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The detection of temperature induced structural changes in T4B and T7 bacteriophages by means of high precision acoustic velocity measurements
U/wasound in Med. & Bid. Printed in the U.S.A.
Vol. 12, No. 6, pp. 51 l-517,
1986
0301~5629/86 0 1986 Pqamon
S3.00 + .OO Press Ltd.
@Original Contribution THE DETECTION OF TEMPERATURE INDUCED STRUCTURAL CHANGES IN T4B AND T7 BACTERIOPHAGES BY MEANS OF HIGH PRECISION ACOUSTIC VELOCITY MEASUREMENTS G.
V.SHILNIKOV,A. A.KHUSAINOV and A.P. SARVAZYAN Institute of Biological Physics, USSR Academy of Science, Pushchino, USSR
and A. R. WILLIAMS Department
of Medical Biophysics, University of Manchester Medical School, Manchester, England (Received 10 September 1985; infinalform
4 February 1986)
Abstract-High precision measurements of the velocity of 7-7.5 MHz ultrasonic waves in suspensions of both T4B and T7 bacteriophages as a function of temperature revealed the presence of a distinct transition in the physiological range of 3545°C. Data from acoustic measurements, sedimentation analysis and electron microscopy enabled us to identify this transition as being caused by the protein component of the phage and not the DNA. This transition does not depend on the position of the long tail fibers and may be part of some normal physiological process within the bacteriophage which presumably enhances its recognition and attachment to its host cell. Key Words: Acoustics,
Ultrasonics,
Ultrasonic toxicity, Bacteriophage,
Acoustic velocity.
a cylindrical tail piece (2) (about 100 X 15 nm) which terminates in an hexagonal base plate (3) to which are attached six “pins” or spikes (4) and six long tail fibers (5) (Gendon et al., 1975). These long tail fibers may be in a retracted or “up” position so that they lie close to the tail and head of the phage particle, or they may be in the extended or “down” position as indicated in Fig. 1. It is known that the long tail fibers play an important role in the initial stages of host cell recognition and the binding of the base plate (3) to the surface of the bacterium. Once the base plate has been attached, the tail piece (2) contracts which results in the injection of the phage DNA into the bacterial cell. The bacteriophage is maximally infective only when all of its long tail fibers are in the “down” position (Greve and Blok, 1975). The configuration of these long tail fibers is determined by the presence or absence of chemical triggers such as L-tryptophan (in the case of T4B), and by the environmental conditions within the suspending medium such as its ionic strength, pH and temperature (Greve and Block, 1973; Boer et al., 198 1; Boontje et al., 1978). The T7 bacteriophage differs from the T-even phages such as T4 in that it has only a short tail piece which cannot contract (Bradley, 1967; Studier, 1972).
INTRODUCI'ION structures of the various bacteriophages have largely been deduced from electron microscopic investigations (Simon and Anderson, 1967; Ring, 1968). More recently, diverse physical techniques such as optical methods (Thomas, 1976; Fidy et al., 1983), electrical birefiingence (Greve and Blok, 1973; Boontje et al., 1978; Boer et al., 1981), microcalorimetry (Arisaka et al., 198 l), sedimentation analysis (Greve and Blok, 1973; 1975), and small angle X-ray diffraction (Rolbin et al., 1982) have also been employed in an endeavour to complement the static electron micrographic images so as to obtain a greater insight into both the structure and the dynamic functioning of bacteriophages. The present paper describes the application of yet another different but highly sensitive physical technique; namely, the measurement of ultrasonic velocity with high precision. This technique measures the bulk elastic properties of small volumes of solution with high accuracy and can yield information about inter- and intramolecular interactions which cannot be obtained by other methods. The T-even bacteriophages (Fig. 1) consist of an icosahedral head (1) (about 100 X 80 nm) attached to The complex
511
512
Ultrasound in Medicine and Biology
Fig. 1. T-even bacteriophage: (1) icosahedral head, (2) tail piece, (3) hexagonal base plate, (4) pins, (5) tail fibres.
