- Email: [email protected]
Infection of protoplasts of Escherichia coli by bacteriophage ∅X174 treated with specific antibody
506
DISCUSSION Infection
of Protoplasts
by Bacteriophage with Specific
of Escherichia
AND
PRELIMINARY
co/i
+X174 Treated Antibody]
Protoplasts of bacteria are resistant to infection by intact bacteriophage (1) but are sensitive to infection by subviral particles (I-3). The mechanism of infection depends on the ability of the bacteriophage nucleic acid to penetrate bacterial cells without cell wall. The occurrence (3-8) of infectious nucleic acid (INA) in both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses introduced a new dimension in virology (9). Herriott (9) considered INA as a possible mechanism to account for the occurrence of virus activity in vivo and in vitro in the presence of excess specific antibody. In the present, paper experiments on surviving phage in the +X174-antiphage system are described, the results of which demonstrate the ability of antiserum-treated $X174 to infect protoplasts of Escherichia coli K12S which are normally resistant to untreated +X174. A stock suspension of highly purified bacteriophage +X174, containing only 114 S particles, was diluted 1:200 in an aqueous solution of 0.1 M KC1 plus 10V3 M tris(hydroxymethyl) aminomethane (KCl-Tris buffer), pH 8.1. Rabbit antiserum (K = 3000 minute-l) was diluted 4.5 x lo4 times in the KCl-Tris buffer. One-tenth milliliter of t,he diluted phage was added to 9.9 ml of the diluted, prewarmed antiserum solution, and the mixture was incubated at 37” for 5 hours. Two control mixtures were also incubated at 37” for 5 hours. The first (No. 1) contained 0.1 ml of the diluted phage plus 9.9 ml of the KClTris buffer, and the second control (No. 2) contained 0.1 ml of the phage plus 9.9 ml normal rabbit serum diluted 4.5 x lo4 times in the KCl-Tris buffer. At the end of the 5hour incubation period, 0.5 ml of 0.1 M Tris was added to 4.5 ml of the phage-antiphage reaction mixture in order to increase the 1 Supported in part by Public Health Service Training Grant No. 23-162. Presented in part at the annual meeting of the American Association of Immunologists, Atlantic City, New Jersey, ,4pril, 1963.
REPORTS
Tris concentration to 0.01 M. To reduce the phage concentration in the phage control suspensions to a level approximating that in the phage-antiphage mixture, the suspensions were diluted 1: 70 in 0.01 M Tris. Some loss of activity occurred spontaneously. Two milliliters of the adjusted (0.01 M Tris) phage-antiphage mixture was then added to 2 ml of a stock E. co& K12S protoplast suspension containing approximately 4 X lo8 protoplasts ml-l. The protoplasts were prepared by lysozyme and disodium ethylenediamine tetraacetate treatment (3). This material, designated Adsorption Mixture A, was incubated at 37”. Adsorption Mixture B consisted of 2 ml of phage control No. 1 suspension plus 2 ml of the stock protoplast suspension. Adsorption Mixture C was identical to Adsorption Mixture B except that it contained control phage suspension No. 2. These were also incubated at 37”. After 10 minutes a sample taken from Adsorption Mixture A was diluted 1:5 in prewarmed protoplast nutrient broth (PNB) (3) and labeled Growth Mixture A. At. the same time another sample was diluted 1:lO in PNB with 2% bovine serum albumin (BSA), and a third sample was diluted 1 :lO in chloroform-water (1 ml chloroform plus 25 ml water). To determine the total number of infectious centers in Adsorption Mixture A, 0.1 ml of t.he PNB-BSA-diluted mixture was plated with E. coli C in 2.5 ml of prot,oplast top agar (3). One-tenth milliliter of the chloroform-water diluted mixture was plated as usual to determine the number of free phages in Adsorption Mixture A. The contents of Adsorption Mixtures B and C were treated exactly as described for Adsorption Mixture A. Eighty minutes after preparat,ion, Growth Mixt’ures A, B, and C were assayed for their content of phage. The results of a typical experiment are shown in Fig. 1. It can be seen that after 10 minutes of incubation, Adsorption Mixture A cont,ained 0.4 X lo3 infected protoplasts ml-l and that after 80 minutes of incubation Growth Mixture A contained 2.97 X lo5 plaque-formers ml-r (relative to Adsorption Mixture A). This represented an average yield of approximately 750
DISCUSSION AND PRELIMINARY
Diluted
REPORTS
I:2
Diluted
Adsorption
Yield
6
4
Mixture
A
60' at 37. C: 5.95 x IO4 *5 2.97x IO'
= 750
I:2
Mixture
J Growth After
507
per
Infected
PROTOPLAST
Growth After
Mixture
B
60' at 37O C: 5.37 x IO3 x5 2.68 x IO4 Yield
= ZERO
FIG. 1. Infection of Escherichia coli K12S protoplasts with surviving bacteriophage +X174. PNB = protoplast nutrient broth with 2% bovine serum albumin. CHCl, = chloroform water.
