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A preliminary study of the high-molecular-weight components of normal and virus-infected Escherichia coli
A Preliminary Study of the High-Molecular-Weight ponents of Normal and Virus-Infected Escbericbia
ComcoZi*
Albert Siegel,2 S. J. Singe9 and S. G. Wildman From the Ketckhoff Laboratories of Biology and the Crellin Laboratories of Chemislr!l, California Znatitute of Technology, Pasadena, California; and from the Department of Botany, University of Caliiornia, Los Angeles, California Received
April
1, 1952
When a bacteriophage particle infects a bacterium, it disappears as a free infectious unit. After a specific latent period, dependent on the host-virus system and on the environmental conditions, the bacterium bursts and releases many new phage particles (1). For approximat.ely the first half of the latent period, which we will refer to as the “eclipse,” no intact infectious bacteriophage particles are recovered upon disruption of the infected bacterium. During the second half of the latent period, bacteriophage particles appear in increasing numbers within the bacterium until the end of the latent period is reached and the bacterium burst.s (2). In a fundamental sense, the process of viral biosynthesis is chemical in nature. In order to understand more clearly the mechanism of this process, the chemical pathways of the synthesis of new virus under the control of the original infecting particle must be understood and defined. Several studies of the chemistry of virus synthesis have been undcrtaken. One approach has been the study of the effect of infection upon the over-all amounts of bacterial protein and nucleic acid (3). Another approach has been to study the origin of bacteriophage carbon, nitrogen, and phosphorus with the aid of isotopes (3, 4, 5). 1 The work reported here was tnken in part from a thesis submitted by Albert Siegel to the Division of Biology of the California Institute of Technology in part,iaI fulfillment of the requirements for the Degree of Doctor of Philosophy, June, 1951. University of California at Los * Present address: Botany Department, Angeles, Los Angeles, Californin. 3 Present address: Chemistry Department, Yale University, New Haven, Con nccticut. 27x
E. COLI COMPONENTS
279
The approach that has been undertaken in the present study is the investigation of the high-molecular-weight components of uninfected and infected bacteria. One of the objects of such an investigation is the detection, isolation, and characterization of high-molecular-weight intermediates which might appear or disappear during the course of virus synthesis. We have therefore undertaken a preliminary investigation using this approach. We have studied the feasibility of extracting and resolving the high-molecular-weight components of bacteria and the possibility of detecting changes which take place in these components as a result of a bacteriophage infection. MATERIALS
AND METHODS
Host-Virus
System
The host-virus system chosen for study was Escherichiu coli strain B and bacteriophage T2, both obtained through the courtesy of Dr. M. Delbrtick of this Institube.
Media The bacteria were cultured in a nutrient broth solution containing 8 g. Difco nutrient broth, 5 g. NaCl, and 1 g. glucose per liter of water. High-titer phage lysates were prepared in a phosphate-buffered, glucose synthetic medium prepared by mixing a sterile inorganic salt solution with sterile 4?& glucose solution in the ratio of 9:l. The salt solution contained Na*HPO,, 7.0 g.; KHzPOd, 2.0 g.; NaCl, 0.5 g.; NH&l, 1.0 g.; CaC12, 15 mg.; water, 1 1.
Electrophoretic and UZtracent~ijugaE Analyses The electrophoretic analyses were performed in a modified Tiselius electrophoresis apparatus designed by S. Swingle (6). When comparing extracts from normal and infected bacteria, two experiments were carried out simultaneously at identical total protein concentrations, usually 1%. The potential gradient in all experiments was 3.47 V./cm. Scanning patterns were taken after 2 hr. migration, unless otherwise indicated. The buffer used in all preparative as well as analytical procedures was 0.1 ionic strength cacod,ylic acid buffer at pH 6.83, consisting of 0.0223 M cacodylic acid, 0.02 M NaOH, and 0.08 M NaCl. The ultracentrifugal analyses were carried out in a synchronous motor, directdrive instrument designed and built in the Chemistry Division of the California Institute of Technology (7). The sedimentation. experiments were performed in the cacodylic acid buffer described above, at a rotor speed of 44,406 r.p.m. Protein determinations given in the text represent the nitrogen content of thoroughly dialyzed material X 6.25. Phosphorus analyses were performed by the Allen calorimetric method (8). Desoxypentose nucleic acid (DNA) analyses were performed by the cysteine hydrochloride calorimetric method of Dische (9). A sample of squid testis desoxypentosenucleic acid, kindly supplied by Dr. A. Rich of this Institute, was used as a standard in the quantitative determinations.
280
SIEGEL, SINGER AND WILDMAX EXPERIMENTAL
The Culture and Collection of Bacteria
The bacteria were grown in 5-gal. jugs containing 13 1. of nutrient broth. Ten to 20 ml. of a suspension of 24-hr.-old bacteria were used to seed the medium. The culture was incubated at 25°C. with vigorous aeration until the concentration of bacteria in the jug had reached 24 X 108cells/ml., as determined with the aid of a Petroff-Hauser bacterial counting chamber. It was determined that at this stage the bacteria are in t.he logarithmic phase of growth, doubling in number every 50 min. The bacteria were collected in a refrigerated Sharples superccntrifuge running at a speed of 30,000 r.p.m. with a flow rate of M I./mm. The bacteria were scraped from the bowl of the centrifuge and were resuspended in cu. 50 ml. of 0.1 ionic strength cacodylic acid buffer. The fresh weight of the bacteria collected in this manner was IO-12 g. The carboy lot of bacteria was washed twice by sedimcnting t,he bacteria in a Servall angle-head centrifuge, Model SS-1, at a force of 10,000 X g for 20 min. and subsequently resuspending the bacteria in buffer. The centrifugal conditions just described were those found to be minimal for completely sedimenting the dense suspensions of bacteria. The Extraction of the Bacterial Soluble High-Molecular-Weight
Components
The bacteria were disrupted in an Eppenbach colloid mill, model &V-S, with the by-pass and outlet assembly replaced by a length of x X sc in. rubber tubing which served to decrease the minimal capacity of the mill from 125 to 30 ml. A washed carboy lot of bacteria was suspended in cu. 30 ml. of buffer to form a viscous slurry which was then submitted to colloid mill action for 30 min. The clearance between the rotor and stator was set at 25-50 p. Ice water was circulated through the jacket of the mill, and during the treatment the temperature of the circulating suspension did not rise above 15°C. All other preparative procedures were carried out in a room maintained at 2°C. The material recovered from the colloid mill is termed the grindate. Figure 1 summarizes the procedure used for preparing the bacterial extract. When the grindate was centrifuged at 10,000 X g for 20 min., the supernatant liquid (supernatant I) was highly turbid, in contrast to the clear solution obtained after sedimenting intact bacteria under these conditions. It was found necessary to centrifuge supernatant I at
E.
COLI
281
COMPONENTS
20,000 X g for 2 hr. in order to obtain a clear solution (supernatant II) suitable for electrophoretic analysis. The distribution of nitrogen among the various fractions is given in Fig. 1. The thoroughly dialyzed bacBacteria grown in 13 1.of nutrient broth to 24 X + 108cells/ml.