The rate of adsorption of bacteriophage particles onto the surface of the host cell (E. cd) is strongly dependent on temperature (Stent and Wollman, 1952). They also found that the maximum rate of adsorption was observed at temperatures corresponding to the physiological range, i.e. 35-40°C (Fig. 2), which coincides with the temperature of the normal habitat of the host cell (-37°C). This temperature dependence of the rate of adsorption of the bacteriophage could be related to corresponding temperature dependent changes of the bacteriophage structure and/or of the host cell surface. The purpose of the present study was therefore to search for structural transitions of the bacteriophage in the region of 35-40°C. The presence of such a transition could be an indication that evolutionary development has optimised the structure of the phage so that it could best fit the surface of the host cell just at physiological temperatures. The thermal behaviour of the phage has been the subject of numerous investigations. Thomas (1976) studied the phage P22 (which is morphologically similar to T7) using Raman spectroscopy and showed that the tail and DNA were stable up to 80°C. Even though Thomas (1976) used a phage concentration of 30 mg/ ml, he was unable to detect any structural transitions occurring below this temperature. However, Fidy et al. (1983) investigated the thermal behaviour of T7 phage by optical density and polarisation of fluorescence techniques over the range 20- 100°C and they described a phase transition occurring between 50 and 60°C. They interpreted this result as evidence for relaxational processes occurring within the DNA. Arisaka et al. (198 1) studied the temperature dependent reorganisation of the tail of the T4 phage using scanning cal-
June 1986, Volume 12, Number 6
orimetry. They observed an endothermic effect having a maximum at 68°C and an exothermic effect having a maximum at 72°C which was accompanied by contraction of the tail structure. However, they did not observe any molecular transitions at physiological temperatures. GreveandBlok(1973; 1975)andBoeretaZ.(1981) investigated the temperature dependence of molecular relaxation times (over the range O-50’%) in T4B, T4D, and T7 phages, in both the presence and absence of tryptophan, using the technique of electrical birefiingence. They observed a small increase in relaxation times between 30 and 40°C but attributed it to a possible error which could arise due to polarisation effects in the electrolyte. They also measured the Kerr coefficient and the rotational diffusion of T4 phage and its mutants at 5, 20, 30, and 35°C and at different pH values. They observed an increased value of the Kerr coefficient of T4D at a temperature of 30-35°C and pH 6-8 which they ascribed to the unfolding of the long tail fibers of the phage particle. The present paper describes a series of high precision ultrasonic velocity measurements on native and modified suspensions of T4B and T7 phage particles over the temperature range 25-50°C. These measurements demonstrated well-defined transitions occurring within the temperature range of 30-40°C. METHODS
AND MATERIALS
Phage culture Bacteriophage T7 and the mutant strain T4B (which requires the presence of L-tryptophan in the
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A’ 10
20 TEMPERATURE
30
40
(‘-3
Fig. 2. Rate of adsorption of T4 bacteriophage onto the surface of E. coli host cell as a function of temperature (according to the data of Stent and Wollman, 1952). Dashed area in the diagram corresponds to temperatures above the limit of the survival of host cell.
Temperature induced structural changes in T4B and T7 bacteriophages 0 G. V. SHILNIKOV et al.
growth medium before its long tail fibers can be lowered) were grown on E. coli by the standard procedures described by Tichonenko et al. (1963). The T7 phage particles were harvested by precipitation in the presence of 60 g/l polyethylene glycol (MW 6,000) followed by repeated low and high speed centrifugation. The T7 phage particles were suspended in 0.00 1 M phosphate buffer pH 7.0 containing 0.00 1 M magnesium sulphate and were repeatedly dialysed against this same buffer as described by Pouta et al. (1974). The T4B phage particles were harvested by repeated low and high speed centrifugation followed by chromatographic separation on a column of DEAE cellulose (Tichonenko et al., 1963). The T4B particles were suspended in 0.05 M phosphate buffer, pH 7.05, and were dialysed against three changes of distilled water during the 48 h period before the acoustic measurements were to be performed. Samples of the phage suspensions after dialysis were examined by electron microscopy and by microbiological titration and they were found to have a normal appearance and to be capable of infecting E. coli. Modification of phage structure Modified phage particles were obtained either by heating for 40 min at 70°C or by osmotic disruption as described by Heriot and Barlow (1957). This article also describes the purification procedure for the removal of the DNA which has been released from the head capsule, and the same procedure was used in the present work. Estimation of concentration Samples of the final phage suspensions were weighed on a Mettler (model HL 52) microanalytical balance and the difference in weight between the phage suspension and the equivalent volume of buffer both before and after drying at 110°C were determined. The error in the estimate of concentration by this technique was less than 2%. The molal extinction coefficient given by Greve and Blok (1973) was also used to obtain an estimate of phage particle concentration from optical density measurements. A phage concentration of 0.5 to 1.0 mg/ml was used for the acoustic velocity measurements and a concentration of 0.5 to 1.5 mg/ml for the sedimentation analysis measurements. Acoustic measurements A differential fixed path interferometer for the precise determination of the velocity of an ultrasonic wave in small volumes of solutions of biological materials has been described earlier by Sarvazyan (1982;
513
