect of phage:cell ratio. Even at high host cell concentrations and over long periods of time, only a small proportion of Ml3 phage adsorb if the number of phage greatly exceeds the number of cells in the adsorption mixture. In the experiment shown in Fig. 2, adsorption was measured over a period of 3 hours, with phage: cell input ratios of 7.0, 3.5, 0.7, and 0.007. The proportion of phage adsorbed was maximal at the input ratios of 0.007 and 0.7, but markedly lower at 3.5 and still lower with 7.0 phage per cell. These and similar results obtained at temperatures from 0” to 37” with both starved and growing cells in various media show that the host cells have a very limited capacity for RI13 adsorption. This type of experiment is not sufficiently accurate to measure exactly how many phage the cell can hold at saturation, nor even to demonstrate beyond a doubt that a saturation point is reached. Other experiments, measuring either the adsorption of S3j-labeled phage or the proportion of cells producing mottled plaques after mixed infection with clear- and turbid-plaque type mutants (Salivar et aZ., 1964) indicated that about 3 phage can adsorb per cell. This probably represents the number of adsorption sites per cell, although other possible explanations for the limited adsorption capacity have not been excluded. Further studies on the number and nature of adsorption sites will be presented elsewhere. Attachment and Penetration Steps In experiments designed to test 11113 adsorption to starved cells at various temperatures, it became obvious that the phagecell complex resulting from adsorption at 0”
INITIAL
STEPS
IN
differed from that formed at 37”. In the 0” complex, the infectivity still resembled that of free phage in its sensitivity to anti-Ml3 serum and to the powerful proteolytic enzyme Nagarse (Matsubara et al., 1958) and in addition was inactivated by blending at 15,000 rpm in a semimicro Waring blendor. On the other hand, the infectivity of the 37” complex was completely insensitive to antiphage serum, Nagarse, or blendor treatment. Treatment with DiYase did not affect either complex, while shaking with chloroform, which kills the cells, destroyed both. We conclude from these results and from experiments with P5-labeled phage, presented later in the paper, that at 0” the RI13 phage only attach to the cell and remain on the surface, while at 37” the phage not only attach, but the infective principle, the DISA (Hofschneider, 1963), penetrates into the cell. The terms attached and penetrated will be used to describe phage in these two states. Table 1 summarizes the sensitivity of free, attached, and penetrated Ml3 phage to various treatments. Conditions Required for Attachment Ml3 phage has been found to attach to starved cells in solutions of any of the following salts: NaCl, NaN03, Na2S04, KCI, NH&l, CaC12, MgC12, and ?\IgSO,. With these salts, the ionic strength and not the particular ions determines the attachment rate, which reaches a maximum at a molar ionic strength of about 0.1. This is shown for NaCl in the experiment illustrated in Fig. 3, where the adsorption rate constant is plotted as a function of salt concentration. TABLE
1
SENSITIVITY OF THE INFECTIVITY OF FREE, ATTACHED: AND PENETRATED Ml3 PHAGE TO TREATMENT WITH ~'ARIOUS AGENTS" Phage
Anti-M13 Chloro- NagarseWaring blendor Dxase serllm form
Free Attached Penetrated
+ + -
a Plus indicates
sign indicates insensitivity.
+ +
+ + sensitivity;
+ -
-
minus
sign
INFECTION
f
WITH
$2
375
Ml3
0.03 005 o.cn 01 0.2 0.4 MOLAR IONIC STRENGTH NaCl
08
FIG. 3. Ml3 attachment rate versus NaCl cons centration. K bacteria were grown to 2.5 X locells/ml in H-broth, washed with distilled water on a Millipore HA filter, and resuspended in distilled water at a concentration of 5 X lo9 cells/ml. NaCl was added to aliquots of the cell suspension to give a series of concentrations from 0.01 to 0.8 M. 8 X IO5 Ml3 particles per milliliter were added to each aliquot, and the cell-phage mixtures were held at 0” for 200 minutes. Unadsorbed phage were measured every 40 minutes by the chloroform method, and average adsorption rate constants were calculated from these measurements.
The same salt requirement exists in broth medium. Changes in pH over the range from 5 to 8 have no influence on the attachment rate. Lower and higher pH values have not been tested. The attachment rate depends on the temperature over the range from 0” to 45” as shown in Fig. 4, in which the data from several experiments are summarized in the form of an Arrhenius plot. The attachment rate increases 2-fold as the temperature is raised from 0” to 25”, remains approximately constant to 37”, then falls off abruptly by 45”. The increase in attachment rate from 0” to 25” corresponds to the decrease in the viscosity of water over the same temperature range. This would be expected if the attachment process were rate limited by diffusion and had essentially no heat of activation, as pointed out by Garen and Puck (1951), who found the same phenomenon with the reversible attachment of Tl phage. The decline in Ml3 attachment rate found at higher temperatures results from inactivation of the attachment sites on the cell, as can be demonstrated by exposing the cells to a temperature of 45”
376
TZAGOLOFF
AND
PRATT
a particular Tl-resistant host strain or to cells killed by ultraviolet light irradiation. All these conditions, as well as adsorption in the presence of lo-” M KCN and in low ionic strength media, have been tried for h113. None were found to promote reversible attachment. The use of either 10M3 M Zn++ or the ?IIl3-resistant strain of K bacteria isolated in this laboratory completely prevented attachment. All the other conditions allowed at least some attachment to occur, but it was not reversible. l\o infective phage could be eluted from the cells over periods of as long as 2 hours at temperatures from 0” to 37”, using either distilled water or any one of several salt solutions as eluting medium.
9 s 2p I5 o.a0.9Y OJ-, 23 0.6t 056
0.0031
I
0.0033
I
I/T
I
0.0035
Conditions Required for Penetration
I
0.0037
attachment rate versus temperature. Ml3 phage were added to a concentration of 8 X lo5 phage/ml to aliquots of starved K bacteria at a concentration of 5 X 109 cells/ml in 0.08 M NaCl. The phage-cell mixtures were held for 150 minutes at temperatures from 0” to 46”, and unadsorbed phage were measured every 30 minutes by the chloroform method. Average attachment rate constants were calculated on the basis of these measurements. The data were obtained in 4 separate experiments, designated by the symbols 0, A, 0, 0, FIG.