Bacteria collected Bacteria aaahed twice in in Sharples super- --+ buffer; 10,000 X g for 20 centrifuge min. sediments bacteria
Carboy lot of bacteria suspended in 30 cc. of buffer and submitted to action of colloid mill 1 Grindate N - 100% Centrifuge 10,000 X g for 20 min.
1
1
Supernatant I N = 43% I Centrifuge 20,OCQ X g for 2 hr.
Sediment I N = 55%
, 1 Supernatant II N = 35% I Overnight dialysis against buffer 1 Bacterial extract N = 30%
1 1 Sediment II N = 8%
FIG. 1. Procedure for preparing bacterial extracts.
terial extract contained approximately SOY0 of the nitrogen of the grindate. Nitrogen analysis as well as trichloroacetic acid precipitation revealed that the bacterial extract consisted primarily of protein.
282
GIEGEL,
Bnzymdic
SINGER
AND
WILDYAN
Activity of the Bacterial Extract
In order to determine whether the soluble proteins of the bacteria had been grossly damaged by the extraction procedure, a series of rapid enzymatic assays were performed on the undialyzed supernatant II solution. When the Thunberg method was used in the same manner as used by Bonner and Wildman (lo), the following dehydrogenases were found to he present and active: lactate, choline, malate, formate, and
A
..1,,
FIG. 2. Electrophoretic scanning patterns of extracts from bacteria in the logarithmic phase of growth. Descending patterns are shown on the left; ascending, right. The three patterns represent three independently prepared extracts. The top figure indicates the method by which the patterns were divided into sectors in order to obtain relative area and mobility data.
glutamate. Adenosine deaminase was also found to be present by determination of liberated ammonia. Evidently, there was little denaturation of protein as evidenced by the retention of numerous enzymatic activities in the bacterial extract. Behavior of the Bacterial Extract upon Electrophoresis The bacterial extract was examined in the electrophoresis apparatus. The results arc shown by scanning patterns in Fig. 2, where it is evident
E.
COLI
COMPONENTS
283
that t.he soluble high-molecular-weight components extracted from Eschetichia coli in the logarithmic phase of growth form a complex multicomponent system. For the purpose of quantitative analysis the patterns were arbitrarily divided into five sectors by drawing perpendicular lines from the minima in the schlieren curves to the base line, as illustrated in Fig. 2. The sectors in the ascending diagram are designated by the letters a to e, starting with the fastest moving sector. The sectors in the descending diagram are designated in the same manner with primed letters, since it is uncertain that the sectors in both patterns represent precisely the same components. In Table I the relative areas of the various sectors and the mobilities, determined from the maxima in each sector, are listed. No more elaborate relative area or mobility measurements seem justifiable for this complex system at the present time. In order to determine the magnitude of variability to be expected in the electrophoretic patterns of independently prepared bacterial extracts, prepared under the same carefully controlled conditions, several bacterial extracts were made and compared with each other. The electrophoretic diagrams obtained in three independent representative experiments are presented in Fig. 2. In gross aspect the results of the three experiments are indeed quite similar, although they differ somewhat in finer detail. The differences, however, are small enough so that the mobility and sector area data of the experiments, listed in Table I, are quite similar in the various separate experiments. The Behavior of Extracts Obtained from Bacteria Infected with Bacteriophage for a Short Period The bacteria to be infected were grown in the same manner and under the same conditions as described above for bacteria which were examined in the uninfected state. When the concentration of bacteria had attained 24 X lOa cells/ml., the bacteria were infected by dumping 5 1. of nutrient broth containing phage particles into the 13 1. of bacterial culture medium. Enough phage particles were added to insure that each bacterium, on the average, was infected with 5 to 10 phage particles. Adequate mixing of phage particles and bacteria was achieved by vigorous shaking of the culture jug during the addition of the infectious suspension. The percentage of infection was determined by withdrawing aliquots from the culture jug immediately before and 10 min. after infection, and determining, by standard bacteriological plating met.hods, the number of bacteria that gave rise to colonies. Under these conditions only the uninfected bacteria gave rise to colonies. With this method of
41 45 63 42 46 64
E t. Ix
Normal Normal Normal “Eclipse” “Eclipse” “Eclipse”
Condition
Quantitative Analysis
TABLE
I
6.4 4.6 3.9 2.2 1.8 2.1
a ~-__-
c
d
c -_
4.149.722.217.616.8 4.649.624.117.115.4 3.453.523.116.214.2 2.346.529.719.3 4.547.528.917.310.6 3.648.628.017.610.5
b
Ascending
8.3
a’
e’
D
b
c
9.1 8.9 8.7
a’
105
b’
9.3 9.5 9.7
9.3 9.4
G’
I
6.4 6.6 6.7 6.7 6.8 5.8
d’
Descending
Bacteria
5.614.611.8 5.714.912.4 5.415.212.810.4 5.914.212.0 5.514.111.9 5.914.412.0
c
-Mobility X
and “Eclipse”
8.3 9.2
d
Ascending
4.251.712.914.517.614.510.9 3.355.915.211.218.214.811.3 3.953.819.111.317.915.011.710.0 3.459.116.213.117.715.011.0 2.556.717.612.6 14.410.8 4.356.618.210.417.514.711.3
d’
Descending b’ c’ -__-______-
Relativezma
of the Electrophoretic Scanning Patterns of Extracts of Normal
4.5 4.3 4.8 4.5 4.1 4.6
e’
4 g FZ $
2
#”
E 3
.F
2 g
E.
COLI
COMPONENTS
285
infection, from 99 to 99.99a/0 of the bacteria was found to have been infected. In order to halt the further course of infection during the latent period, sodium cyanide, at a final concentration of 0.01 M, was added to the culture medium 21 min. after infection. Under the conditions of the experiment, the latent period of infection lasts for cu. 45 min. Doermann (2) has shown that if metabolic poisons are added to bacteria during the second half of the latent period, when they already contain newly synthesized intact phage particles, the bacteria will lyse. Such treatment, however, will not lyse uninfected or “eclipse” bacteria. Thus, it is possible to arrest phage development during the eclipse period. When cyanide was added to bacteria, cultured under the standard conditions used in these experiments, 22 or 23 min. after infection, the turbidity in the growth jugs decreased rapidly, indicating that the end of the “eclipse” stage of infection had been reached, whereas there was no decrease in turbidity when the poison was added 21 min. after infection. There were two reasons for poisoning the bacteria just prior to the end of the “eclipse”: (a) At this time the maximum change in the composition of the bacterial extracts could be expected without the complication of the presence df intact intracellular phage particles; and (b) the possibility of changes taking place subsequent to poisoning was reduced, since any bacteria which continued the course of infection past the “eclipse” would be lost because of lysis in the presence of cyanide. Infected bacteria were collected immediately after poisoning and were extracted by the same method described for uninfected bacteria. In experiments where extracts of uninfected and infected bacteria were to be compared, the two types of bacterial extracts were prepared at the same time, with equivalent conditions being maintained throughout the experiment. In the case of the uninfected bacteria, 5 1. of sterile broth were added to the culture jug at the same time that the bacteria in a parallel jug were being infected. Analysis of the controls revealed that the addition of the extra broth as well as cyanide treatment caused no discernible changes in the electrophoretic scanning patterns of the uninfectsed bacterial extracts. The electrophoretic analyses of the extracts of “eclipse” bacteria, obtained in three separate experiments, are presented in Fig. 3. The patterns are seen to be quite similar in gross aspect to those obtained upowelectrophoresis of the extractsof uninfected bacteria. Thesescanning patterns were quantitatively analyzed as to relative area distribution
286
SIEGEL,
SINGER
AND
WILDMAN
and sector mobility in the same manner as that described above for the patterns of the extracts of uninfected bacteria. The data so obtained are presented in Table I, where it is again seen that the distribution of soluble high-molecular-weight components has not been grossly altered during the “eclipse” stage of infection. However, there is a difference between the patterns of the extracts of the two types of bacteria which is marked and which has been confirmed in numerous experiments. There is a de-
AIL I
c
FIG. 3. Electrophoretic scanning patterns of extrwts of bacteria in the “eclipse” stage of infection. Descending patterns are shown on the left; ascending, right. The three patterns represent three independently prepared extracts.
