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The ultraviolet action spectrum of a Bacillus megatherium bacteriophage
The Ultraviolet Action Spectrum of a Bacillus megatherium Bacteriophage’ R. M. Franklin2, M. Friedman, and R. B. Setlow From the Biophysics Division,
Sloane Physics Laboratory, Haven, Connecticut
Received
February
Yale University,
New
11, 1953
INTRODUCTION
The study of the relative effectiveness of various wavelengths of ultraviolet light in inactivating a virus may provide a useful physical index of an organism, yielding information on the structure of the virus and on the action of the light. Action spectra for many viruses have been reported in the literature (1, 2). The most detailed action spectrum reported is that of dried T-l (3), showing high inactivation at 2652 A. and 2805 A., but low inactivation at 1942 A. The virus M-5 has as its host several strains of Bacillus megatherium (4). It is viable after drying in low concentrations of broth. Because its physical properties and biological behavior are quite different from that of T-l, it is useful to compare the two action spectra. All bacterial viruses which have been chemically analyzed so far consist almost entirely of desoxyribonucleic acid and protein (5). In this paper we assume that M-5 has a chemical content similar to that of the other bacterial viruses although no chemical analyses have as yet been performed on any of the M series of phage. EXPERIMENTAL Either 0.02 or 0.05 ml. of a solution of M-5 in a 0.01% tryptose (Difco)-yeast extract (Difco) broth was dried down on glass cover slips in a vacuum desiccator. There was a lO-20% survival of phage in this drying process. There was no loss of titer in transferring the virus from the usual 1% broth to the 0.01% broth. When the 1942 A. Hg line was used, the virus was dried in O.OOl~O broth to keep absorption at a minimum. Even at this low concentration the survival after drying was 1 This investigation was supported in part by the Office of Saval 2 National Science Foundation Predoctoral Fellow. 259
Research.
260
FRANKLIN,
FRIEDMAN AND SETLOW
lO-20%. The phage titer was adjusted so that in the assay there would be 150-200 plaques per plate. The dried samples were placed in an irradiation chamber having a quartz window. All irradiations were done in a vacuum at room temperature (20-30°C.). After irradiation the samples were dissolved in 1 ml. of 1% broth and plated versus the host B. megatherium by a technique similar to that used for the T-series of phage (4). All plates were done in duplicate. The ultraviolet monochromator used in these experiments had a medium-pressure mercury arc source, two aluminum mirrors of 10 in. diameter, and a large water prism with thin quartz faces of 10 in. diameter (3). A GL-935 phototube was used in measuring the light intensity. This was calibrated against a thermopile, and the thermopile calibrated against a standard carbon-filament lamp. The photocell response could thus be expressed in absolute intensity units as ergs/sq. mm./sec. Intensity values ranged from 9 ergs/sq. mm./sec. at 2652 A. to 0.1 erg/sq. mm./sec. at 1942 A. Several corrections must be made to the measured energy values. The quartz window on the radiation chamber causes an energy loss due to reflection, amounting to approximately 10% at each wavelength. Secondly, a correction was made for the absorption by the small amount of dried broth present. At 1942 A., 40% of the apparent absorption was due to scattered light, and an appropriate correction was made. Five micrograms of broth was dried down on a quartz cover slip, and its absorption spectrum was measured with a Beckman spectrophotometer. The maximum absorption found for the 0.01% broth film was 30% at 2260 A. At 2650 A. the absorption was 29%. The O.OOl~Obroth film absorbed about 10% at 1942 A. Errors due to nonuniformity in the broth layer are small and since they enter into a correction term which is itself quite small, they may be neglected. RESULTS AND DISCUSSION
Figure 1 shows typical dose-survival curves, which follow an exponential relationship, at least to survival values of 10 %. The absorption due to the broth makes the survival curves deviate from the exponential survival values. The expression for the curve is ln(n/no) = --a~ where n is the number of survivors after a dose of energy E ergs/sq. mm.; 120 is the initial number; and 0 is a constant for a given wavelength, known as the inactivation cross section. At 37% survival, ln(n/n,) = -1 so that u = l/tg~. The expression assumes the use of a uniform beam of light. Reciprocity of time and intensity was assumed for this experiment since it is known to hold for the inactivation of T-l phage (6). The action spectrum can be expressed as the variation of u with wave-
length. For example, from Fig. 1, the 37% dose for X of 2700 A. is 147 ergs/sq. mm. Then u = l/147 = 6.81 X 10m3sq. mm/erg for X = 2700 A. The complete action spectrum is shown in Fig. 2. The prominent peaks are at 2537 and 2805 A., and the light becomes ineffective for in-
SPECTRUM
OF
BACILLUS
MEGATHERIUM
BACTERIOPHAGE
261
activation around 3ooO A. These peaks may be considered a resolution of the action on the nucleic acid (2537 A.) and the protein (2805 A.) components. These peaks are more pronounced in the M-5 action spectrum as compared to that of T-l (3). This may be due to the shift in
FIG. 1. Typical
survival
inactivation curves for M-5. The logarithm of the per cent is plotted versus the dose of ultraviolet light for several wavelengths.