4. Ml3
for $5 hour and then measuring adsorption at 30”. The transient exposure to 45” destroys the cell’s ability to adsorb phage without affecting their colony-forming ability. The Irreversibility
of Ml3 Attuchnaent
When infective phage can be eluted from cells to which they have attached, the attachment is termed reversible. This has been studied most thoroughly with Tl phage, by Garen and Puck (1951), Garen (1954), and Christensen and Tolmach (1955). These authors discovered several experimental conditions under which Tl attached only in a completely reversible manner. The conditions included: adsorption at 0”; adsorption in the presence of 1OF M sodium azide or 10V3 M Zn++; and adsorption to cells of
Penetration was earlier defined operationally as the process by which attached h113 phage became resistant to treatment with anti-Ml3 serum and the enzyme Nagarse and to high speed blending. Of these characteristics, only the serum resistance could be determined quickly and accurately enough to serve as a measure of penetration in the studies described below. It will be assumed that when serum resistance has developed, the penetration is complete. Penetration has been measured in various media and found to occur at approximately the same rate with either growing cells in broth or starved cells in 0.05 M ammonium phosphate buffer, pH 6.8. Other buffers and salt solutions also permitted penetration, but at lower rates. 10s3 M Zn++, which prevented h213 attachment, had no inhibitory effect on penetration. Despite the fact that 1113 penetration can occur efficiently in starved cellqenergy supplied by cell metabolism is essential for the process, as has been shown by the use of metabolic inhibitors. When either 1OF M KCIV or lo-* M sodium azide was added to starved cells and then phage were added several minutes later, the phage which attached did not penetrate until the inhibitor had been removed. Starved cells apparently have sufficient endogenous metabolism to supply the energy needed for penetration, but the
INITIAL
STEPS IN INFECTION
WITH
377
Ml3
I0.0031 (BEFORE SERUM TREATMENT)
FIG. 5
0.0033 I/T
0.0035
FIG. 6
FIG. 5. Kinetics of Ml3 penetration at temperatures from 0” to 45”. Ml3 phage at 3 X lo8 infective particles/ml were allowed 2 hours at 0” to attach to starved K bacteria suspended at 5 X log cells/ml in 0.08 M NaCl. The mixture was then diluted 500-fold into each of 7 tubes containing equal parts of H-broth and 0.05 M ammonium phosphate buffer, pH 6.8, at temperatures from 0” to 45”. The progress of penetration with time at each temperature was measured by treating aliquots from each tube with anti-Ml3 serum, k’ = 20, for 5 minutes at 37”, then diluting and assaying for serum-resistant plaque formers. FIG. 6. Ml3 penetration rate as a function of temperature. The relative penetration rate constants were determined by comparing the initial slopes of the curves in Fig. 5. supply is blocked by addition of KCN or sodium azide. Penetration of attached phage has been carried out in the presence of DNase and of the proteolytic enzyme Nagarse. The DNase had no effect on penetration, demonstrating that the phage DNA is never exposed to the medium during the process. Nagarse, in contrast, destroyed most of the phage before penetration could occur. This indicates that attached phage, like free phage, are sensitive to the enzyme, and that inactivation occurs more rapidly than penetration. Temperature affects the penetration process dramatically, as demonstrated by the experiment shown in Fig. 5. Phage attachment was allowed to take place at O”, and the phage cell complexes were then diluted into aliquots of an optimum penetration medium at temperatures from 0” to 45”. Penetration was measured as a function of time. No detectable penetration took place at O”, but the rate was easily rneasured at Go, and increased more than 20.fold between Go and 45”. The relative reaction rates at the different temperatures, deenergy
termined from the initial slopes of the curves in Fig. 5, are shown in an Arrhenius plot in Fig. 6. The Arrhenius constant, or energy of activation, determined over the range from 15” to 30” is approximately 25,000 cal. At 37”, attached phage penetrated with a halftime of about 2 minutes and a minimum time of less than 20 seconds. Experiments with Xs5-Labeled Phage Waring blendor experiments of the type designed by Hershey and Chase (1952) were carried out with both attached and penetrated Ml3 phage, to determine whether the phage protein and DNA were inside or outside of the host cell at each stage. S35 was used to label the protein, while the phage infectivity was used as a label for the DNA. That is, the DNA was considered to be outside the cell if the infectivity could be removed by blendor treatment, and inside if it could not be removed. a. Attached phage. EY5-labeled Ml3 phage, prepared as described under Materials and Methods, were allowed to attach to starved I< cells. The temperature was kept at 0”
378
TZAGOLOFF
to prevent penetration. The phage-cell complexes were centrifuged to remove unattached phage, resuspended in distilled water at O”, and blended at 15,000 rpm in a semimicro Waring blendor cooled with ice. The cells and the blending medium were then separated by centrifugation and each was assayed for phage and P5. It was found that the infectivity of attached phage was completely destroyed by blendor treatment. Of the S35, all but about 2% was stripped from the cells by blending as shown in Fig. 7A. Therefore, both the DI\‘A and protein of
0
20
% OF
I
40 I
S35
I
60 I
I
80 I
I
100 I
FIG. 7. Removal of SWabeled phage protein by blending phage-bacterium complexes after attachment or penetration. A. Blending after attachment. EP-labeled phage were mixed at a multiplicity of 0.8 phage/ cell with starved K bacteria at 5 X 109 cells/ml in ice cold 0.08 M NaCl. Ninety minutes were allowed for attachment to occur. Unattached phage were removed by centrifugation and washing at a low temperature. The phage-cell complexes were resuspended in distilled water at 0” and blended as described in the text. After blending, the cells were centrifuged, washed, and resuspended in water. Aliquots of the resuspended pellet and the supernatant fluid were counted with an endwindow Geiger counter. The values “7, of W” given in the graph are relative to total S35 prior to blending. B. Blending after penetration. Labeled phage were mixed at a multiplicity of 0.58 phage/cell with starved K bacteria suspended at 5 X lo9 cells/ml in 0.08 M NaCl + 0.05 M ammonium phosphate buffer, pH 6.8. The mixture was incubated for 2 hours at 30” to allow attachment and penetration to occur. Unadsorbed phage were removed by centrifugation and washing at room temperature, and the phage-cell complexes were resuspended in distilled water. Blending and the subsequent counts for radioactivity were carried out as in part A, except that the blendor was cooled with tap water instead of ice.