crease in the fastest moving, a and a’, components in the scanning patterns of the extracts of the “eclipse” bacteria.
The Significance of the Decrease of the a,and a’ Sectors of the Electrophoresis Patterns of the Extracts of “Eclipse” Bacteria The apparent mobility of the a component, about -18 X 10e6 cm./sec./v./cm., suggested that it might be due t.o free desoxypentose nucleic acid (DNA),
since this very large anoclic mobi1it.y is characteristic
E.
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COMPONENTS
287
of thymus DNA examined under similar conditions (11). In order to investigate this possibility directly, it was decided to remove samples from appropriate sections of the cell after an electrophoresis experiment and analyze these samples for DNA content. Accordingly, Expts. 63 (normal) and 64 (infected) were performed simultaneously, and electrophoresis was permitted to continue for 1 hr. longer than the usual 2-hr. migration time in order to increase the resolution among the components. The electrophoresis diagrams at the end of 3 hr. are shown in Fig. 4. Then, with each experiment, a fine metal capillary was carefully lowered by means of a fixed rack and pinion device into the ascending limb of each cell, and accurately positioned
FIG. 4. Electrophoretic patterns of Expts. 63 (normal bacteria, lower patterns) and 64 (infected bacteria, upper patterns). Vertical lines above the patterns indicate the positions to which capillary was brought for removal of sample. Capillary entered cell in direction indicated by arrows contiguous to these lines. Arrows below the patterns indicate the direction of migration and the starting positions. Descending patterns are on the left, ascending on the right.
with respect to the schlieren boundaries in the limb with the aid of the optical system. The location of the capillary in the ascending limb is, indicated in Fig. 4. The capillary was placed so that appreciable amounts of only the fastest-migrating (a) component were withdrawn, The boundaries in the cell were observed during the withdrawal of the fraction, and no appreciable disturbance occurred below the tip of the capillary. The withdrawal of the sample was continued until the a-component boundary descended to the tip of the capillary. The volumes of the samples so removed were within about 35!&of one another in the two experiments, as is indicated by the weights of the fractions given in col. 2 of Table II. The results of DNA analyses given in ~01s.3 and 4
288
SIEGEL,
SINGER
AND
WILDMAN
of Table II indicate that there was about half as much DNA in this ascending limb fraction from the infected extract as from the normal. Furthermore, over 80% of the nitrogen of sector a can be accounted for as DNA nitrogen in both the normal and infected samples. This finding confirms the hypothesis that the a component in these extracts is largely freely migrating DNA, and that this free DNA is diminished relative to the total nitrogen of the extract in the infected sample as compared with the normal. At the same time, samples were removed from the descending limbs of the two experiments in a similar manner. The capillary was lowered a safe distance short of the a’-component boundary, as indicated in Fig. 4. The volumes of the fractions withdrawn from the two descending TABLE DNA
Contents
II
of Electrophoresis
Fractions Descending fraction
Ascending fraction Expt.
No. 63: normal No. 64: “eclipse”
wt. of %Ullple withdrawn
DNA/ml.
Et$ fraction/ 1 N in tota extracr
wt. of SCUllpIe
withdrawn
8.
Pg.
4%.
g.
0.95 0.92
330 170
240 110
1.42 1.39
DNA/ml.
DNA of isolated fraction/ N in totaT extracr
le.
KS.
140 200
106 130
a No, 63 contained 1.39 mg. N/ml.; No. 64, 1.55 mg. N/ml. The figuresin ~01s. 4 and 7 are therefore corrected for this small difference in N contents of the original solutions.
limbs were within 3% of one another. Any significant amount of DNA found in these fractions undoubtedly migrated more slowly than free DNA, and was presumably bound to protein (see below). The results of DNA analyses for these fractions are given in ~01s.6 and 7 of Table II, and they indicate that somewhat more DNA was present in this descending limb fraction in the infected sample than in the normal. Enough of the normal extract No. 63 was available to repeat the electrophoresis experiment with it in an independent run. Fractions were removed in as similar a manner as possible to that already described, and the results of the DNA analyses for the ascending and descending limb fractions were similar to those given in the first row of Table II. This indicates that the sampling technique was adequately reproducible and reliable.
E.
COLI
289
COMPONENTS
The Behavior of Extracts of Normal and Infected Bacteria Upon Analytical Ultracentrifugation4
The decrease in the amount of freely migrating DNA during the “eclipse” phase of infection was also observed when the extracts of normal and “eclipse” bacteria were examined in the analytical ultracentrifuge. The sedimentation diagrams are presented in Fig. 5, and the sedimentation constants of the various peaks are recorded in Table III. We have not yet investigated the relationships between the various peaks in the ultracentrifuge patterns in a given extract and the peaks in the corresponding electrophoresis diagrams, except for the case of freely migrating DNA. The sharp, spikelike peak labeled DNA in the ultra-
b lid nil [II 1250
580
380
FIG. 5. Ultracentrifuge patterns of extracts of: (a) normal bacteria, (5) infected “eclipse” bacteria. Sedimentation proceeds to the left, and the numbers under the diagrams indicate the time in seconds after the rotor attained full speed.
centrifuge diagrams of Fig. 5 is significantly shorter in the ultracentrifuge diagram of the extract of “eclipse” bacteria than in the normal. The material represented by this sharp boundary has been identified in other experiments as free DNA (13). Any quantitative estimate of the diminution of this peak, however, is rendered difficult by the sharpness of the peak. Because only a few experiments have as yet been performed in the ultracentrifuge, the sedimentation patterns were not divided into sectors 4 Preparations of the soluble high-molecular-weight components of E. coli and other bacteria have been made independently by Pardee et al. (12). The ultracentrifuge diagrams obtained by these authors for their preparations are practically identical with those presented in this paper.