1983a, b). Briefly, it consists of a matched pair of acoustic cells (resonators) each holding 0.1 ml of solution which are thermostated within the same wellinsulated chamber. Each cell is connected to a separate electronic circuit which automatically estimates two values; these are (1) the frequency of the chosen resonance of the cell within the range 7-7.5 MHz by measuring the point of inflection on the phase-frequency curve; and (2) the value of the derivative of the curve at this point. This derivative value is proportional to the quality factor of the resonator. The value of the resonance frequency is a linear function of the ultrasonic velocity within the liquid filling the cell, and the value of the quality factor is a known function of ultrasonic attenuation within the investigated liquid. Conventional methods of ultrasound velocity measurement using resonators is based on the measurement of the difference between the frequencies of close resonant peaks. However, in our case it is not the absolute value of the velocity, but only a small difference between the ultrasonic velocities of two nearly identical solutions or a small change in velocity due to some physico-chemical process, which is being measured. It is therefore sufficient to measure only the shifts in frequency of one close resonance peak. The reference cell is used for temperature compensation. When measurements were performed at different temperatures, the matched pair of cells were warmed at a rate not greater than 0.1 “C/min and were maintained at the required temperature for 20-30 min before any measurements were made. The thermostating system provided temperature stability of about t-0.025”C while the difference between the temperatures of the measuring and reference cells was less than 0.001 “C. The inherent variability in the measurement of relative changes of acoustic velocity using this technique is about lO-‘j of the absolute value. The range of working frequencies is 7-7.5 MHz. Sedimentation analysis Measurement of the sedimentation coefficients of both phages were obtained by means of an analytical ultracentrifuge (model 3 170) using a rotor having a two sector cell. A Schlieren optical system was used to determine the position of the symmetrical boundary. Measurements were performed at 20 and 40°C; for the latter the rotor and sample were prewarmed to 40°C before loading into the ultracentrifuge. The precision for maintaining the temperature within the ultracentrifuge was +0.5”C. Sedimentation rates were obtained at 6,000 and 10,000 rpm, and at a variety of phage concentrations. All sedimentation coefficients were corrected to S20,w
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June 1986, Volume 12, Number 6
(sedimentation constant normalized to 20°C and the viscosity of water) and the values of a partial specific volume of 0.625and the experimentally determined viscosity were included.
RESULTS AND DISCUSSION The velocity
of ultrasound in a solution depends compressibility and density of that solution and provides information concerning the volume and intrinsic compressibility of the solute as well as solute-solute and solute-solvent interactions (Sar-
on the adiabatic
vazyan, 1983b). The results of ultrasonic measurements in dilute solutions can be presented in terms of a relative specific concentrational increment of ultrasound velocity (A) which is defined as
I
.
25
35
45
55
TEMPEJlATURE ("C)
Fig. 4. Temperature dependence of the relative specific concentrational increment of ultrasound velocity (A) for the suspension of bacteriophage T4B in its native (0) and osmotically shocked ( - ) state. The measurement errors are smaller than the size of the data points.
A = (U - Uo) u, ’ where U and U. are the ultrasonic velocities in the solution and solvent, respectively, and c is the concentration of the solute. In dilute solution, where solute-solute interaction can be neglected, the ultrasound velocity is a linear function of concentration and the value for A is a constant depending on the bulk elastic properties of the solute and its hydration shell (Sarvazyan, 1983a; Sarvazyan and Kharakoz, 1977). Figure 3 shows a concentration dependence of ultrasound velocity in a suspension of phage T4B. The ultrasound velocity increment is independent of the concentration of phage over
/
/. 7
2
3
. 4
. 5
. 6
CONCENTRATION (,&ml)
Fig. 3. The difference between ultrasonic velocities in the bacteriophage T4B suspension and in the solvent as a function of bacteriophage concentration.
the investigated range of 0.3 to 6 mg/ml. Also the shape of the temperature dependence of A as shown in Figs. 4 and 5, which will be discussed below, remained the same even when the phage concentration was varied. This indicates that over the investigated range of temperatures and concentrations there are no artefacts introduced by possible aggregation of the phage particles. Figure 4 presents the effects of varying the temperature on the acoustic parameter A for native and osmotically shocked T4B phage, respectively. The temperature dependence of ultrasound velocity in an aqueous solution of a substance having no conformational transitions should be a monotonically decreasing line because an increase in temperature increases both the compressibility of the water of hydration and the intermolecular compressibilities of macromolecules and their complexes (Sarvazyan and Kharakoz, 1977; Sarvazyan, 198313).The monotonic increase in compressibility results in a corresponding monotonic decrease in the velocity of the ultrasonic waves. However, Fig. 4 shows that there is an additional transition which occurs between 35 and 45°C and results in an increase in the acoustic velocity increment. There are two physical processes which could be responsible for this observed increase of ultrasound velocity. These are (1) an increase in the amount of the hydration water due to rearrangement of the structural elements of the phage resulting in an increase in the accessible surface for solvation; and/or (2) structural transitions in the phage decreasing the compressibility of some of its elements. These rearrangements and/or transitions must be reflecting some normal physiological process since it covers the range of temperatures
Temperature induced structural changes in T4B and T7 bacteriophages 0 G. V. SHILNIKOV et al.