A-TAlYlJ
T\T\.mm rlttA11
attached phage must be located on the outside of the host cell. Cell viability was unaffected by the blending but was reduced to about 15 % by the suspension in ice-cold distilled water preparatory to blending. Water was used because the blendor treatment did not remove the P5 from cells suspended in salt-containing media. b. Penetrated phage. S35-labeled phage were allowed to penetrate starved K cells at 30” as described in the legend to Fig. 7B. Unadsorbed phage were removed and the phage-cell complexes were blended as described above for attached phage, except that the cells were cooled only to about 15” for blending. The cell survival was over 70 %, which is considerably higher than in the blending experiment with attached phage. The lower survival in that experiment was due to the lower temperatures maintained during both the adsorption and blending periods. The infectivity of penetrated phage proved to be insensitive to blending. This result, taken in conjunction with the fact that the infectivity of penetrated phage is also insensitive to DBase, Nagarse, and anti-Ml3 serum, shows that the phage DNA has entered the cell. Blending removed about 80% of the S35 label from the cells after penetration had occurred, as shown in Fig. 7B. This is significantly less than the 98% removal by blending after phage attachment. Both results were reproducible, suggesting that upon penetration at least a fraction of the S35becomes more intimately associated with the cell than at the attachment stage. The 20 % of the S3j left with the cells after blending is probably still outside the cell, as shown by the following experiment, in which all but 6% of the S3j of penetrated phage came off the cells in the presence of the proteolytic enzyme Nagarse. Labeled phage were allowed to penetrate as in the experiment described in Fig. 7B. The phagecell complexes were washed and resuspended at a titer of 1.2 X log complexes per milliliter in phosphate buffer, pH 7. The suspension was divided into two parts. Xagarse was added to one part to a concentration of 10 mg/ml; the second part was kept as a control. Both were incubated at 37” for 1 hour,
INITIAL
STEPS
IN
INFECTION
WITH
Ml3
379
(Puck et al., 1951; Garen and Puck, 1951). It seems likely in the case of 1113, as suggested by these authors for Tl, that the ions in the adsorption medium promote g 80 \ attachment by damping out the charges \ Il-70 H \ which would otherwise repel phage from cell, and that the attachment itself could be an electrostatic binding. We have no direct evidence, however, for the nature of the bond. The Ml3 attachment rate constant is at least lOO-fold lower than that for the larger T-even phages, suggesting that with Ml3 a much lower proportion of the phage-cell collisions results in attachment. This low collision efficiency can be explained by the I I I I J I I 0 IO 20 30 40 50 60 relative paucity of MIS-specific adsorption MINUTES INCUBATION sites on the cell surface. The evidence obFIG. 8. Effect of Nagarse on the elution of Sa5 tained for Ml3 shows that a K cell has the after penetration of labeled phage. The experiment capacity to adsorb only about 3 phage, and was carried out as described in the text. Elution therefore probably has only that number of of Sa6 counts was determined by centrifuging and adsorption sites. The host cells for T2 washing the cells and counting the Ss5 still associphage, on the other hand, have at least ated with them. hundreds of adsorption sites (Watson, 1950). n213 differs from all the T phages that with no loss of infected cells. EY5 counts have been carefully examined (Tolmach, eluted from the cells into the medium in 1957) and from 4X174 (Fujimura and both the Nagarse and control samples, as Kaesberg, 1962) in that it apparently does shown in Fig. 8. However, Nagarse increased not attach reversibly to the host cell. Conboth the rate and extent of S35 elution, ditions used successfully in demonstrating indicating that the protein of the penetrated reversible attachment in these other phage phage remained accessible to the enzyme, systems were found either to completely and therefore on the outside of the cell. prevent 3113 attachment or to give rise only to irreversible attachment. While the DISCUSSION From the results presented in this paper, possibility remains that reversible attachment could be demonstrated under some the following picture emerges for the initial still untried conditions, it would seem unsteps in Ml3 phage infection. likely to be involved as a first step in inThe first detectable step is an irreversible fection. There is no evidence from kinetic attachment of the phage to the surface of experiments to suggest that Ml3 attachment the host cell. The molar ionic strength of the adsorption medium exerts a marked in- involves two steps, and there is no “need” fluence on this step. Attachment takes place for a reversible RI13 attachment such as at maximum rate in any one of several salt Garen (1954) postulated for Tl. With Tl, it appeared that a high proportion of the solutions at molar ionic strength of about 0.1, but does not occur measurably in dis- collisions between phage and cell lead to irreversible attachment, despite the very tilled water. Temperature, on the other hand, influences attachment only to the high energy of activation of that step. Garen pointed out that this could be explained if extent that it affects the phage diffusion reversible attachment, which has a low rate. Thus, in respect to the temperature effect and ionic requirements, the irreversible energy of activation, occurred as the first attachment of ?\I13 corresponds closely to step and served to hold phage and cell in the reversible attachment of phage Tl contact long enough to allow the irreversible
380
TZAGOLOFF
step to occur. Whatever the validity of this model for Tl, reversible adsorption would not be needed to play the same role in Ml3 adsorption, since there the irreversible attachment itself has a low energy of activation. The second detectable step in infection with Ml3 is penetration of the phage DNA into the host cell. As with phage T2 (Hershey and Chase, 1952) the bulk of the S35 in labeled phage, and so presumably the bulk of the phage protein, remains outside the cell when the DNA penetrates. Hence penetration is not in any sense a reversal of the phage release from infected cells, where the whole phage passes through the cell wall (Hofschneider and Preuss, 1963). RI13 phage penetration, like that of Tl (Christensen and Tolmach, 1955) and T5 (Lanni, 1960) depends on energy supplied by the host cell metabolism. With M13, the process can be carried out in less than 20 seconds at 37”. We do not have sufficient evidence to suggest how the phage DXA is disengaged from the rod-shaped protein coat in such a short period of time. REFERENCES ADAMS, M. H. (1950). Methods of study of bacterial viruses. Methods Med. Res. 2, l-73. ADAMS, M. H. (1959). “Bacteriophages.” Interscience, New York. CHRISTENSEN, J. R., and TOLMACH, L. J. (1955). On the early stages of infection of Escherichia coli B by bacteriophage Tl. Arch. Biochem. Biophys. 57, 195-207. EDGAR, R. S. (1958). Mapping experiments with rII and h mutants of bacteriophage T4D. Virology 6, 215225. FUJIMURA, R., and KAESBERG, P. (1962). The adsorption of bacteriophage $X174 to its host. Biophys. J. 2, 433-449. GAREN, A. (1954). Thermodynamic and kinetic studies on the attachment of Tl bacteriophage to bacteria. Biochim. Biophys. Acta 14, 163-172. GAREN, A,, and PUCK, T. T. (1951). The first two steps of the invasion of host cells by bacterial virsuses. II. J. Exptl. Med. 94, 177-189.