290
SIEGEL,
SINGER
AND ~ILDM.~N
for quantitative area measurements. As in the electrophoresis experiments, the patterns of the normal and “eclipse” extracts appear similar in gross aspects. It is not yet known whether differences observed in the two patterns other than in the DNA peaks are reproducible and significant. The Release of DNh from Intracellular
Bacteriophage Particles
An attempt was made to detect gross changes in the extracts of bacteria which had been infected for longer than the “eclipse” period, and which consequently contained intact phage particles. Since cyanide causes bacteria to lyse during this advanced stage of infection, the bacteria were rapidly collected in the Sharples supercentrifuge without prior poisoning. It is possible to examine the extracts of bacteria which have been infected for longer than the normal latent period of 45 min. TABLE Sedimentation
Constants
III
of the High-Molecular-Weight Extracts
Components
oj Bacterial
Expt.
No. 65, normal.. No. 66, “eclipse”.
since the wild-type strain of T2 shows the phenomenon of lysis inhibition (14). Figure 6 illustrates the electrophoretic scanning patterns of an extract of bacteria which had been infected for an average of 100 min., where it can be seen that there has been a striking increase in the area occupied by the a and a’ sectors. Since intact phage particles were present in the bacteria at the time of disruption, the question arose as to whether this increase in the a and a’ sectors resulted from a bacterial product or through the disruption of intracellular phage particles. That the a and a’ sectors did not represent intact phage particles was clear from the high mobility of these components and also from the fact that intact phage particles would have been sedimented from suspension during the preparation of the extract. To test the possibility that the increase in the a and a’ sectors in this instance was at least in part due to a phage
E. COLI COMPONENTS
291
disruption product, a carboy lot of washed, uninfected bacteria was mixed with a suspension of phage particles in the ratio of 1 bacterium to 100 phage particles just prior to colloid mill treatment. When the extract of this mixture was prepared according to the standard procedure used throughout these experiments, a solution was obtained which upon electrophoresis yielded a scanning pattern which was similar to that observed for extracts of bacteria infected for 100 min. in that a marked increase in the a and a’ components was evident, indicating that the increase was at least in part due to a phage disruption product. Purified phage concentrates themselves were then submitted to colloid mill t,reatment. A component was isolated from the disruption products of bacteriophage with the same mobility characteristics as the a and a’
CL FIG. 6. Electrophoretic scanning for 100 min. The descending patt,ern
pattern of an ext.rac’t of bacteria is on the left; ascending, right.
infected
components present in the extracts of bacteria infected for 100 min. This isolated component was identified as phage DNA (13). DISCUSSION
If we consider first the results obtained with uninfected bacteria, it appears that the DNA may be roughly divided into two categories, “free” and “bound,” on the basis of electrophoretic mobility. DNA found in a, region of the electrophoresis cell out of which (in a noninteracting system) free DNA should have migrated, is termed “bound.” The significance of the distinction between “free” and “bound” DNA is not precisely known at present. Several possible explanations may be suggested: (a) The “bound” DNA may actually be part of a nucleic acidproteiri complex which is not in rapid exchange with the free DNA; (b) all of the bacterial DNA may be in equilibrium with a nucleic acidprotein complex, such t.hat under a particular set of conditions a certain amount of DNA is free, and the rest bound. If such an equilibrium
292
SIEGEL,
SINGER
IND
WILDMAN
existed, interactions such as have been noted with mixtures of nucleic acids and albumins (15, 16) might have been observed during electrophoresis of the bacterial extracts although these effects might be obscured by the large amounts of noninteracting proteins in the extracts. At the pH and ionic strength, 6.8 and 0.1, respectively, at which our experiments were performed, however, it is doubtful that such an equilibrium could exist; (c) it must further be appreciated that the physicalchemical conditions in the extract may be different from those within the bacteria, and, as a consequence,the amounts of “free” and “bound” DNA in the extracts, while reproducible and significant, may not reflect the actual DNA distribution within the bacteria. Nonetheless, while the effects mentioned in (b) and (c) might be of importance, these would complicate the interpretation of, but not detract from the significance of, the observed differences in the amounts of “free” and “bound” DNA in extracts of “eclipse’‘-infected bacteria as compared with the amounts of “free” and “bound” DNA in normal extracts. Extracts from “eclipse’‘-infected bacteria at the same total N content as normal samples reveal a striking decreasein the “free” DNA fraction without any corresponding decrease in the “bound” DNA. Cohen (3) has demonstrated that in infected bacteria there is an increased net synthesis of protein N relative to the net synthesis of DNA as compared with uninfected bacteria. It might be expected, therefore, that if infected and normal extracts were compared at the same total N content, that less f&l DNA would be found in the infected sample than in the normal. This effect, however, (which we might term “the protein dilution of the DNA”) cannot be the sole explanation of the results which we have obtained, since the amount of “bound” DNA in the infected sample, as compared with the normal, did not decrease in the same proportion as did the “free” DNA. The methods of preparing bacterial extracts which are described in this paper should be of interest in a variety of problems dealing with the high-molecular-weight components of bacteria. In this paper, these methods have been applied in a preliminary study to the problem of viral synthesis. A refinement of these methods in combination with other techniques can be employed in an effort to assessthe significance of the change in the nucleic acid economy of the bacterium during the early stages of virus infection.
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SUMMARY
1. Methods have been developed for the extraction of the soluble high-molecular-weight components of Escherichia COG.The extracts have been characterized by electrophoretic and ultracentrifugal analysis. 2. Soluble high-molecular-weight components were extracted from bacteria infected with the T2 strain of bacteriophage in the “eclipse” stage of infection. Electrophoretic comparison of these extracts at the same total nitrogen content with those of normal uninfected bacteria suggests that the amount of “free” desoxypentose nucleic acid decreases during the early stages of infection without a corresponding decrease in the amount of “bound” desoxypentose nucleic acid. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
E~LLIS, E. L., AND DELBR~~CK, M., J. Gen. Physiol. 22, 365 (1939). DOERMANN, A. H., Carnegie Inst. Wash. Yrbk. 47, 176 (1948). COHEN, S. S., Federation Proc. 10, 585 (1951). LABAW, L. W., MOSLEY, V. M., AND WYKOFF, R. W. G., J. Back 80,511 (1950). KOZLOFF, L., PUTNAM, F. W., AND EVANS, E. A., JR., in Viruses 1950, pp. 55-63. Calif. Inst. of Tech. Pasadena, 1950. SWINQLE, S., Rev. Sci. Instruments 18, 128 (1947). SINGER, S. J., AND CAMPBELL, D. H., J. Am. Chem. Sot. 74, 1794 (1952). ALLEN, R. J. L., Biochem. J. 34, 858 (1940). DISCHE, Z., Proc. Sot. Ezptl. Biol. Med. 66, 217 (1944). BONNER, J., AND WILDMAN, S. G., Arch. Biochem. 10, 497 (1946). STENHAGEN, E., AND THEORELL, T., Trans. Faraday Sot. 36, 743 (1939). SCHACHMAN, H. K., PARDEE, A., AND STANIER, R., Arch. Biochem. Biophys. 38, 245 (1952). SIEGEL, A. AND SINGER, S. J., Biochim. et Biophys. Acta, in press. DOERMANN, A. H., J. Bact. 66, 257 (1948). LONGSWORTH, L. G., AND MACINNES, D. A., J. Gen. Physiol. 26, 607 (1942). GOLDWASSER, E., AND PUTNAM, F. W., J. Phys. & Colloid Chem. 64.79 (1950).