over which the bacteriophage is maximally infective. It is therefore of interest to attempt to identify which of the structural components comprising the bacteriophage could be responsible for this presumed transition. Osmotically shocked phage particles do not contain any DNA and yet the magnitude of the temperature dependent transition is almost identical with that of the native bacteriophage. This transition must therefore be occurring within the protein components and not within the DNA. The magnitude of the transition (roughly a 10% increase in acoustic velocity increment) indicates that it must either involve a large structural element such as a head capsule or a tail piece or else be associated with a phenomenon which is known to cause a large increase of hydration such as the production or uncovering of a large number of charged groups (Sarvazyan, 1983a; 1983b). Conformational changes within a small structural element such as the long tail fibers (which only comprise about 3% of the phage structure) as they move from the up to the down position would not be large enough to account for a change in velocity of this magnitude. The role of the tail piece in the observed transition was revealed by the investigation of the temperature dependence of ultrasound velocity in suspensions of T7 bacteriophage. The difference in the structure of T7 and T4B bacteriophages is mainly in the dimensions of the tail piece which is extremely short in T7. Figure 5 presents the effect of varying the temperature on the acoustic parameter A for T7 bacteriophage. Despite its short tail piece it also exhibited a distinct transition in the temperature range 35 to 40°C. The presence of such a transition in the same temperature range as that shown by the T4B bacteriophage again implies that .
?
0,35c
a X ti
0,3oc
! PI
0,25C
20
30
40
5’
TEMPERA!NRE(W) Fig. 5. Temperature dependence of the relative specific concentrational increment of ultrasound velocity (A) for a suspension of bacteriophage T7. The measurement errors are smaller than the size of the data points.
25°C
515
40°C
Fig. 6. Electron micrographic images of bacteriophage T4B before and after the observed temperature transition.
this phenomenon plays an important role in host cell recognition and/or infection. If the observed temperature-induced transition is caused by a rearrangement of the phage elements which expose new molecular surfaces to the solvent and thus increases hydration, then it could also result in a substantial change in the shape of the phage. A series of
electron micrographic investigations were therefore performed on phage T4B fixed with 2% formaldehyde in the presence and absence of tryptophan over the temperature range 20-40°C. Figure 6 shows that within the limitations of the negative staining technique, there were no significant changes either in the dimensions of the phage or in its morphology. These observations confirm the measurements of Greve and Blok ( 1973; 1975) who found that the coefficient of rotational diffusion determined by electric birefringence remained constant over the temperature range 20-50°C for a mutant of phage T4B which had no long tail fibers. Thus, if any temperature-dependent structural transitions are occurring within the head or tail piece of the bacteriophage, they are not detectable by electron microscopy and they have little effect on its hydrodynamic behaviour. Greve and Blok (1973; 1975) also investigated the temperature dependence of the coefficient of rotational diffusion for T4B phage both in the absence of tryptophan (i.e. with the long tail fibers in the up position) and in the presence of tryptophan (i.e. with the long tail fibers in the down position). They observed a large change in the presence of tryptophan over the temperature range 30-40°C which they attributed to lowering of the long tail fibers. However, they also observed a smaller change over the same temperature range in the absence of tryptophan which they attributed to an
516
Ultrasound in Medicine and Biology
June 1986, Volume 12, Number 6
Table 1. Values of the sedimentation constants for bacteriophage T4B normalized to 20°C and the viscosity of water Position of long tail libres Temperature This article Results of Greve and Blok (1973; 1975)
down
up 20°C 1017 + 20 s 1023 f 12 S
40°C 950 + 20 s
20°C 856 f 20 S 818 + 11 s
40°C 806 + 20 s
S = Svedberg units