the point of maximum sensitivity of the nucleic acid from 2650 A. in T-l to 2537 A. in M-5. Of especial interest is the large value of u at 1942 A. The work on T-l showed that this wavelength was quite ineffective in inactivating the virus (3). It is difficult to interpret these data in terms of virus structure
262
FRANKLIN,
FRIEDMAN
AND
SETLOW
since we do not know how far the low wavelength light penetrates into the virus. The high inactivation of M-5 could be due to the direct rupture of peptide bonds since the peptide absorption maximum is near 1942 A. (7).
pE .c
7-
bB 3 b3-
FIG. 2. The relative effectiveness of various wavelengths of ultraviolet light in inactivating M-5 bacteriophage. The vertical lines at 2706 and 2300 A. represent standard errors. The cross sections are plotted on a logarithmic scale since the shape of the curve is then independent of concentration.
The cross sections for T-l (3) are very much larger than the corresponding cross sections for M-5, the values differing by a factor of almost four in the region of 2690 A. in spite of the fact that M-5 is the larger of the two viruses.8 From the electron micrograph of M-5 (Fig. 3) the head diameter is 709 A. and the tail is 3999 X 159 A. It has a mass * The published values of the T-l ultraviolet inactivation cross sections indicate a sensitivity equal to that of M-5 (6). However, later work by Fluke (3), corroborated by one of the authors, showed that these values were too small by a factor of four.
SI’ECTRVM
OF
BACILLUS
MEG.4T~IERIT’M
B.\CTF,RIOPII.\GE
26.1
ttpproximately 3.0 times larger than T-l (8). Evidelltly size cannot, be used as a cariterion for radiation sensitivity of \jrusrs. Furthermore, t,he larger (WSS sections in T-l indicate a greater prc~lu~hilit~y of inactivation.
If the sensitivity of the individual wits is assumed to he the same wt’ must cwlwlrltle that there are more sellsiti\e units it1 T-1 thall in RL.3. Sevrrnl attempts \vere made to obtain a purified sample of M-5 t)>
2G4
FRANKLIN,
FRIEDMAN AND SETLOR
differential centrifugation. Unfortunately these were not successful so t,hat we could not get an absorption spectrum and therefore are unable t,o report. on t,he qlla.ntum efficiency of t’he inart8ivntjion. ACKNOWLEDGMENTS The authors wish to thank Dr. W. H. Gaylord, Jr., for his assistance in preparing the electron micrograph, and Professor E. C. Pollard for many helpful discussions. SUMMARY
The relative efficiency of the ultraviolet light for inactivating a dry bacterial virus (M-5) has been determined in the wavelength region 1942-3000 A. The data indicate that photons absorbed in protein and nucleic acid components can result in inactivation. From a comparison with T-l virus, it has been suggested that there are more sensitive units in T-l than in M-5. REFERENCES 1. 2. 3. 4. 5.
LOOFBOUROR,J. R., Growth, 12 (suppl.), 77 (1948). HOLLAENDER, A., AND OLIPHANT, J. W., J. Bacterial. 48, 447 (1944). FLUKE, D. J., Ph.D. Thesis, Yale Univ., New Haven, Conn., 1950. FRIEVMAN, M., J. Bacterial., in press. EVAXS, E. A., Biochemical Studies of Bacterial Viruses. The University of Chicago Press, Chicago, Ill., 1952. 6. FLUKE, D. J., AND POLLARD, E. C., Science 110, 274 (1949). 7. HAnI, J. S., AND PLATT, J. R., J. Chenz. Phys. 20, 335 11952). 8. WYCKOFF,R. W. G., Electron Microscopy. Interscience Pul)lishers, New York, 1949.