ComcoZi*
Albert Siegel,2 S. J. Singe9 and S. G. Wildman From the Ketckhoff Laboratories of Biology and the Crellin Laboratories of Chemislr!l, California Znatitute of Technology, Pasadena, California; and from the Department of Botany, University of Caliiornia, Los Angeles, California Received
April
1, 1952
When a bacteriophage particle infects a bacterium, it disappears as a free infectious unit. After a specific latent period, dependent on the host-virus system and on the environmental conditions, the bacterium bursts and releases many new phage particles (1). For approximat.ely the first half of the latent period, which we will refer to as the “eclipse,” no intact infectious bacteriophage particles are recovered upon disruption of the infected bacterium. During the second half of the latent period, bacteriophage particles appear in increasing numbers within the bacterium until the end of the latent period is reached and the bacterium burst.s (2). In a fundamental sense, the process of viral biosynthesis is chemical in nature. In order to understand more clearly the mechanism of this process, the chemical pathways of the synthesis of new virus under the control of the original infecting particle must be understood and defined. Several studies of the chemistry of virus synthesis have been undcrtaken. One approach has been the study of the effect of infection upon the over-all amounts of bacterial protein and nucleic acid (3). Another approach has been to study the origin of bacteriophage carbon, nitrogen, and phosphorus with the aid of isotopes (3, 4, 5). 1 The work reported here was tnken in part from a thesis submitted by Albert Siegel to the Division of Biology of the California Institute of Technology in part,iaI fulfillment of the requirements for the Degree of Doctor of Philosophy, June, 1951. University of California at Los * Present address: Botany Department, Angeles, Los Angeles, Californin. 3 Present address: Chemistry Department, Yale University, New Haven, Con nccticut. 27x
E. COLI COMPONENTS
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The approach that has been undertaken in the present study is the investigation of the high-molecular-weight components of uninfected and infected bacteria. One of the objects of such an investigation is the detection, isolation, and characterization of high-molecular-weight intermediates which might appear or disappear during the course of virus synthesis. We have therefore undertaken a preliminary investigation using this approach. We have studied the feasibility of extracting and resolving the high-molecular-weight components of bacteria and the possibility of detecting changes which take place in these components as a result of a bacteriophage infection. MATERIALS
AND METHODS
Host-Virus
System
The host-virus system chosen for study was Escherichiu coli strain B and bacteriophage T2, both obtained through the courtesy of Dr. M. Delbrtick of this Institube.
Media The bacteria were cultured in a nutrient broth solution containing 8 g. Difco nutrient broth, 5 g. NaCl, and 1 g. glucose per liter of water. High-titer phage lysates were prepared in a phosphate-buffered, glucose synthetic medium prepared by mixing a sterile inorganic salt solution with sterile 4?& glucose solution in the ratio of 9:l. The salt solution contained Na*HPO,, 7.0 g.; KHzPOd, 2.0 g.; NaCl, 0.5 g.; NH&l, 1.0 g.; CaC12, 15 mg.; water, 1 1.
Electrophoretic and UZtracent~ijugaE Analyses The electrophoretic analyses were performed in a modified Tiselius electrophoresis apparatus designed by S. Swingle (6). When comparing extracts from normal and infected bacteria, two experiments were carried out simultaneously at identical total protein concentrations, usually 1%. The potential gradient in all experiments was 3.47 V./cm. Scanning patterns were taken after 2 hr. migration, unless otherwise indicated. The buffer used in all preparative as well as analytical procedures was 0.1 ionic strength cacod,ylic acid buffer at pH 6.83, consisting of 0.0223 M cacodylic acid, 0.02 M NaOH, and 0.08 M NaCl. The ultracentrifugal analyses were carried out in a synchronous motor, directdrive instrument designed and built in the Chemistry Division of the California Institute of Technology (7). The sedimentation. experiments were performed in the cacodylic acid buffer described above, at a rotor speed of 44,406 r.p.m. Protein determinations given in the text represent the nitrogen content of thoroughly dialyzed material X 6.25. Phosphorus analyses were performed by the Allen calorimetric method (8). Desoxypentose nucleic acid (DNA) analyses were performed by the cysteine hydrochloride calorimetric method of Dische (9). A sample of squid testis desoxypentosenucleic acid, kindly supplied by Dr. A. Rich of this Institute, was used as a standard in the quantitative determinations.
280
SIEGEL, SINGER AND WILDMAX EXPERIMENTAL
The Culture and Collection of Bacteria
The bacteria were grown in 5-gal. jugs containing 13 1. of nutrient broth. Ten to 20 ml. of a suspension of 24-hr.-old bacteria were used to seed the medium. The culture was incubated at 25°C. with vigorous aeration until the concentration of bacteria in the jug had reached 24 X 108cells/ml., as determined with the aid of a Petroff-Hauser bacterial counting chamber. It was determined that at this stage the bacteria are in t.he logarithmic phase of growth, doubling in number every 50 min. The bacteria were collected in a refrigerated Sharples superccntrifuge running at a speed of 30,000 r.p.m. with a flow rate of M I./mm. The bacteria were scraped from the bowl of the centrifuge and were resuspended in cu. 50 ml. of 0.1 ionic strength cacodylic acid buffer. The fresh weight of the bacteria collected in this manner was IO-12 g. The carboy lot of bacteria was washed twice by sedimcnting t,he bacteria in a Servall angle-head centrifuge, Model SS-1, at a force of 10,000 X g for 20 min. and subsequently resuspending the bacteria in buffer. The centrifugal conditions just described were those found to be minimal for completely sedimenting the dense suspensions of bacteria. The Extraction of the Bacterial Soluble High-Molecular-Weight
Components
The bacteria were disrupted in an Eppenbach colloid mill, model &V-S, with the by-pass and outlet assembly replaced by a length of x X sc in. rubber tubing which served to decrease the minimal capacity of the mill from 125 to 30 ml. A washed carboy lot of bacteria was suspended in cu. 30 ml. of buffer to form a viscous slurry which was then submitted to colloid mill action for 30 min. The clearance between the rotor and stator was set at 25-50 p. Ice water was circulated through the jacket of the mill, and during the treatment the temperature of the circulating suspension did not rise above 15°C. All other preparative procedures were carried out in a room maintained at 2°C. The material recovered from the colloid mill is termed the grindate. Figure 1 summarizes the procedure used for preparing the bacterial extract. When the grindate was centrifuged at 10,000 X g for 20 min., the supernatant liquid (supernatant I) was highly turbid, in contrast to the clear solution obtained after sedimenting intact bacteria under these conditions. It was found necessary to centrifuge supernatant I at
E.
COLI
281
COMPONENTS
20,000 X g for 2 hr. in order to obtain a clear solution (supernatant II) suitable for electrophoretic analysis. The distribution of nitrogen among the various fractions is given in Fig. 1. The thoroughly dialyzed bacBacteria grown in 13 1.of nutrient broth to 24 X + 108cells/ml.