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PRATT
HERSHEY, A. D., and CHASE, M. (1952). Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36, 39-56. HOFFMANN-BERLING, H., D~~RIVALD, H., and BEULKE, I. (1963). Ein fadiger DNB-Phage (fd) und ein spharischer RNA-Phage (fr) wirtsspezifisch fur mannliche Stamme von E. coli. III. Biologisches Verhalten von fd und fr. 2. Naturforsch. 18b, 893-898. HOFSCHNEIDER, P. H. (1963). Untersuchungen iiber “kleine” E. coli K12 Bakteriophagen. 1. Mitt. : Die Isolierung und einige Eigenschaften der “kleinen” Bakteriophagen M12, M13, und M20. 2. Naturforsch. 18b, 203-205. HOFSCHNEIDER, P. H., and PRETJSS, A. (1963). Ml3 bacteriophage liberation from intact bacteria as revealed by electron microscopy. J. Mol. Biol. 7,450-451. LANNI, Y. T. (1960). Invasion by bacteriophage T5. I. Some basic kinetic features. Virology 10, 50-513. MATSUBARA, H., HAGIHARA, B., XAKAI, M., KOMAKI, T., YONETANI, T., and OKI:NVKI, K. (1958). Crystalline bacterial proteinase. II. General properties of crystalline proteinase of Bacillus subtilis N’. J. Biochem. (Tokyo) 45, 251-258. PARANCHYCH, W., and GRAHAM, A. F. (1962). Isolation and properties of an RNA-containing bacteriophage. J. Cellular Comp. Physiol. 60, 199-208. PUCK, T. T., GAREN, A., and CLINE, J. (1951). The mechanism of virus attachment to host cells. I. The role of ions in the primary reaction. J. Exptl. Med. 93, 6688. SALIVAR, W. O., TZAGOLOFF, H., and PRATT, D. (1964). Some physical-chemical and biological properties of the rod-shaped coliphage M13. Virology 24, 359-371. STENT, G. S., and WOLLMAN, E. L. (1952). On the two-step nature of bacteriophage adsorption. Biochim. Biophys. Acta 8, 260-269. TOLMACH, L. J. (1957). Attachment and penetration of cells by viruses. Advan. Virus Res. 4, 63-110. WATSON, J. I>. (1950). The properties of x-ray inactivated bacteriophage: I. Inactivation by direct effect. J. Bacterial. 60, 697-718.
24, 372380
(19G4)
The Initial
Steps in Infection
HELEP\’ TZAGOLOFF Department
of Bacteriology,
with
AND
Coliphage
DAVID
of Wisconsin,
University Accepted July
M13’
PRATT Madison,
Wisconsin
15, 1964
Infection of Escherichia coli with the rod-shaped DNA bacteriophage Ml3 begins with an irreversible attachment of the phage to one of a very limited number of sites on the cell surface. The attachment rate is maximal in media of molar ionic strength of about 0.1. Temperature influences the rate only to the extent that it affects the rate of phage diffusion. Following attachment, the phage DNA penetrates into the cell, leaving behind over 90% of the protein. The penetration process is extremely temperature sensitive, with an energy of activation of about 25,000 Cal, can occur in less than 20 seconds at 37”, and depends on the host cell metabolism. Thus, the initial steps of infection with Ml3 fit into the general pattern known for other bacteriophages. The penetration is not analogous to the release of Ml3 phage from infected cells, where intact virus particles pass through the cell wall. INTRODUCTION
The rod-shaped male-specific phages of Escherichia coli can escape from the intact host cell by passage through the cell wall (Hoffmann-Berling et al., 1963; Hofschneider and Preuss, 1963). To determine whether the phage invades the host by an analogous mechanism, and to establish the optimum conditions for adsorption, we have examined the nature of the initial steps of WI13 infection. The term adsorption will be used here in its broadest sense, to mean fixation of the phage to the bacterium so that the two will sediment together (Adams, 1959). The terms attachment and penetration will be given more detailed operational definitions later in the paper. MATERIALS
AND
METHODS
The accompanying paper by Salivar et al. (1964) describes the growth, purification, and assay of MI3 phage stocks, the preparation of anti-Ml3 rabbit sera, and most of the media used in experiments with M13. 1 Supported by grants 5Tl CM 686 and AI 05627 from the IJnited States Public Health Service and an institutional grant. from t,he American Cancer Society.
Bacteria. E. coli strain 112-12 (Ah) (Edgar, 1958) was used in all experiments. This is a K12 F+ strain which will hereafter be designated simply as K. Unless otherwise specified, K bacteria were prepared for adsorption experiments as follows. An overnight culture in H-broth was diluted 5O@fold into fresh H-broth and grown with aeration (on a shaker) at 37” to 5 X 108 cells/ml. The cells were centrifuged, resuspended in an equal volume of 0.08 M NaCl, and starved with aeration at 37” for 45 minutes. The starved culture was centrifuged and the pellet resuspended at 5 X 109 cells/ml in 0.08 M NaCl for phage attachment, or in 0.08 64 NaCl + 0.05 M ammonium phosphate buffer, pH 6.8, for attachment and penetration. Preparation of SWabeled phage. Carrierfree radioactive sulfur, as HzSO+ was obtained from Oak Ridge National Laboratories. The labeled phage stock was prepared in essentially the same manner as standard Ml3 stocks except that a modified M9 medium, low in sulfur (see Media), was substituted for the 3XD medium described by Salivar et al. (1964). S35was added to the 372
INITIAL
STEPS
IN
medium to a specific activity of 8 mc/mg at the onset of bacterial growth. The radioactive phage were purified by 3 cycles of differential centrifugation in distilled water. In the resulting stock, 90 % of the S35was precipitable by anti-Ml3 serum, and there were approximately 1 X 10-s radioactive disintegrations per minute per infective phage particle. Enzymes. Crystalline bacterial proteinase, Nagarse, was obtained from the Enzyme Development Corporation (64 Wall Street, New York City). Treatment of free, attached, and penetrated phage was usually carried out at 37” in phosphate buffer, pH 7. The enzyme was used at the very high concentration of 10 mg/ml to ensure rapid digestion of the phage protein. Such treatment did not affect the host cell viability. Crystalline deoxyribonuclease I, DNase from beef pancreas (Worthington Biochemical Corporation Freehold, New Jersey) was used at 30” at a concentration of 50 pg/ml in 0.05 M RIgS04. Media. The following media were used. Modified 119 medium: 7 g Na2HP04, 3 g KH2P04, 1 g SH,Cl, 0..5 g NaCl, 1 liter distilled water, 1 ml 0.1 M CaCl?, 0.25 ml 1 M MgSO4, 0.3 ml 0.01 M FeC13, 10 ml 20% glucose, plus 10 rg/ml each of the following nonsulfur-containing amino acids : threonine, leucine, arginine, glutamic acid, aspartic acid, phenylalanine, serine, isoleucine, valine, alanine, and proline. 0.05 M ammonium phosphate buffer, pH 6.8: 4.0 g (IYH&HPO,, 2.3 g SH~HZPO~, 1 liter distilled water. Assaying for attached phage. Attachment was usually determined indirectly by measuring the proportion of the input phage still remaining unattached at any given time. An aliquot of the adsorption mixture was either shaken gently with a few drops of chloroform and then assayed for surviving phage, or centrifuged and the supernatant fluid assayed for phage. Attachment was measured directly in other experiments, either by centrifuging the adsorption mixture and assaying for plaque formers in the resulting pellet, or by allowing the attached phage to penetrate and then measuring penetrated phage as described below. The