error in their measurement system but which (in the light of our results) may have been a real effect. Acoustic results have shown that the temperature induced transition could not be explained just in terms of the lowering of the long tail fibers as proposed by Greve and Blok and we therefore performed sedimentation analysis experiments to link the acoustic and hydrodynamic results. We have measured sedimentation constants of bacteriophage T4B at 20 and 40°C both in the presence and in absence of tryptophan, that is with the long tail fibers in their up and down position, respectively. Table 1 is a compilation of our own and Greve and Blok’s (1973; 1975) data on the hydrodynamic properties of bacteriophage T4B. All the results are normalised to 20°C and to the viscosity of water. In all of the sedimentation analysis experiments only one peak was observed which indicated that our samples were homogeneous and we did not have aggregation. Table 1 shows that the constants of sedimentation for T4B phage in the absence of tryptophan measured at 20 and 40°C differed from each other by a value greater than the measurement error even though the long tail fibers were still in their up position. This indicates that the transition detected by acoustic velocity measurements in the range 35-45°C (which reflects changes in the hydration and/or intrinsic compressibility of the phage) is associated with a detectable change in the hydrodynamic properties of the phage. It is possible that this same thermally-induced change in hydrodynamic properties of T4B in the absence of tryptophan was also detected by Greve and Blok ( 1973; 1975) but was interpreted by them as an experimental error. An important conclusion which can be made from our sedimentation data shown in Table 1 is that the temperature transitions seems to be independent of the position of the long tail fibers. This conclusion is made from the consideration of the values of the ratio of sedimentation constants of the phage with the long tail fibers in up position at two temperatures and the same ratio for the down position: JS0.w(UP 20°C) = I o7 5-203 (UP 40°C) *
and S20,w(down 20°C) = 1.06. S20,w(down 40°C) These two ratios coincide well within the experimental error. Thus, our high precision ultrasonic velocity measurements have enabled us to detect a change in the state of hydration and/or the intrinsic compressibility of both T4B and T7 bacteriophages within the temperature range 35-40°C. This change is not associated with the DNA or the tail piece of the phage since osmotically shocked phages or short-tailed phages yield similar results. This change can also be detected whether the long tail fibers are in their up or down positions. By elimination, we deduce that this change is most probably associated with some unidentified structural rearrangement within the protein of the head capsule which may be important in maximising the infectivity of the phage to its host cell. Acknowledgments-The authors are grateful to Drs 0. N. Ozolin and S. G. Kamzolova for culturing the phage particles. A. R. Williams wishes to thank the Royal Society for selecting him to participate in the Inter-Academy Exchange Program, and the Academy of Sciences, USSR, and the Institute of Biological Physics in Pushchino for making his visit possible and productive.
REFERENCES Arisaka F., Engel J. and Klump H. (198 1) Contraction and dissociation of the bacteriophage T4 tail sheath induced by heat and urea. In Bacteriophage Assembly, pp. 365-379. A. R. Liss, N.Y. Boer J., Beck-Boot J. and Greve J. (1981) The influence of bacteriophage T4D short tail fibers on long tail fiber extension. Virology 110, 344-348. Boontje W., Greve J. and Blok J. (1978) Transient electric birefringence of T-even bacteriophages. IV. T2LO and T6 with extended tail fibers. Biopolymers 17, 2689-2702. Bradley D. E. ( 1967) Ultrastructure of the bacteriophages and bacteriocins. Bact. Rev. 31,230-3 14. Fidy J., Mauss G., Pataki K., Chamron J. and Ronto Gy. (1983) Flourescence label studies of the phase transition of T7. Biophys. Struct. Mech. 10, 109-l 19. Gendon U. S., Poglazov B. F. and Tichonenko T. J. (1975) Nuclear Acids and Virion Proteins, 98- 150. Nauka, Moscow. Greve J. and Blok J. (1973) Transient electric birefringence of Teven bacteriophages. I. T4B in the absence of tryptophan and fiberless T4 particles. Biopolymers 12, 2607-2622. Greve J. and Blok J. (1975) Transient electric birefringence of T-
Temperature induced structural changes in T4B and T7 bacteriophages 0 G. V. even bacteriophages. II. T4B in the presence of tryptophan and T4D. Biopolymers 14, 139- 154. Heriot R. M. and Barlow J. L. (1957) The protein coats or “ghosts” of coliphage T2. Gen. Physiol. 40, 809-825. King J. ( 1968) Assembly of the tail of bacteriophage T4. J. Mol. Biol. 32,23 l-262. Pouta H., RahwsdorfH. J., Pai S. H., Hi&i-Kauffmann M., Herrlich P. and Schweiger M. (1974) Control of gene expression in bacteriophage T7. Mol. Gen. Genet. 134,281-287. Rolbin J. A., Swergun D. J., Feigin L. A., Gaspar Sh. and Ronto D. ( 1982) Bacteriophage T7 structure according to the data of smallangle X-ray scattering. Docl. Acad. Nauk USSR 225, 1497-1500. Sarvazyan A. P. (1982) Development of methods of precise ultrasonic measurements in small volumes of liquids. Ultrasonics 20, 15l154. Sarvazyan A. P. (1983a) Ultrasonic velocimetry of biological substances. Molekulyarnaya Biologiya 17, 739-750. Sarvazyan A. P. (1983b) Propagation of ultrasound in solutions of biological substances. In Ultrasound Interactions in Biology and
SHILNIKOV
et al.