plaque-formers per infected protoplast. On the other hand, Adsorption Mixture B contained no infected protoplasts after 10 minutes’ incubation and the amount of phage did not increase. Similarly, no infection of protoplasts was observed in Adsorption Mixture C and no growth occurred in Growth Mixture C (not shown in Fig. 1). These results indicate that the protoplasts were not infected by the control phage which had not been exposed to specific antiserum. This experiment was repeated three times with essentially the same results. The antiserum-treated phage was exposed to DNase to test the effect of this enzyme on the ability of the serum-treated phage to infect E. coli K12S protop1ast.s. Pancreatic DNase in either distilled water or 0.05 M Tris buffer, pH 7.1, was incubated at a concentration of 0.25, 2.5, and 25 pg ml-l with control phage and with serumtreated phage at 37” for intervals up to 120 minutes. DNase at, these concentrations had no effect on the infectivity of either untreated phage or of serum-treated phage. Previous studies have shown that INA from various sources will infect cells nor-
mally resistant to intact virus (3, 7, 10). Several investigators (3, 7, 8) have shown that DNA chemically isolated from bacteriophage +X174 is infectious for strains of E. cc& normally resistant to intact +X174. The results obtained in the present investigation showed clearly that a fraction of +X174 treated with specific antiserum can infect E. coli K12S protoplasts. The protoplasts were insusceptible to infection with +X174 that had not been treated previously with specific antiserum. The resistance of the serum-treated phage to DNase indicates that the material which was infectious for K12S protoplasts must not be free DNA. Possibly this material is DNA associated with some viral protein component. Recently we have shown that .+X174 bacteriophage surviving neutralization by specific antiserum is sensitive to almost complete inactivation by lupus erythematosus serum which probably contained antiDNA antibodies (to be published). The results of the present experiments and of those with lupus erythematosus sera support the hypothesis that a change occurs in the phys-
ical state of at least some of the phagc cluring neutralization, resulting in either exposure or partial liberation of DKA. Marc generally, Ih’A, either partially liberated or in damaged capsids, arising through contact of a virus with specific antibody, may be responsible for viral infectivity in the presence of antibody, thus explaining a number of clinical conditions presently considered to be anomalous. REFERENCES 1. FRASER, D., MAHLER, H. R., SHUG, A. L., and THO~IAS, ,A. C., JR., Proc. Natl. Acad. Sci. U.S.
43,939-947 (1957). 2. LEDERBERO, J., and ST. CLAIR,
J., J. Bnctcriol.
75,143-160 (1958). G. D., and SIXSHEIMER, R. I,., 1. Mol. Biol. 2, 297-305 (1960). 4. GIERER, A., and SCHRAMM, G., Nature 177, 7023. GUTHRIE,
703 (1956). 5. COLTER, J. S., BIRD,
Nature
H. H., and BROUTK;,
R. A.,
179,859~860 (1957).
6. ALEXANDER, H. E., KOCH, G., MOUNTAIN, I. M., and VAN DAMME, O., J. Exptl. Med. 108, 493-
506 (1958). M., TAKETO, 8., and TAKAGI, Y., Biophys. Acta 45, 199-200 (1960). 8. HUPPERT, J., WAHI,, R., and EMERIQGE-BLUU, I+ Biochim. Biophus. Acta 55, W-291 (1961). 9. HERRIOTT, R. M., Science 134, 256-260 (1961). 10. HOLLAND, J. J., MCLAREI\‘, L. C., and STVERTOPI’, J. T., Proc. Sot. Exptl. Biol. Med. 100, 843845 (1959). BERNARD U. BOWMAN, JH.~ ROBERT A. PATNoDe Departmrut of Microbiology University oj Oklahoma Medical Center Oklahoma City, Oklahoma Accepted August S, 1963 7. SEKIWCI~I,
Biochzm.