INFECTION
WITH
Ml3
373
details of these methods are described in the (Adams, appendix to “Bacteriophages” 1959). Assaying for penetrated phage. The adsorption mixture was treated with anti-Ml3 serum at K = 20 for 5 minutes at 37” and then diluted and assayed for surviving plaque formers. The serum inactivates both free phage and those phage which have attached but not yet penetrated. RESULTS
Rate of Ml3 Adsorption a. Elffeet of host cell concentration. Adsorption of Ml3 phage to the host cells proceeds extremely slowly at the cell concentrations routinely used for measuring adsorption of other phages (Adams, 1950). The adsorption rate may be increased, however, by raising the host cell concentration, as shown in Fig. 1. The proportion of phage remaining unadsorbed after 9 minutes under optimum conditions is plotted as a function of cell concentration. It can be seen that the
O.Olb ! I I I I 1 I I I I I I , I
1x108lxd
IXIO’O CELL CONCENTRATION/ml
FIG. 1. Ml3 adsorption as a function of host cell concentration. K bacteria were grown to 5 X lo* cells/ml in H-broth, chilled, centrifuged, and resuspended in cold broth at a concentration of 1.4 X lOI cells/ml, as measured by assaying for colony formers and confirmed by microscopic count. Dilutions were made in cold broth to obtain a series of 9 concentrations from 3.5 X 10’ cells/ml to 1.4 X 10’0 cells/ml. The cell suspensions were warmed to 37”, infected with 3.5 X 107M13 phage/ ml, and aerated by vigorous shaking. After 9 minutes, unadsorbed phage were measured by the chloroform method and adsorbed phage by serum as described under Materials and treatment, Methods.
374
TZAGOLOFF
adsorption rate increases directly in proportion to cell concentration over the range tested, 3.5 X 1O’to 1.4 X lOlo cells/ml. This means that the frequency of collisions between phage and adsorption site limits the rate of Ml3 adsorption over this entire range. This is unlike the adsorption of T4 and +X174 phages, where collisions limit the adsorption rate only at low cell concentrations, and some second step becomes rate limiting at higher concentrations (Stent and Wollman, 1952; Fujimura and Kaesberg, 1962). Either there is no such second step for RI13, or else the first step proceeds so slowly that the second never becomes rate limiting. The adsorption rate constant, K, calculated from the experiment shown in Fig. 1 is approximately 3 X lo-l1 ml/min, which is lOO-fold lower than that for T-even phages under similar conditions (Adams, 1950) and also less than that for the small phages 4X174 (Fujimura and Kaesberg, 1962) and R17 (Paranchych and Graham, 1962). To compensate for the low adsorption rate, further adsorption experiments described
FIG. 2. Ml3 adsorption as a function of the number of input phage per cell. Aliquots of starved K cells at 5 X 109 cells/ml in 0.08 M N&l+ 0.05 M ammonium phosphate buffer, pH 6.8, were mixed with Ml3 at phage:cell ratios of 7, 3.5, 0.7, and 0.007. The mixtures were incubated at 30” for 3 hours, and unadsorbed phage were measured every 15 minutes by the chloroform method.
AND
PRATT
in this paper were carried out at a high cell concentration, 5 X lo9 cells/ml. The cells were always starved to increase their ability to survive for long periods at this high concentration under the different conditions of medium and temperature used. With these starved cells, Ml3 adsorption rates were somewhat lower than with growing cells, due at least in part to mechanical damage sustained by the adsorption sites during the washings carried out before and after starvation. b. E$ect of phage:cell ratio. Even at high host cell concentrations and over long periods of time, only a small proportion of Ml3 phage adsorb if the number of phage greatly exceeds the number of cells in the adsorption mixture. In the experiment shown in Fig. 2, adsorption was measured over a period of 3 hours, with phage: cell input ratios of 7.0, 3.5, 0.7, and 0.007. The proportion of phage adsorbed was maximal at the input ratios of 0.007 and 0.7, but markedly lower at 3.5 and still lower with 7.0 phage per cell. These and similar results obtained at temperatures from 0” to 37” with both starved and growing cells in various media show that the host cells have a very limited capacity for RI13 adsorption. This type of experiment is not sufficiently accurate to measure exactly how many phage the cell can hold at saturation, nor even to demonstrate beyond a doubt that a saturation point is reached. Other experiments, measuring either the adsorption of S3j-labeled phage or the proportion of cells producing mottled plaques after mixed infection with clear- and turbid-plaque type mutants (Salivar et aZ., 1964) indicated that about 3 phage can adsorb per cell. This probably represents the number of adsorption sites per cell, although other possible explanations for the limited adsorption capacity have not been excluded. Further studies on the number and nature of adsorption sites will be presented elsewhere. Attachment and Penetration Steps In experiments designed to test 11113 adsorption to starved cells at various temperatures, it became obvious that the phagecell complex resulting from adsorption at 0”
INITIAL
STEPS
IN
differed from that formed at 37”. In the 0” complex, the infectivity still resembled that of free phage in its sensitivity to anti-Ml3 serum and to the powerful proteolytic enzyme Nagarse (Matsubara et al., 1958) and in addition was inactivated by blending at 15,000 rpm in a semimicro Waring blendor. On the other hand, the infectivity of the 37” complex was completely insensitive to antiphage serum, Nagarse, or blendor treatment. Treatment with DiYase did not affect either complex, while shaking with chloroform, which kills the cells, destroyed both. We conclude from these results and from experiments with P5-labeled phage, presented later in the paper, that at 0” the RI13 phage only attach to the cell and remain on the surface, while at 37” the phage not only attach, but the infective principle, the DISA (Hofschneider, 1963), penetrates into the cell. The terms attached and penetrated will be used to describe phage in these two states. Table 1 summarizes the sensitivity of free, attached, and penetrated Ml3 phage to various treatments. Conditions Required for Attachment Ml3 phage has been found to attach to starved cells in solutions of any of the following salts: NaCl, NaN03, Na2S04, KCI, NH&l, CaC12, MgC12, and ?\IgSO,. With these salts, the ionic strength and not the particular ions determines the attachment rate, which reaches a maximum at a molar ionic strength of about 0.1. This is shown for NaCl in the experiment illustrated in Fig. 3, where the adsorption rate constant is plotted as a function of salt concentration. TABLE
1
SENSITIVITY OF THE INFECTIVITY OF FREE, ATTACHED: AND PENETRATED Ml3 PHAGE TO TREATMENT WITH ~'ARIOUS AGENTS" Phage
Anti-M13 Chloro- NagarseWaring blendor Dxase serllm form
Free Attached Penetrated
+ + -
a Plus indicates
sign indicates insensitivity.