Bacteria collected Bacteria aaahed twice in in Sharples super- --+ buffer; 10,000 X g for 20 centrifuge min. sediments bacteria
Carboy lot of bacteria suspended in 30 cc. of buffer and submitted to action of colloid mill 1 Grindate N - 100% Centrifuge 10,000 X g for 20 min.
1
1
Supernatant I N = 43% I Centrifuge 20,OCQ X g for 2 hr.
Sediment I N = 55%
, 1 Supernatant II N = 35% I Overnight dialysis against buffer 1 Bacterial extract N = 30%
1 1 Sediment II N = 8%
FIG. 1. Procedure for preparing bacterial extracts.
terial extract contained approximately SOY0 of the nitrogen of the grindate. Nitrogen analysis as well as trichloroacetic acid precipitation revealed that the bacterial extract consisted primarily of protein.
282
GIEGEL,
Bnzymdic
SINGER
AND
WILDYAN
Activity of the Bacterial Extract
In order to determine whether the soluble proteins of the bacteria had been grossly damaged by the extraction procedure, a series of rapid enzymatic assays were performed on the undialyzed supernatant II solution. When the Thunberg method was used in the same manner as used by Bonner and Wildman (lo), the following dehydrogenases were found to he present and active: lactate, choline, malate, formate, and
A
..1,,
FIG. 2. Electrophoretic scanning patterns of extracts from bacteria in the logarithmic phase of growth. Descending patterns are shown on the left; ascending, right. The three patterns represent three independently prepared extracts. The top figure indicates the method by which the patterns were divided into sectors in order to obtain relative area and mobility data.
glutamate. Adenosine deaminase was also found to be present by determination of liberated ammonia. Evidently, there was little denaturation of protein as evidenced by the retention of numerous enzymatic activities in the bacterial extract. Behavior of the Bacterial Extract upon Electrophoresis The bacterial extract was examined in the electrophoresis apparatus. The results arc shown by scanning patterns in Fig. 2, where it is evident
E.
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283
that t.he soluble high-molecular-weight components extracted from Eschetichia coli in the logarithmic phase of growth form a complex multicomponent system. For the purpose of quantitative analysis the patterns were arbitrarily divided into five sectors by drawing perpendicular lines from the minima in the schlieren curves to the base line, as illustrated in Fig. 2. The sectors in the ascending diagram are designated by the letters a to e, starting with the fastest moving sector. The sectors in the descending diagram are designated in the same manner with primed letters, since it is uncertain that the sectors in both patterns represent precisely the same components. In Table I the relative areas of the various sectors and the mobilities, determined from the maxima in each sector, are listed. No more elaborate relative area or mobility measurements seem justifiable for this complex system at the present time. In order to determine the magnitude of variability to be expected in the electrophoretic patterns of independently prepared bacterial extracts, prepared under the same carefully controlled conditions, several bacterial extracts were made and compared with each other. The electrophoretic diagrams obtained in three independent representative experiments are presented in Fig. 2. In gross aspect the results of the three experiments are indeed quite similar, although they differ somewhat in finer detail. The differences, however, are small enough so that the mobility and sector area data of the experiments, listed in Table I, are quite similar in the various separate experiments. The Behavior of Extracts Obtained from Bacteria Infected with Bacteriophage for a Short Period The bacteria to be infected were grown in the same manner and under the same conditions as described above for bacteria which were examined in the uninfected state. When the concentration of bacteria had attained 24 X lOa cells/ml., the bacteria were infected by dumping 5 1. of nutrient broth containing phage particles into the 13 1. of bacterial culture medium. Enough phage particles were added to insure that each bacterium, on the average, was infected with 5 to 10 phage particles. Adequate mixing of phage particles and bacteria was achieved by vigorous shaking of the culture jug during the addition of the infectious suspension. The percentage of infection was determined by withdrawing aliquots from the culture jug immediately before and 10 min. after infection, and determining, by standard bacteriological plating met.hods, the number of bacteria that gave rise to colonies. Under these conditions only the uninfected bacteria gave rise to colonies. With this method of
41 45 63 42 46 64
E t. Ix
Normal Normal Normal “Eclipse” “Eclipse” “Eclipse”
Condition
Quantitative Analysis
TABLE
I
6.4 4.6 3.9 2.2 1.8 2.1
a ~-__-
c
d
c -_
4.149.722.217.616.8 4.649.624.117.115.4 3.453.523.116.214.2 2.346.529.719.3 4.547.528.917.310.6 3.648.628.017.610.5
b
Ascending
8.3
a’
e’
D
b
c
9.1 8.9 8.7
a’
105
b’
9.3 9.5 9.7
9.3 9.4
G’
I
6.4 6.6 6.7 6.7 6.8 5.8
d’
Descending
Bacteria
5.614.611.8 5.714.912.4 5.415.212.810.4 5.914.212.0 5.514.111.9 5.914.412.0
c
-Mobility X
and “Eclipse”
8.3 9.2
d
Ascending
4.251.712.914.517.614.510.9 3.355.915.211.218.214.811.3 3.953.819.111.317.915.011.710.0 3.459.116.213.117.715.011.0 2.556.717.612.6 14.410.8 4.356.618.210.417.514.711.3
d’
Descending b’ c’ -__-______-
Relativezma
of the Electrophoretic Scanning Patterns of Extracts of Normal
4.5 4.3 4.8 4.5 4.1 4.6
e’
4 g FZ $
2
#”
E 3
.F
2 g
E.
COLI
COMPONENTS
285
infection, from 99 to 99.99a/0 of the bacteria was found to have been infected. In order to halt the further course of infection during the latent period, sodium cyanide, at a final concentration of 0.01 M, was added to the culture medium 21 min. after infection. Under the conditions of the experiment, the latent period of infection lasts for cu. 45 min. Doermann (2) has shown that if metabolic poisons are added to bacteria during the second half of the latent period, when they already contain newly synthesized intact phage particles, the bacteria will lyse. Such treatment, however, will not lyse uninfected or “eclipse” bacteria. Thus, it is possible to arrest phage development during the eclipse period. When cyanide was added to bacteria, cultured under the standard conditions used in these experiments, 22 or 23 min. after infection, the turbidity in the growth jugs decreased rapidly, indicating that the end of the “eclipse” stage of infection had been reached, whereas there was no decrease in turbidity when the poison was added 21 min. after infection. There were two reasons for poisoning the bacteria just prior to the end of the “eclipse”: (a) At this time the maximum change in the composition of the bacterial extracts could be expected without the complication of the presence df intact intracellular phage particles; and (b) the possibility of changes taking place subsequent to poisoning was reduced, since any bacteria which continued the course of infection past the “eclipse” would be lost because of lysis in the presence of cyanide. Infected bacteria were collected immediately after poisoning and were extracted by the same method described for uninfected bacteria. In experiments where extracts of uninfected and infected bacteria were to be compared, the two types of bacterial extracts were prepared at the same time, with equivalent conditions being maintained throughout the experiment. In the case of the uninfected bacteria, 5 1. of sterile broth were added to the culture jug at the same time that the bacteria in a parallel jug were being infected. Analysis of the controls revealed that the addition of the extra broth as well as cyanide treatment caused no discernible changes in the electrophoretic scanning patterns of the uninfectsed bacterial extracts. The electrophoretic analyses of the extracts of “eclipse” bacteria, obtained in three separate experiments, are presented in Fig. 3. The patterns are seen to be quite similar in gross aspect to those obtained upowelectrophoresis of the extractsof uninfected bacteria. Thesescanning patterns were quantitatively analyzed as to relative area distribution
286
SIEGEL,
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AND
WILDMAN
and sector mobility in the same manner as that described above for the patterns of the extracts of uninfected bacteria. The data so obtained are presented in Table I, where it is again seen that the distribution of soluble high-molecular-weight components has not been grossly altered during the “eclipse” stage of infection. However, there is a difference between the patterns of the extracts of the two types of bacteria which is marked and which has been confirmed in numerous experiments. There is a de-
AIL I
c
FIG. 3. Electrophoretic scanning patterns of extrwts of bacteria in the “eclipse” stage of infection. Descending patterns are shown on the left; ascending, right. The three patterns represent three independently prepared extracts.