Sloane Physics Laboratory, Haven, Connecticut
Received
February
Yale University,
New
11, 1953
INTRODUCTION
The study of the relative effectiveness of various wavelengths of ultraviolet light in inactivating a virus may provide a useful physical index of an organism, yielding information on the structure of the virus and on the action of the light. Action spectra for many viruses have been reported in the literature (1, 2). The most detailed action spectrum reported is that of dried T-l (3), showing high inactivation at 2652 A. and 2805 A., but low inactivation at 1942 A. The virus M-5 has as its host several strains of Bacillus megatherium (4). It is viable after drying in low concentrations of broth. Because its physical properties and biological behavior are quite different from that of T-l, it is useful to compare the two action spectra. All bacterial viruses which have been chemically analyzed so far consist almost entirely of desoxyribonucleic acid and protein (5). In this paper we assume that M-5 has a chemical content similar to that of the other bacterial viruses although no chemical analyses have as yet been performed on any of the M series of phage. EXPERIMENTAL Either 0.02 or 0.05 ml. of a solution of M-5 in a 0.01% tryptose (Difco)-yeast extract (Difco) broth was dried down on glass cover slips in a vacuum desiccator. There was a lO-20% survival of phage in this drying process. There was no loss of titer in transferring the virus from the usual 1% broth to the 0.01% broth. When the 1942 A. Hg line was used, the virus was dried in O.OOl~O broth to keep absorption at a minimum. Even at this low concentration the survival after drying was 1 This investigation was supported in part by the Office of Saval 2 National Science Foundation Predoctoral Fellow. 259
Research.
260
FRANKLIN,
FRIEDMAN AND SETLOW
lO-20%. The phage titer was adjusted so that in the assay there would be 150-200 plaques per plate. The dried samples were placed in an irradiation chamber having a quartz window. All irradiations were done in a vacuum at room temperature (20-30°C.). After irradiation the samples were dissolved in 1 ml. of 1% broth and plated versus the host B. megatherium by a technique similar to that used for the T-series of phage (4). All plates were done in duplicate. The ultraviolet monochromator used in these experiments had a medium-pressure mercury arc source, two aluminum mirrors of 10 in. diameter, and a large water prism with thin quartz faces of 10 in. diameter (3). A GL-935 phototube was used in measuring the light intensity. This was calibrated against a thermopile, and the thermopile calibrated against a standard carbon-filament lamp. The photocell response could thus be expressed in absolute intensity units as ergs/sq. mm./sec. Intensity values ranged from 9 ergs/sq. mm./sec. at 2652 A. to 0.1 erg/sq. mm./sec. at 1942 A. Several corrections must be made to the measured energy values. The quartz window on the radiation chamber causes an energy loss due to reflection, amounting to approximately 10% at each wavelength. Secondly, a correction was made for the absorption by the small amount of dried broth present. At 1942 A., 40% of the apparent absorption was due to scattered light, and an appropriate correction was made. Five micrograms of broth was dried down on a quartz cover slip, and its absorption spectrum was measured with a Beckman spectrophotometer. The maximum absorption found for the 0.01% broth film was 30% at 2260 A. At 2650 A. the absorption was 29%. The O.OOl~Obroth film absorbed about 10% at 1942 A. Errors due to nonuniformity in the broth layer are small and since they enter into a correction term which is itself quite small, they may be neglected. RESULTS AND DISCUSSION
Figure 1 shows typical dose-survival curves, which follow an exponential relationship, at least to survival values of 10 %. The absorption due to the broth makes the survival curves deviate from the exponential survival values. The expression for the curve is ln(n/no) = --a~ where n is the number of survivors after a dose of energy E ergs/sq. mm.; 120 is the initial number; and 0 is a constant for a given wavelength, known as the inactivation cross section. At 37% survival, ln(n/n,) = -1 so that u = l/tg~. The expression assumes the use of a uniform beam of light. Reciprocity of time and intensity was assumed for this experiment since it is known to hold for the inactivation of T-l phage (6). The action spectrum can be expressed as the variation of u with wave-
length. For example, from Fig. 1, the 37% dose for X of 2700 A. is 147 ergs/sq. mm. Then u = l/147 = 6.81 X 10m3sq. mm/erg for X = 2700 A. The complete action spectrum is shown in Fig. 2. The prominent peaks are at 2537 and 2805 A., and the light becomes ineffective for in-
SPECTRUM
OF
BACILLUS
MEGATHERIUM
BACTERIOPHAGE
261
activation around 3ooO A. These peaks may be considered a resolution of the action on the nucleic acid (2537 A.) and the protein (2805 A.) components. These peaks are more pronounced in the M-5 action spectrum as compared to that of T-l (3). This may be due to the shift in
FIG. 1. Typical
survival
inactivation curves for M-5. The logarithm of the per cent is plotted versus the dose of ultraviolet light for several wavelengths.