+ +
+ + sensitivity;
+ -
-
minus
sign
INFECTION
f
WITH
$2
375
Ml3
0.03 005 o.cn 01 0.2 0.4 MOLAR IONIC STRENGTH NaCl
08
FIG. 3. Ml3 attachment rate versus NaCl cons centration. K bacteria were grown to 2.5 X locells/ml in H-broth, washed with distilled water on a Millipore HA filter, and resuspended in distilled water at a concentration of 5 X lo9 cells/ml. NaCl was added to aliquots of the cell suspension to give a series of concentrations from 0.01 to 0.8 M. 8 X IO5 Ml3 particles per milliliter were added to each aliquot, and the cell-phage mixtures were held at 0” for 200 minutes. Unadsorbed phage were measured every 40 minutes by the chloroform method, and average adsorption rate constants were calculated from these measurements.
The same salt requirement exists in broth medium. Changes in pH over the range from 5 to 8 have no influence on the attachment rate. Lower and higher pH values have not been tested. The attachment rate depends on the temperature over the range from 0” to 45” as shown in Fig. 4, in which the data from several experiments are summarized in the form of an Arrhenius plot. The attachment rate increases 2-fold as the temperature is raised from 0” to 25”, remains approximately constant to 37”, then falls off abruptly by 45”. The increase in attachment rate from 0” to 25” corresponds to the decrease in the viscosity of water over the same temperature range. This would be expected if the attachment process were rate limited by diffusion and had essentially no heat of activation, as pointed out by Garen and Puck (1951), who found the same phenomenon with the reversible attachment of Tl phage. The decline in Ml3 attachment rate found at higher temperatures results from inactivation of the attachment sites on the cell, as can be demonstrated by exposing the cells to a temperature of 45”
376
TZAGOLOFF
AND
PRATT
a particular Tl-resistant host strain or to cells killed by ultraviolet light irradiation. All these conditions, as well as adsorption in the presence of lo-” M KCN and in low ionic strength media, have been tried for h113. None were found to promote reversible attachment. The use of either 10M3 M Zn++ or the ?IIl3-resistant strain of K bacteria isolated in this laboratory completely prevented attachment. All the other conditions allowed at least some attachment to occur, but it was not reversible. l\o infective phage could be eluted from the cells over periods of as long as 2 hours at temperatures from 0” to 37”, using either distilled water or any one of several salt solutions as eluting medium.
9 s 2p I5 o.a0.9Y OJ-, 23 0.6t 056
0.0031
I
0.0033
I
I/T
I
0.0035
Conditions Required for Penetration
I
0.0037
attachment rate versus temperature. Ml3 phage were added to a concentration of 8 X lo5 phage/ml to aliquots of starved K bacteria at a concentration of 5 X 109 cells/ml in 0.08 M NaCl. The phage-cell mixtures were held for 150 minutes at temperatures from 0” to 46”, and unadsorbed phage were measured every 30 minutes by the chloroform method. Average attachment rate constants were calculated on the basis of these measurements. The data were obtained in 4 separate experiments, designated by the symbols 0, A, 0, 0, FIG.
4. Ml3
for $5 hour and then measuring adsorption at 30”. The transient exposure to 45” destroys the cell’s ability to adsorb phage without affecting their colony-forming ability. The Irreversibility
of Ml3 Attuchnaent
When infective phage can be eluted from cells to which they have attached, the attachment is termed reversible. This has been studied most thoroughly with Tl phage, by Garen and Puck (1951), Garen (1954), and Christensen and Tolmach (1955). These authors discovered several experimental conditions under which Tl attached only in a completely reversible manner. The conditions included: adsorption at 0”; adsorption in the presence of 1OF M sodium azide or 10V3 M Zn++; and adsorption to cells of
Penetration was earlier defined operationally as the process by which attached h113 phage became resistant to treatment with anti-Ml3 serum and the enzyme Nagarse and to high speed blending. Of these characteristics, only the serum resistance could be determined quickly and accurately enough to serve as a measure of penetration in the studies described below. It will be assumed that when serum resistance has developed, the penetration is complete. Penetration has been measured in various media and found to occur at approximately the same rate with either growing cells in broth or starved cells in 0.05 M ammonium phosphate buffer, pH 6.8. Other buffers and salt solutions also permitted penetration, but at lower rates. 10s3 M Zn++, which prevented h213 attachment, had no inhibitory effect on penetration. Despite the fact that 1113 penetration can occur efficiently in starved cellqenergy supplied by cell metabolism is essential for the process, as has been shown by the use of metabolic inhibitors. When either 1OF M KCIV or lo-* M sodium azide was added to starved cells and then phage were added several minutes later, the phage which attached did not penetrate until the inhibitor had been removed. Starved cells apparently have sufficient endogenous metabolism to supply the energy needed for penetration, but the
INITIAL
STEPS IN INFECTION
WITH
377
Ml3
I0.0031 (BEFORE SERUM TREATMENT)
FIG. 5
0.0033 I/T
0.0035
FIG. 6
FIG. 5. Kinetics of Ml3 penetration at temperatures from 0” to 45”. Ml3 phage at 3 X lo8 infective particles/ml were allowed 2 hours at 0” to attach to starved K bacteria suspended at 5 X log cells/ml in 0.08 M NaCl. The mixture was then diluted 500-fold into each of 7 tubes containing equal parts of H-broth and 0.05 M ammonium phosphate buffer, pH 6.8, at temperatures from 0” to 45”. The progress of penetration with time at each temperature was measured by treating aliquots from each tube with anti-Ml3 serum, k’ = 20, for 5 minutes at 37”, then diluting and assaying for serum-resistant plaque formers. FIG. 6. Ml3 penetration rate as a function of temperature. The relative penetration rate constants were determined by comparing the initial slopes of the curves in Fig. 5. supply is blocked by addition of KCN or sodium azide. Penetration of attached phage has been carried out in the presence of DNase and of the proteolytic enzyme Nagarse. The DNase had no effect on penetration, demonstrating that the phage DNA is never exposed to the medium during the process. Nagarse, in contrast, destroyed most of the phage before penetration could occur. This indicates that attached phage, like free phage, are sensitive to the enzyme, and that inactivation occurs more rapidly than penetration. Temperature affects the penetration process dramatically, as demonstrated by the experiment shown in Fig. 5. Phage attachment was allowed to take place at O”, and the phage cell complexes were then diluted into aliquots of an optimum penetration medium at temperatures from 0” to 45”. Penetration was measured as a function of time. No detectable penetration took place at O”, but the rate was easily rneasured at Go, and increased more than 20.fold between Go and 45”. The relative reaction rates at the different temperatures, deenergy