?Supported by a United States Public Health Service Predoetoral fellowship. This paper is taken from a dissertation submitted to the Graduate School, University of Oklahoma Medical Center, in May, 1963, in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Conservation an Abortive
of Vaccinial Cycle
DNA during
of Multiplication
5 - Fluorodeoxyuridine (FUDR) inhibits the synthesis of DNA by interfering with the enzyme that controls the conversion of synthesis has proved useful for distinguish-
ing DNA viruses from tllorc that contain RNA (g-4) and also for the kinetic analysis of DNA synt~hesis in cells infected with the former viruses (2, 5, 6). In sucli infect4 cells, it might be cxpect,cd that inhibition of DNA synthesis would lead to a failure of infect,ious virus production and that there would, in consequence, be no cmcrgcnce of virus from “eclipse.” Examination of published results in the case of vaccinia virus (which cont,ains DNA) reveals, however, that a variable amount of this virus may be formed in the presence of FUDR (2, 3, 7). Since failure to achieve any inhibition at all has also been reported (,7), it seemed possible that virus production, if it occurred, might be due to the presence of thymidine in the ccl1 environment. Even in medium lacking thymidine, however, the author has consistently obtained yields of vaccinia virus, in the presence of FUDR, of the same order as those associated with the infected cells at the start of the experiment. It was considered of interest, to determine whether such a result was due to the persistence of the infecting virus in an unalt,ered form, or could be correlated with de no~o DXA synthesis permitted either by TABLE
1
EFFECT OF FUDR ox THE SYNTHESIS DNA AND THE PR~DIJCTION OF INFECTIOUS VIRUS Titer of virusa (PFU/ml X 103)
Incubation
OF
CPM”
time
(hours) Control
FUDR
Azide (lo-2 Y)
4B 4.2 4.5 24
xi
Control
FUDR
~~
0 G 11
24
46 5 50 240
5401 11280
4
-
9693
NSr iv!
i&
~1Plaque-forming units on chick embryo fibroblast monolayers. Virus from 8.3 X lo* cells. h Counts per minute over background (420 per minut,e). Mat.erial from 7.5 X lo5 cells. Cells were fixed with alcohol-acetic, treated with 0.01% RNase for 30 minutes at 37”, washed three times with cold 57, trichloroacetic acid, resuspended in water, and sonically disrupted. Counting was carried out in a liquid scintillation counter for a ueriod of 5 minutes per sample. c NS = Not significantly greater than barkground.
DISCUSSION Infection
of Protoplasts
by Bacteriophage with Specific
of Escherichia
AND
PRELIMINARY
co/i
+X174 Treated Antibody]
Protoplasts of bacteria are resistant to infection by intact bacteriophage (1) but are sensitive to infection by subviral particles (I-3). The mechanism of infection depends on the ability of the bacteriophage nucleic acid to penetrate bacterial cells without cell wall. The occurrence (3-8) of infectious nucleic acid (INA) in both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses introduced a new dimension in virology (9). Herriott (9) considered INA as a possible mechanism to account for the occurrence of virus activity in vivo and in vitro in the presence of excess specific antibody. In the present, paper experiments on surviving phage in the +X174-antiphage system are described, the results of which demonstrate the ability of antiserum-treated $X174 to infect protoplasts of Escherichia coli K12S which are normally resistant to untreated +X174. A stock suspension of highly purified bacteriophage +X174, containing only 114 S particles, was diluted 1:200 in an aqueous solution of 0.1 M KC1 plus 10V3 M tris(hydroxymethyl) aminomethane (KCl-Tris buffer), pH 8.1. Rabbit antiserum (K = 3000 minute-l) was diluted 4.5 x lo4 times in the KCl-Tris buffer. One-tenth milliliter of t,he diluted phage was added to 9.9 ml of the diluted, prewarmed antiserum solution, and the mixture was incubated at 37” for 5 hours. Two control mixtures were also incubated at 37” for 5 hours. The first (No. 1) contained 0.1 ml of the diluted phage plus 9.9 ml of the KClTris buffer, and the second control (No. 2) contained 0.1 ml of the phage plus 9.9 ml normal rabbit serum diluted 4.5 x lo4 times in the KCl-Tris buffer. At the end of the 5hour incubation period, 0.5 ml of 0.1 M Tris was added to 4.5 ml of the phage-antiphage reaction mixture in order to increase the 1 Supported in part by Public Health Service Training Grant No. 23-162. Presented in part at the annual meeting of the American Association of Immunologists, Atlantic City, New Jersey, ,4pril, 1963.