the point of maximum sensitivity of the nucleic acid from 2650 A. in T-l to 2537 A. in M-5. Of especial interest is the large value of u at 1942 A. The work on T-l showed that this wavelength was quite ineffective in inactivating the virus (3). It is difficult to interpret these data in terms of virus structure
262
FRANKLIN,
FRIEDMAN
AND
SETLOW
since we do not know how far the low wavelength light penetrates into the virus. The high inactivation of M-5 could be due to the direct rupture of peptide bonds since the peptide absorption maximum is near 1942 A. (7).
pE .c
7-
bB 3 b3-
FIG. 2. The relative effectiveness of various wavelengths of ultraviolet light in inactivating M-5 bacteriophage. The vertical lines at 2706 and 2300 A. represent standard errors. The cross sections are plotted on a logarithmic scale since the shape of the curve is then independent of concentration.
The cross sections for T-l (3) are very much larger than the corresponding cross sections for M-5, the values differing by a factor of almost four in the region of 2690 A. in spite of the fact that M-5 is the larger of the two viruses.8 From the electron micrograph of M-5 (Fig. 3) the head diameter is 709 A. and the tail is 3999 X 159 A. It has a mass * The published values of the T-l ultraviolet inactivation cross sections indicate a sensitivity equal to that of M-5 (6). However, later work by Fluke (3), corroborated by one of the authors, showed that these values were too small by a factor of four.
SI’ECTRVM
OF
BACILLUS
MEG.4T~IERIT’M
B.\CTF,RIOPII.\GE
26.1
ttpproximately 3.0 times larger than T-l (8). Evidelltly size cannot, be used as a cariterion for radiation sensitivity of \jrusrs. Furthermore, t,he larger (WSS sections in T-l indicate a greater prc~lu~hilit~y of inactivation.
If the sensitivity of the individual wits is assumed to he the same wt’ must cwlwlrltle that there are more sellsiti\e units it1 T-1 thall in RL.3. Sevrrnl attempts \vere made to obtain a purified sample of M-5 t)>
2G4
FRANKLIN,
FRIEDMAN AND SETLOR
differential centrifugation. Unfortunately these were not successful so t,hat we could not get an absorption spectrum and therefore are unable t,o report. on t,he qlla.ntum efficiency of t’he inart8ivntjion. ACKNOWLEDGMENTS The authors wish to thank Dr. W. H. Gaylord, Jr., for his assistance in preparing the electron micrograph, and Professor E. C. Pollard for many helpful discussions. SUMMARY
The relative efficiency of the ultraviolet light for inactivating a dry bacterial virus (M-5) has been determined in the wavelength region 1942-3000 A. The data indicate that photons absorbed in protein and nucleic acid components can result in inactivation. From a comparison with T-l virus, it has been suggested that there are more sensitive units in T-l than in M-5. REFERENCES 1. 2. 3. 4. 5.
LOOFBOUROR,J. R., Growth, 12 (suppl.), 77 (1948). HOLLAENDER, A., AND OLIPHANT, J. W., J. Bacterial. 48, 447 (1944). FLUKE, D. J., Ph.D. Thesis, Yale Univ., New Haven, Conn., 1950. FRIEVMAN, M., J. Bacterial., in press. EVAXS, E. A., Biochemical Studies of Bacterial Viruses. The University of Chicago Press, Chicago, Ill., 1952. 6. FLUKE, D. J., AND POLLARD, E. C., Science 110, 274 (1949). 7. HAnI, J. S., AND PLATT, J. R., J. Chenz. Phys. 20, 335 11952). 8. WYCKOFF,R. W. G., Electron Microscopy. Interscience Pul)lishers, New York, 1949.