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Medicine (Edited by R. Millner, E. Rosenfeld and U. Cobet), pp.
195-202. Plenum Press, New York. Sarvazyan A. P. and Kharakoz D. P. (1977) Acoustic investigation of the conformational changes of protein in water solutions. In Molecular and Cellular Biophysics (Edited by G. M. Frank), pp. 93- 106. Nauka, Moscow. Simon L. D. and Anderson T. F. ( 1967) The Infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope. Virology 32, 279-297. Stent G. S. and Wollman E. L. (1952) On the two-step nature of bacteriophage adsorption. Biochim. et Biophys. Ada 8,260-269. Studier F. W. (1972) Bacteriophage T7-genetic and biochemical analysis of this simple phage gives information about basic genetic processes. Science 176,367. Thomas G. J. (1976) Raman spectroscopy and virus research. Appl. Spectroscopy 30,483-494.
Tichonenko T. J., Koudelka L. and Borshpolec S. J. (1963) Concentration and purification of bacteriophages by column chromotography. Microbiology 22, 723-726.
Vol. 12, No. 6, pp. 51 l-517,
1986
0301~5629/86 0 1986 Pqamon
S3.00 + .OO Press Ltd.
@Original Contribution THE DETECTION OF TEMPERATURE INDUCED STRUCTURAL CHANGES IN T4B AND T7 BACTERIOPHAGES BY MEANS OF HIGH PRECISION ACOUSTIC VELOCITY MEASUREMENTS G.
V.SHILNIKOV,A. A.KHUSAINOV and A.P. SARVAZYAN Institute of Biological Physics, USSR Academy of Science, Pushchino, USSR
and A. R. WILLIAMS Department
of Medical Biophysics, University of Manchester Medical School, Manchester, England (Received 10 September 1985; infinalform
4 February 1986)
Abstract-High precision measurements of the velocity of 7-7.5 MHz ultrasonic waves in suspensions of both T4B and T7 bacteriophages as a function of temperature revealed the presence of a distinct transition in the physiological range of 3545°C. Data from acoustic measurements, sedimentation analysis and electron microscopy enabled us to identify this transition as being caused by the protein component of the phage and not the DNA. This transition does not depend on the position of the long tail fibers and may be part of some normal physiological process within the bacteriophage which presumably enhances its recognition and attachment to its host cell. Key Words: Acoustics,
Ultrasonics,
Ultrasonic toxicity, Bacteriophage,
Acoustic velocity.
a cylindrical tail piece (2) (about 100 X 15 nm) which terminates in an hexagonal base plate (3) to which are attached six “pins” or spikes (4) and six long tail fibers (5) (Gendon et al., 1975). These long tail fibers may be in a retracted or “up” position so that they lie close to the tail and head of the phage particle, or they may be in the extended or “down” position as indicated in Fig. 1. It is known that the long tail fibers play an important role in the initial stages of host cell recognition and the binding of the base plate (3) to the surface of the bacterium. Once the base plate has been attached, the tail piece (2) contracts which results in the injection of the phage DNA into the bacterial cell. The bacteriophage is maximally infective only when all of its long tail fibers are in the “down” position (Greve and Blok, 1975). The configuration of these long tail fibers is determined by the presence or absence of chemical triggers such as L-tryptophan (in the case of T4B), and by the environmental conditions within the suspending medium such as its ionic strength, pH and temperature (Greve and Block, 1973; Boer et al., 198 1; Boontje et al., 1978). The T7 bacteriophage differs from the T-even phages such as T4 in that it has only a short tail piece which cannot contract (Bradley, 1967; Studier, 1972).