crease in the fastest moving, a and a’, components in the scanning patterns of the extracts of the “eclipse” bacteria.
The Significance of the Decrease of the a,and a’ Sectors of the Electrophoresis Patterns of the Extracts of “Eclipse” Bacteria The apparent mobility of the a component, about -18 X 10e6 cm./sec./v./cm., suggested that it might be due t.o free desoxypentose nucleic acid (DNA),
since this very large anoclic mobi1it.y is characteristic
E.
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COMPONENTS
287
of thymus DNA examined under similar conditions (11). In order to investigate this possibility directly, it was decided to remove samples from appropriate sections of the cell after an electrophoresis experiment and analyze these samples for DNA content. Accordingly, Expts. 63 (normal) and 64 (infected) were performed simultaneously, and electrophoresis was permitted to continue for 1 hr. longer than the usual 2-hr. migration time in order to increase the resolution among the components. The electrophoresis diagrams at the end of 3 hr. are shown in Fig. 4. Then, with each experiment, a fine metal capillary was carefully lowered by means of a fixed rack and pinion device into the ascending limb of each cell, and accurately positioned
FIG. 4. Electrophoretic patterns of Expts. 63 (normal bacteria, lower patterns) and 64 (infected bacteria, upper patterns). Vertical lines above the patterns indicate the positions to which capillary was brought for removal of sample. Capillary entered cell in direction indicated by arrows contiguous to these lines. Arrows below the patterns indicate the direction of migration and the starting positions. Descending patterns are on the left, ascending on the right.
with respect to the schlieren boundaries in the limb with the aid of the optical system. The location of the capillary in the ascending limb is, indicated in Fig. 4. The capillary was placed so that appreciable amounts of only the fastest-migrating (a) component were withdrawn, The boundaries in the cell were observed during the withdrawal of the fraction, and no appreciable disturbance occurred below the tip of the capillary. The withdrawal of the sample was continued until the a-component boundary descended to the tip of the capillary. The volumes of the samples so removed were within about 35!&of one another in the two experiments, as is indicated by the weights of the fractions given in col. 2 of Table II. The results of DNA analyses given in ~01s.3 and 4
288
SIEGEL,
SINGER
AND
WILDMAN
of Table II indicate that there was about half as much DNA in this ascending limb fraction from the infected extract as from the normal. Furthermore, over 80% of the nitrogen of sector a can be accounted for as DNA nitrogen in both the normal and infected samples. This finding confirms the hypothesis that the a component in these extracts is largely freely migrating DNA, and that this free DNA is diminished relative to the total nitrogen of the extract in the infected sample as compared with the normal. At the same time, samples were removed from the descending limbs of the two experiments in a similar manner. The capillary was lowered a safe distance short of the a’-component boundary, as indicated in Fig. 4. The volumes of the fractions withdrawn from the two descending TABLE DNA
Contents
II
of Electrophoresis
Fractions Descending fraction
Ascending fraction Expt.
No. 63: normal No. 64: “eclipse”
wt. of %Ullple withdrawn
DNA/ml.
Et$ fraction/ 1 N in tota extracr
wt. of SCUllpIe
withdrawn
8.
Pg.
4%.
g.
0.95 0.92
330 170
240 110
1.42 1.39
DNA/ml.
DNA of isolated fraction/ N in totaT extracr
le.
KS.
140 200
106 130
a No, 63 contained 1.39 mg. N/ml.; No. 64, 1.55 mg. N/ml. The figuresin ~01s. 4 and 7 are therefore corrected for this small difference in N contents of the original solutions.
limbs were within 3% of one another. Any significant amount of DNA found in these fractions undoubtedly migrated more slowly than free DNA, and was presumably bound to protein (see below). The results of DNA analyses for these fractions are given in ~01s.6 and 7 of Table II, and they indicate that somewhat more DNA was present in this descending limb fraction in the infected sample than in the normal. Enough of the normal extract No. 63 was available to repeat the electrophoresis experiment with it in an independent run. Fractions were removed in as similar a manner as possible to that already described, and the results of the DNA analyses for the ascending and descending limb fractions were similar to those given in the first row of Table II. This indicates that the sampling technique was adequately reproducible and reliable.
E.
COLI
289
COMPONENTS
The Behavior of Extracts of Normal and Infected Bacteria Upon Analytical Ultracentrifugation4
The decrease in the amount of freely migrating DNA during the “eclipse” phase of infection was also observed when the extracts of normal and “eclipse” bacteria were examined in the analytical ultracentrifuge. The sedimentation diagrams are presented in Fig. 5, and the sedimentation constants of the various peaks are recorded in Table III. We have not yet investigated the relationships between the various peaks in the ultracentrifuge patterns in a given extract and the peaks in the corresponding electrophoresis diagrams, except for the case of freely migrating DNA. The sharp, spikelike peak labeled DNA in the ultra-
b lid nil [II 1250
580
380
FIG. 5. Ultracentrifuge patterns of extracts of: (a) normal bacteria, (5) infected “eclipse” bacteria. Sedimentation proceeds to the left, and the numbers under the diagrams indicate the time in seconds after the rotor attained full speed.
centrifuge diagrams of Fig. 5 is significantly shorter in the ultracentrifuge diagram of the extract of “eclipse” bacteria than in the normal. The material represented by this sharp boundary has been identified in other experiments as free DNA (13). Any quantitative estimate of the diminution of this peak, however, is rendered difficult by the sharpness of the peak. Because only a few experiments have as yet been performed in the ultracentrifuge, the sedimentation patterns were not divided into sectors 4 Preparations of the soluble high-molecular-weight components of E. coli and other bacteria have been made independently by Pardee et al. (12). The ultracentrifuge diagrams obtained by these authors for their preparations are practically identical with those presented in this paper.
290
SIEGEL,
SINGER
AND ~ILDM.~N
for quantitative area measurements. As in the electrophoresis experiments, the patterns of the normal and “eclipse” extracts appear similar in gross aspects. It is not yet known whether differences observed in the two patterns other than in the DNA peaks are reproducible and significant. The Release of DNh from Intracellular
Bacteriophage Particles
An attempt was made to detect gross changes in the extracts of bacteria which had been infected for longer than the “eclipse” period, and which consequently contained intact phage particles. Since cyanide causes bacteria to lyse during this advanced stage of infection, the bacteria were rapidly collected in the Sharples supercentrifuge without prior poisoning. It is possible to examine the extracts of bacteria which have been infected for longer than the normal latent period of 45 min. TABLE Sedimentation
Constants
III
of the High-Molecular-Weight Extracts
Components
oj Bacterial
Expt.