termined from the initial slopes of the curves in Fig. 5, are shown in an Arrhenius plot in Fig. 6. The Arrhenius constant, or energy of activation, determined over the range from 15” to 30” is approximately 25,000 cal. At 37”, attached phage penetrated with a halftime of about 2 minutes and a minimum time of less than 20 seconds. Experiments with Xs5-Labeled Phage Waring blendor experiments of the type designed by Hershey and Chase (1952) were carried out with both attached and penetrated Ml3 phage, to determine whether the phage protein and DNA were inside or outside of the host cell at each stage. S35 was used to label the protein, while the phage infectivity was used as a label for the DNA. That is, the DNA was considered to be outside the cell if the infectivity could be removed by blendor treatment, and inside if it could not be removed. a. Attached phage. EY5-labeled Ml3 phage, prepared as described under Materials and Methods, were allowed to attach to starved I< cells. The temperature was kept at 0”
378
TZAGOLOFF
to prevent penetration. The phage-cell complexes were centrifuged to remove unattached phage, resuspended in distilled water at O”, and blended at 15,000 rpm in a semimicro Waring blendor cooled with ice. The cells and the blending medium were then separated by centrifugation and each was assayed for phage and P5. It was found that the infectivity of attached phage was completely destroyed by blendor treatment. Of the S35, all but about 2% was stripped from the cells by blending as shown in Fig. 7A. Therefore, both the DI\‘A and protein of
0
20
% OF
I
40 I
S35
I
60 I
I
80 I
I
100 I
FIG. 7. Removal of SWabeled phage protein by blending phage-bacterium complexes after attachment or penetration. A. Blending after attachment. EP-labeled phage were mixed at a multiplicity of 0.8 phage/ cell with starved K bacteria at 5 X 109 cells/ml in ice cold 0.08 M NaCl. Ninety minutes were allowed for attachment to occur. Unattached phage were removed by centrifugation and washing at a low temperature. The phage-cell complexes were resuspended in distilled water at 0” and blended as described in the text. After blending, the cells were centrifuged, washed, and resuspended in water. Aliquots of the resuspended pellet and the supernatant fluid were counted with an endwindow Geiger counter. The values “7, of W” given in the graph are relative to total S35 prior to blending. B. Blending after penetration. Labeled phage were mixed at a multiplicity of 0.58 phage/cell with starved K bacteria suspended at 5 X lo9 cells/ml in 0.08 M NaCl + 0.05 M ammonium phosphate buffer, pH 6.8. The mixture was incubated for 2 hours at 30” to allow attachment and penetration to occur. Unadsorbed phage were removed by centrifugation and washing at room temperature, and the phage-cell complexes were resuspended in distilled water. Blending and the subsequent counts for radioactivity were carried out as in part A, except that the blendor was cooled with tap water instead of ice.
A-TAlYlJ
T\T\.mm rlttA11
attached phage must be located on the outside of the host cell. Cell viability was unaffected by the blending but was reduced to about 15 % by the suspension in ice-cold distilled water preparatory to blending. Water was used because the blendor treatment did not remove the P5 from cells suspended in salt-containing media. b. Penetrated phage. S35-labeled phage were allowed to penetrate starved K cells at 30” as described in the legend to Fig. 7B. Unadsorbed phage were removed and the phage-cell complexes were blended as described above for attached phage, except that the cells were cooled only to about 15” for blending. The cell survival was over 70 %, which is considerably higher than in the blending experiment with attached phage. The lower survival in that experiment was due to the lower temperatures maintained during both the adsorption and blending periods. The infectivity of penetrated phage proved to be insensitive to blending. This result, taken in conjunction with the fact that the infectivity of penetrated phage is also insensitive to DBase, Nagarse, and anti-Ml3 serum, shows that the phage DNA has entered the cell. Blending removed about 80% of the S35 label from the cells after penetration had occurred, as shown in Fig. 7B. This is significantly less than the 98% removal by blending after phage attachment. Both results were reproducible, suggesting that upon penetration at least a fraction of the S35becomes more intimately associated with the cell than at the attachment stage. The 20 % of the S3j left with the cells after blending is probably still outside the cell, as shown by the following experiment, in which all but 6% of the S3j of penetrated phage came off the cells in the presence of the proteolytic enzyme Nagarse. Labeled phage were allowed to penetrate as in the experiment described in Fig. 7B. The phagecell complexes were washed and resuspended at a titer of 1.2 X log complexes per milliliter in phosphate buffer, pH 7. The suspension was divided into two parts. Xagarse was added to one part to a concentration of 10 mg/ml; the second part was kept as a control. Both were incubated at 37” for 1 hour,
INITIAL
STEPS
IN
INFECTION
WITH
Ml3
379