INTRODUCI'ION structures of the various bacteriophages have largely been deduced from electron microscopic investigations (Simon and Anderson, 1967; Ring, 1968). More recently, diverse physical techniques such as optical methods (Thomas, 1976; Fidy et al., 1983), electrical birefiingence (Greve and Blok, 1973; Boontje et al., 1978; Boer et al., 1981), microcalorimetry (Arisaka et al., 198 l), sedimentation analysis (Greve and Blok, 1973; 1975), and small angle X-ray diffraction (Rolbin et al., 1982) have also been employed in an endeavour to complement the static electron micrographic images so as to obtain a greater insight into both the structure and the dynamic functioning of bacteriophages. The present paper describes the application of yet another different but highly sensitive physical technique; namely, the measurement of ultrasonic velocity with high precision. This technique measures the bulk elastic properties of small volumes of solution with high accuracy and can yield information about inter- and intramolecular interactions which cannot be obtained by other methods. The T-even bacteriophages (Fig. 1) consist of an icosahedral head (1) (about 100 X 80 nm) attached to The complex
511
512
Ultrasound in Medicine and Biology
Fig. 1. T-even bacteriophage: (1) icosahedral head, (2) tail piece, (3) hexagonal base plate, (4) pins, (5) tail fibres.
The rate of adsorption of bacteriophage particles onto the surface of the host cell (E. cd) is strongly dependent on temperature (Stent and Wollman, 1952). They also found that the maximum rate of adsorption was observed at temperatures corresponding to the physiological range, i.e. 35-40°C (Fig. 2), which coincides with the temperature of the normal habitat of the host cell (-37°C). This temperature dependence of the rate of adsorption of the bacteriophage could be related to corresponding temperature dependent changes of the bacteriophage structure and/or of the host cell surface. The purpose of the present study was therefore to search for structural transitions of the bacteriophage in the region of 35-40°C. The presence of such a transition could be an indication that evolutionary development has optimised the structure of the phage so that it could best fit the surface of the host cell just at physiological temperatures. The thermal behaviour of the phage has been the subject of numerous investigations. Thomas (1976) studied the phage P22 (which is morphologically similar to T7) using Raman spectroscopy and showed that the tail and DNA were stable up to 80°C. Even though Thomas (1976) used a phage concentration of 30 mg/ ml, he was unable to detect any structural transitions occurring below this temperature. However, Fidy et al. (1983) investigated the thermal behaviour of T7 phage by optical density and polarisation of fluorescence techniques over the range 20- 100°C and they described a phase transition occurring between 50 and 60°C. They interpreted this result as evidence for relaxational processes occurring within the DNA. Arisaka et al. (198 1) studied the temperature dependent reorganisation of the tail of the T4 phage using scanning cal-
June 1986, Volume 12, Number 6
orimetry. They observed an endothermic effect having a maximum at 68°C and an exothermic effect having a maximum at 72°C which was accompanied by contraction of the tail structure. However, they did not observe any molecular transitions at physiological temperatures. GreveandBlok(1973; 1975)andBoeretaZ.(1981) investigated the temperature dependence of molecular relaxation times (over the range O-50’%) in T4B, T4D, and T7 phages, in both the presence and absence of tryptophan, using the technique of electrical birefiingence. They observed a small increase in relaxation times between 30 and 40°C but attributed it to a possible error which could arise due to polarisation effects in the electrolyte. They also measured the Kerr coefficient and the rotational diffusion of T4 phage and its mutants at 5, 20, 30, and 35°C and at different pH values. They observed an increased value of the Kerr coefficient of T4D at a temperature of 30-35°C and pH 6-8 which they ascribed to the unfolding of the long tail fibers of the phage particle. The present paper describes a series of high precision ultrasonic velocity measurements on native and modified suspensions of T4B and T7 phage particles over the temperature range 25-50°C. These measurements demonstrated well-defined transitions occurring within the temperature range of 30-40°C. METHODS
AND MATERIALS
Phage culture Bacteriophage T7 and the mutant strain T4B (which requires the presence of L-tryptophan in the
093.
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F! i
,
O,l-
s !j
A’ 10
20 TEMPERATURE
30
40
(‘-3
Fig. 2. Rate of adsorption of T4 bacteriophage onto the surface of E. coli host cell as a function of temperature (according to the data of Stent and Wollman, 1952). Dashed area in the diagram corresponds to temperatures above the limit of the survival of host cell.