No. 65, normal.. No. 66, “eclipse”.
since the wild-type strain of T2 shows the phenomenon of lysis inhibition (14). Figure 6 illustrates the electrophoretic scanning patterns of an extract of bacteria which had been infected for an average of 100 min., where it can be seen that there has been a striking increase in the area occupied by the a and a’ sectors. Since intact phage particles were present in the bacteria at the time of disruption, the question arose as to whether this increase in the a and a’ sectors resulted from a bacterial product or through the disruption of intracellular phage particles. That the a and a’ sectors did not represent intact phage particles was clear from the high mobility of these components and also from the fact that intact phage particles would have been sedimented from suspension during the preparation of the extract. To test the possibility that the increase in the a and a’ sectors in this instance was at least in part due to a phage
E. COLI COMPONENTS
291
disruption product, a carboy lot of washed, uninfected bacteria was mixed with a suspension of phage particles in the ratio of 1 bacterium to 100 phage particles just prior to colloid mill treatment. When the extract of this mixture was prepared according to the standard procedure used throughout these experiments, a solution was obtained which upon electrophoresis yielded a scanning pattern which was similar to that observed for extracts of bacteria infected for 100 min. in that a marked increase in the a and a’ components was evident, indicating that the increase was at least in part due to a phage disruption product. Purified phage concentrates themselves were then submitted to colloid mill t,reatment. A component was isolated from the disruption products of bacteriophage with the same mobility characteristics as the a and a’
CL FIG. 6. Electrophoretic scanning for 100 min. The descending patt,ern
pattern of an ext.rac’t of bacteria is on the left; ascending, right.
infected
components present in the extracts of bacteria infected for 100 min. This isolated component was identified as phage DNA (13). DISCUSSION
If we consider first the results obtained with uninfected bacteria, it appears that the DNA may be roughly divided into two categories, “free” and “bound,” on the basis of electrophoretic mobility. DNA found in a, region of the electrophoresis cell out of which (in a noninteracting system) free DNA should have migrated, is termed “bound.” The significance of the distinction between “free” and “bound” DNA is not precisely known at present. Several possible explanations may be suggested: (a) The “bound” DNA may actually be part of a nucleic acidproteiri complex which is not in rapid exchange with the free DNA; (b) all of the bacterial DNA may be in equilibrium with a nucleic acidprotein complex, such t.hat under a particular set of conditions a certain amount of DNA is free, and the rest bound. If such an equilibrium
292
SIEGEL,
SINGER
IND
WILDMAN
existed, interactions such as have been noted with mixtures of nucleic acids and albumins (15, 16) might have been observed during electrophoresis of the bacterial extracts although these effects might be obscured by the large amounts of noninteracting proteins in the extracts. At the pH and ionic strength, 6.8 and 0.1, respectively, at which our experiments were performed, however, it is doubtful that such an equilibrium could exist; (c) it must further be appreciated that the physicalchemical conditions in the extract may be different from those within the bacteria, and, as a consequence,the amounts of “free” and “bound” DNA in the extracts, while reproducible and significant, may not reflect the actual DNA distribution within the bacteria. Nonetheless, while the effects mentioned in (b) and (c) might be of importance, these would complicate the interpretation of, but not detract from the significance of, the observed differences in the amounts of “free” and “bound” DNA in extracts of “eclipse’‘-infected bacteria as compared with the amounts of “free” and “bound” DNA in normal extracts. Extracts from “eclipse’‘-infected bacteria at the same total N content as normal samples reveal a striking decreasein the “free” DNA fraction without any corresponding decrease in the “bound” DNA. Cohen (3) has demonstrated that in infected bacteria there is an increased net synthesis of protein N relative to the net synthesis of DNA as compared with uninfected bacteria. It might be expected, therefore, that if infected and normal extracts were compared at the same total N content, that less f&l DNA would be found in the infected sample than in the normal. This effect, however, (which we might term “the protein dilution of the DNA”) cannot be the sole explanation of the results which we have obtained, since the amount of “bound” DNA in the infected sample, as compared with the normal, did not decrease in the same proportion as did the “free” DNA. The methods of preparing bacterial extracts which are described in this paper should be of interest in a variety of problems dealing with the high-molecular-weight components of bacteria. In this paper, these methods have been applied in a preliminary study to the problem of viral synthesis. A refinement of these methods in combination with other techniques can be employed in an effort to assessthe significance of the change in the nucleic acid economy of the bacterium during the early stages of virus infection.
E. COLI COMPONENTS
293
SUMMARY
1. Methods have been developed for the extraction of the soluble high-molecular-weight components of Escherichia COG.The extracts have been characterized by electrophoretic and ultracentrifugal analysis. 2. Soluble high-molecular-weight components were extracted from bacteria infected with the T2 strain of bacteriophage in the “eclipse” stage of infection. Electrophoretic comparison of these extracts at the same total nitrogen content with those of normal uninfected bacteria suggests that the amount of “free” desoxypentose nucleic acid decreases during the early stages of infection without a corresponding decrease in the amount of “bound” desoxypentose nucleic acid. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
E~LLIS, E. L., AND DELBR~~CK, M., J. Gen. Physiol. 22, 365 (1939). DOERMANN, A. H., Carnegie Inst. Wash. Yrbk. 47, 176 (1948). COHEN, S. S., Federation Proc. 10, 585 (1951). LABAW, L. W., MOSLEY, V. M., AND WYKOFF, R. W. G., J. Back 80,511 (1950). KOZLOFF, L., PUTNAM, F. W., AND EVANS, E. A., JR., in Viruses 1950, pp. 55-63. Calif. Inst. of Tech. Pasadena, 1950. SWINQLE, S., Rev. Sci. Instruments 18, 128 (1947). SINGER, S. J., AND CAMPBELL, D. H., J. Am. Chem. Sot. 74, 1794 (1952). ALLEN, R. J. L., Biochem. J. 34, 858 (1940). DISCHE, Z., Proc. Sot. Ezptl. Biol. Med. 66, 217 (1944). BONNER, J., AND WILDMAN, S. G., Arch. Biochem. 10, 497 (1946). STENHAGEN, E., AND THEORELL, T., Trans. Faraday Sot. 36, 743 (1939). SCHACHMAN, H. K., PARDEE, A., AND STANIER, R., Arch. Biochem. Biophys. 38, 245 (1952). SIEGEL, A. AND SINGER, S. J., Biochim. et Biophys. Acta, in press. DOERMANN, A. H., J. Bact. 66, 257 (1948). LONGSWORTH, L. G., AND MACINNES, D. A., J. Gen. Physiol. 26, 607 (1942). GOLDWASSER, E., AND PUTNAM, F. W., J. Phys. & Colloid Chem. 64.79 (1950).