REPORTS
Tris concentration to 0.01 M. To reduce the phage concentration in the phage control suspensions to a level approximating that in the phage-antiphage mixture, the suspensions were diluted 1: 70 in 0.01 M Tris. Some loss of activity occurred spontaneously. Two milliliters of the adjusted (0.01 M Tris) phage-antiphage mixture was then added to 2 ml of a stock E. co& K12S protoplast suspension containing approximately 4 X lo8 protoplasts ml-l. The protoplasts were prepared by lysozyme and disodium ethylenediamine tetraacetate treatment (3). This material, designated Adsorption Mixture A, was incubated at 37”. Adsorption Mixture B consisted of 2 ml of phage control No. 1 suspension plus 2 ml of the stock protoplast suspension. Adsorption Mixture C was identical to Adsorption Mixture B except that it contained control phage suspension No. 2. These were also incubated at 37”. After 10 minutes a sample taken from Adsorption Mixture A was diluted 1:5 in prewarmed protoplast nutrient broth (PNB) (3) and labeled Growth Mixture A. At. the same time another sample was diluted 1:lO in PNB with 2% bovine serum albumin (BSA), and a third sample was diluted 1 :lO in chloroform-water (1 ml chloroform plus 25 ml water). To determine the total number of infectious centers in Adsorption Mixture A, 0.1 ml of t.he PNB-BSA-diluted mixture was plated with E. coli C in 2.5 ml of prot,oplast top agar (3). One-tenth milliliter of the chloroform-water diluted mixture was plated as usual to determine the number of free phages in Adsorption Mixture A. The contents of Adsorption Mixtures B and C were treated exactly as described for Adsorption Mixture A. Eighty minutes after preparat,ion, Growth Mixt’ures A, B, and C were assayed for their content of phage. The results of a typical experiment are shown in Fig. 1. It can be seen that after 10 minutes of incubation, Adsorption Mixture A cont,ained 0.4 X lo3 infected protoplasts ml-l and that after 80 minutes of incubation Growth Mixture A contained 2.97 X lo5 plaque-formers ml-r (relative to Adsorption Mixture A). This represented an average yield of approximately 750
DISCUSSION AND PRELIMINARY
Diluted
REPORTS
I:2
Diluted
Adsorption
Yield
6
4
Mixture
A
60' at 37. C: 5.95 x IO4 *5 2.97x IO'
= 750
I:2
Mixture
J Growth After
507
per
Infected
PROTOPLAST
Growth After
Mixture
B
60' at 37O C: 5.37 x IO3 x5 2.68 x IO4 Yield
= ZERO
FIG. 1. Infection of Escherichia coli K12S protoplasts with surviving bacteriophage +X174. PNB = protoplast nutrient broth with 2% bovine serum albumin. CHCl, = chloroform water.
plaque-formers per infected protoplast. On the other hand, Adsorption Mixture B contained no infected protoplasts after 10 minutes’ incubation and the amount of phage did not increase. Similarly, no infection of protoplasts was observed in Adsorption Mixture C and no growth occurred in Growth Mixture C (not shown in Fig. 1). These results indicate that the protoplasts were not infected by the control phage which had not been exposed to specific antiserum. This experiment was repeated three times with essentially the same results. The antiserum-treated phage was exposed to DNase to test the effect of this enzyme on the ability of the serum-treated phage to infect E. coli K12S protop1ast.s. Pancreatic DNase in either distilled water or 0.05 M Tris buffer, pH 7.1, was incubated at a concentration of 0.25, 2.5, and 25 pg ml-l with control phage and with serumtreated phage at 37” for intervals up to 120 minutes. DNase at, these concentrations had no effect on the infectivity of either untreated phage or of serum-treated phage. Previous studies have shown that INA from various sources will infect cells nor-
mally resistant to intact virus (3, 7, 10). Several investigators (3, 7, 8) have shown that DNA chemically isolated from bacteriophage +X174 is infectious for strains of E. cc& normally resistant to intact +X174. The results obtained in the present investigation showed clearly that a fraction of +X174 treated with specific antiserum can infect E. coli K12S protoplasts. The protoplasts were insusceptible to infection with +X174 that had not been treated previously with specific antiserum. The resistance of the serum-treated phage to DNase indicates that the material which was infectious for K12S protoplasts must not be free DNA. Possibly this material is DNA associated with some viral protein component. Recently we have shown that .+X174 bacteriophage surviving neutralization by specific antiserum is sensitive to almost complete inactivation by lupus erythematosus serum which probably contained antiDNA antibodies (to be published). The results of the present experiments and of those with lupus erythematosus sera support the hypothesis that a change occurs in the phys-
ical state of at least some of the phagc cluring neutralization, resulting in either exposure or partial liberation of DKA. Marc generally, Ih’A, either partially liberated or in damaged capsids, arising through contact of a virus with specific antibody, may be responsible for viral infectivity in the presence of antibody, thus explaining a number of clinical conditions presently considered to be anomalous. REFERENCES 1. FRASER, D., MAHLER, H. R., SHUG, A. L., and THO~IAS, ,A. C., JR., Proc. Natl. Acad. Sci. U.S.