(Puck et al., 1951; Garen and Puck, 1951). It seems likely in the case of 1113, as suggested by these authors for Tl, that the ions in the adsorption medium promote g 80 \ attachment by damping out the charges \ Il-70 H \ which would otherwise repel phage from cell, and that the attachment itself could be an electrostatic binding. We have no direct evidence, however, for the nature of the bond. The Ml3 attachment rate constant is at least lOO-fold lower than that for the larger T-even phages, suggesting that with Ml3 a much lower proportion of the phage-cell collisions results in attachment. This low collision efficiency can be explained by the I I I I J I I 0 IO 20 30 40 50 60 relative paucity of MIS-specific adsorption MINUTES INCUBATION sites on the cell surface. The evidence obFIG. 8. Effect of Nagarse on the elution of Sa5 tained for Ml3 shows that a K cell has the after penetration of labeled phage. The experiment capacity to adsorb only about 3 phage, and was carried out as described in the text. Elution therefore probably has only that number of of Sa6 counts was determined by centrifuging and adsorption sites. The host cells for T2 washing the cells and counting the Ss5 still associphage, on the other hand, have at least ated with them. hundreds of adsorption sites (Watson, 1950). n213 differs from all the T phages that with no loss of infected cells. EY5 counts have been carefully examined (Tolmach, eluted from the cells into the medium in 1957) and from 4X174 (Fujimura and both the Nagarse and control samples, as Kaesberg, 1962) in that it apparently does shown in Fig. 8. However, Nagarse increased not attach reversibly to the host cell. Conboth the rate and extent of S35 elution, ditions used successfully in demonstrating indicating that the protein of the penetrated reversible attachment in these other phage phage remained accessible to the enzyme, systems were found either to completely and therefore on the outside of the cell. prevent 3113 attachment or to give rise only to irreversible attachment. While the DISCUSSION From the results presented in this paper, possibility remains that reversible attachment could be demonstrated under some the following picture emerges for the initial still untried conditions, it would seem unsteps in Ml3 phage infection. likely to be involved as a first step in inThe first detectable step is an irreversible fection. There is no evidence from kinetic attachment of the phage to the surface of experiments to suggest that Ml3 attachment the host cell. The molar ionic strength of the adsorption medium exerts a marked in- involves two steps, and there is no “need” fluence on this step. Attachment takes place for a reversible RI13 attachment such as at maximum rate in any one of several salt Garen (1954) postulated for Tl. With Tl, it appeared that a high proportion of the solutions at molar ionic strength of about 0.1, but does not occur measurably in dis- collisions between phage and cell lead to irreversible attachment, despite the very tilled water. Temperature, on the other hand, influences attachment only to the high energy of activation of that step. Garen pointed out that this could be explained if extent that it affects the phage diffusion reversible attachment, which has a low rate. Thus, in respect to the temperature effect and ionic requirements, the irreversible energy of activation, occurred as the first attachment of ?\I13 corresponds closely to step and served to hold phage and cell in the reversible attachment of phage Tl contact long enough to allow the irreversible
380
TZAGOLOFF
step to occur. Whatever the validity of this model for Tl, reversible adsorption would not be needed to play the same role in Ml3 adsorption, since there the irreversible attachment itself has a low energy of activation. The second detectable step in infection with Ml3 is penetration of the phage DNA into the host cell. As with phage T2 (Hershey and Chase, 1952) the bulk of the S35 in labeled phage, and so presumably the bulk of the phage protein, remains outside the cell when the DNA penetrates. Hence penetration is not in any sense a reversal of the phage release from infected cells, where the whole phage passes through the cell wall (Hofschneider and Preuss, 1963). RI13 phage penetration, like that of Tl (Christensen and Tolmach, 1955) and T5 (Lanni, 1960) depends on energy supplied by the host cell metabolism. With M13, the process can be carried out in less than 20 seconds at 37”. We do not have sufficient evidence to suggest how the phage DXA is disengaged from the rod-shaped protein coat in such a short period of time. REFERENCES ADAMS, M. H. (1950). Methods of study of bacterial viruses. Methods Med. Res. 2, l-73. ADAMS, M. H. (1959). “Bacteriophages.” Interscience, New York. CHRISTENSEN, J. R., and TOLMACH, L. J. (1955). On the early stages of infection of Escherichia coli B by bacteriophage Tl. Arch. Biochem. Biophys. 57, 195-207. EDGAR, R. S. (1958). Mapping experiments with rII and h mutants of bacteriophage T4D. Virology 6, 215225. FUJIMURA, R., and KAESBERG, P. (1962). The adsorption of bacteriophage $X174 to its host. Biophys. J. 2, 433-449. GAREN, A. (1954). Thermodynamic and kinetic studies on the attachment of Tl bacteriophage to bacteria. Biochim. Biophys. Acta 14, 163-172. GAREN, A,, and PUCK, T. T. (1951). The first two steps of the invasion of host cells by bacterial virsuses. II. J. Exptl. Med. 94, 177-189.
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PRATT
HERSHEY, A. D., and CHASE, M. (1952). Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36, 39-56. HOFFMANN-BERLING, H., D~~RIVALD, H., and BEULKE, I. (1963). Ein fadiger DNB-Phage (fd) und ein spharischer RNA-Phage (fr) wirtsspezifisch fur mannliche Stamme von E. coli. III. Biologisches Verhalten von fd und fr. 2. Naturforsch. 18b, 893-898. HOFSCHNEIDER, P. H. (1963). Untersuchungen iiber “kleine” E. coli K12 Bakteriophagen. 1. Mitt. : Die Isolierung und einige Eigenschaften der “kleinen” Bakteriophagen M12, M13, und M20. 2. Naturforsch. 18b, 203-205. HOFSCHNEIDER, P. H., and PRETJSS, A. (1963). Ml3 bacteriophage liberation from intact bacteria as revealed by electron microscopy. J. Mol. Biol. 7,450-451. LANNI, Y. T. (1960). Invasion by bacteriophage T5. I. Some basic kinetic features. Virology 10, 50-513. MATSUBARA, H., HAGIHARA, B., XAKAI, M., KOMAKI, T., YONETANI, T., and OKI:NVKI, K. (1958). Crystalline bacterial proteinase. II. General properties of crystalline proteinase of Bacillus subtilis N’. J. Biochem. (Tokyo) 45, 251-258. PARANCHYCH, W., and GRAHAM, A. F. (1962). Isolation and properties of an RNA-containing bacteriophage. J. Cellular Comp. Physiol. 60, 199-208. PUCK, T. T., GAREN, A., and CLINE, J. (1951). The mechanism of virus attachment to host cells. I. The role of ions in the primary reaction. J. Exptl. Med. 93, 6688. SALIVAR, W. O., TZAGOLOFF, H., and PRATT, D. (1964). Some physical-chemical and biological properties of the rod-shaped coliphage M13. Virology 24, 359-371. STENT, G. S., and WOLLMAN, E. L. (1952). On the two-step nature of bacteriophage adsorption. Biochim. Biophys. Acta 8, 260-269. TOLMACH, L. J. (1957). Attachment and penetration of cells by viruses. Advan. Virus Res. 4, 63-110. WATSON, J. I>. (1950). The properties of x-ray inactivated bacteriophage: I. Inactivation by direct effect. J. Bacterial. 60, 697-718.