43,939-947 (1957). 2. LEDERBERO, J., and ST. CLAIR,
J., J. Bnctcriol.
75,143-160 (1958). G. D., and SIXSHEIMER, R. I,., 1. Mol. Biol. 2, 297-305 (1960). 4. GIERER, A., and SCHRAMM, G., Nature 177, 7023. GUTHRIE,
703 (1956). 5. COLTER, J. S., BIRD,
Nature
H. H., and BROUTK;,
R. A.,
179,859~860 (1957).
6. ALEXANDER, H. E., KOCH, G., MOUNTAIN, I. M., and VAN DAMME, O., J. Exptl. Med. 108, 493-
506 (1958). M., TAKETO, 8., and TAKAGI, Y., Biophys. Acta 45, 199-200 (1960). 8. HUPPERT, J., WAHI,, R., and EMERIQGE-BLUU, I+ Biochim. Biophus. Acta 55, W-291 (1961). 9. HERRIOTT, R. M., Science 134, 256-260 (1961). 10. HOLLAND, J. J., MCLAREI\‘, L. C., and STVERTOPI’, J. T., Proc. Sot. Exptl. Biol. Med. 100, 843845 (1959). BERNARD U. BOWMAN, JH.~ ROBERT A. PATNoDe Departmrut of Microbiology University oj Oklahoma Medical Center Oklahoma City, Oklahoma Accepted August S, 1963 7. SEKIWCI~I,
Biochzm.
?Supported by a United States Public Health Service Predoetoral fellowship. This paper is taken from a dissertation submitted to the Graduate School, University of Oklahoma Medical Center, in May, 1963, in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Conservation an Abortive
of Vaccinial Cycle
DNA during
of Multiplication
5 - Fluorodeoxyuridine (FUDR) inhibits the synthesis of DNA by interfering with the enzyme that controls the conversion of synthesis has proved useful for distinguish-
ing DNA viruses from tllorc that contain RNA (g-4) and also for the kinetic analysis of DNA synt~hesis in cells infected with the former viruses (2, 5, 6). In sucli infect4 cells, it might be cxpect,cd that inhibition of DNA synthesis would lead to a failure of infect,ious virus production and that there would, in consequence, be no cmcrgcnce of virus from “eclipse.” Examination of published results in the case of vaccinia virus (which cont,ains DNA) reveals, however, that a variable amount of this virus may be formed in the presence of FUDR (2, 3, 7). Since failure to achieve any inhibition at all has also been reported (,7), it seemed possible that virus production, if it occurred, might be due to the presence of thymidine in the ccl1 environment. Even in medium lacking thymidine, however, the author has consistently obtained yields of vaccinia virus, in the presence of FUDR, of the same order as those associated with the infected cells at the start of the experiment. It was considered of interest, to determine whether such a result was due to the persistence of the infecting virus in an unalt,ered form, or could be correlated with de no~o DXA synthesis permitted either by TABLE
1
EFFECT OF FUDR ox THE SYNTHESIS DNA AND THE PR~DIJCTION OF INFECTIOUS VIRUS Titer of virusa (PFU/ml X 103)
Incubation
OF
CPM”
time
(hours) Control
FUDR
Azide (lo-2 Y)
4B 4.2 4.5 24
xi
Control
FUDR
~~
0 G 11
24
46 5 50 240
5401 11280
4
-
9693
NSr iv!
i&
~1Plaque-forming units on chick embryo fibroblast monolayers. Virus from 8.3 X lo* cells. h Counts per minute over background (420 per minut,e). Mat.erial from 7.5 X lo5 cells. Cells were fixed with alcohol-acetic, treated with 0.01% RNase for 30 minutes at 37”, washed three times with cold 57, trichloroacetic acid, resuspended in water, and sonically disrupted. Counting was carried out in a liquid scintillation counter for a ueriod of 5 minutes per sample. c NS = Not significantly greater than barkground.