Temperature induced structural changes in T4B and T7 bacteriophages 0 G. V. SHILNIKOV et al.
growth medium before its long tail fibers can be lowered) were grown on E. coli by the standard procedures described by Tichonenko et al. (1963). The T7 phage particles were harvested by precipitation in the presence of 60 g/l polyethylene glycol (MW 6,000) followed by repeated low and high speed centrifugation. The T7 phage particles were suspended in 0.00 1 M phosphate buffer pH 7.0 containing 0.00 1 M magnesium sulphate and were repeatedly dialysed against this same buffer as described by Pouta et al. (1974). The T4B phage particles were harvested by repeated low and high speed centrifugation followed by chromatographic separation on a column of DEAE cellulose (Tichonenko et al., 1963). The T4B particles were suspended in 0.05 M phosphate buffer, pH 7.05, and were dialysed against three changes of distilled water during the 48 h period before the acoustic measurements were to be performed. Samples of the phage suspensions after dialysis were examined by electron microscopy and by microbiological titration and they were found to have a normal appearance and to be capable of infecting E. coli. Modification of phage structure Modified phage particles were obtained either by heating for 40 min at 70°C or by osmotic disruption as described by Heriot and Barlow (1957). This article also describes the purification procedure for the removal of the DNA which has been released from the head capsule, and the same procedure was used in the present work. Estimation of concentration Samples of the final phage suspensions were weighed on a Mettler (model HL 52) microanalytical balance and the difference in weight between the phage suspension and the equivalent volume of buffer both before and after drying at 110°C were determined. The error in the estimate of concentration by this technique was less than 2%. The molal extinction coefficient given by Greve and Blok (1973) was also used to obtain an estimate of phage particle concentration from optical density measurements. A phage concentration of 0.5 to 1.0 mg/ml was used for the acoustic velocity measurements and a concentration of 0.5 to 1.5 mg/ml for the sedimentation analysis measurements. Acoustic measurements A differential fixed path interferometer for the precise determination of the velocity of an ultrasonic wave in small volumes of solutions of biological materials has been described earlier by Sarvazyan (1982;
513
1983a, b). Briefly, it consists of a matched pair of acoustic cells (resonators) each holding 0.1 ml of solution which are thermostated within the same wellinsulated chamber. Each cell is connected to a separate electronic circuit which automatically estimates two values; these are (1) the frequency of the chosen resonance of the cell within the range 7-7.5 MHz by measuring the point of inflection on the phase-frequency curve; and (2) the value of the derivative of the curve at this point. This derivative value is proportional to the quality factor of the resonator. The value of the resonance frequency is a linear function of the ultrasonic velocity within the liquid filling the cell, and the value of the quality factor is a known function of ultrasonic attenuation within the investigated liquid. Conventional methods of ultrasound velocity measurement using resonators is based on the measurement of the difference between the frequencies of close resonant peaks. However, in our case it is not the absolute value of the velocity, but only a small difference between the ultrasonic velocities of two nearly identical solutions or a small change in velocity due to some physico-chemical process, which is being measured. It is therefore sufficient to measure only the shifts in frequency of one close resonance peak. The reference cell is used for temperature compensation. When measurements were performed at different temperatures, the matched pair of cells were warmed at a rate not greater than 0.1 “C/min and were maintained at the required temperature for 20-30 min before any measurements were made. The thermostating system provided temperature stability of about t-0.025”C while the difference between the temperatures of the measuring and reference cells was less than 0.001 “C. The inherent variability in the measurement of relative changes of acoustic velocity using this technique is about lO-‘j of the absolute value. The range of working frequencies is 7-7.5 MHz. Sedimentation analysis Measurement of the sedimentation coefficients of both phages were obtained by means of an analytical ultracentrifuge (model 3 170) using a rotor having a two sector cell. A Schlieren optical system was used to determine the position of the symmetrical boundary. Measurements were performed at 20 and 40°C; for the latter the rotor and sample were prewarmed to 40°C before loading into the ultracentrifuge. The precision for maintaining the temperature within the ultracentrifuge was +0.5”C. Sedimentation rates were obtained at 6,000 and 10,000 rpm, and at a variety of phage concentrations. All sedimentation coefficients were corrected to S20,w
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June 1986, Volume 12, Number 6
(sedimentation constant normalized to 20°C and the viscosity of water) and the values of a partial specific volume of 0.625and the experimentally determined viscosity were included.
RESULTS AND DISCUSSION The velocity
of ultrasound in a solution depends compressibility and density of that solution and provides information concerning the volume and intrinsic compressibility of the solute as well as solute-solute and solute-solvent interactions (Sar-
on the adiabatic
vazyan, 1983b). The results of ultrasonic measurements in dilute solutions can be presented in terms of a relative specific concentrational increment of ultrasound velocity (A) which is defined as
I
.
25
35
45
55
TEMPEJlATURE ("C)
Fig. 4. Temperature dependence of the relative specific concentrational increment of ultrasound velocity (A) for the suspension of bacteriophage T4B in its native (0) and osmotically shocked ( - ) state. The measurement errors are smaller than the size of the data points.
A = (U - Uo) u, ’ where U and U. are the ultrasonic velocities in the solution and solvent, respectively, and c is the concentration of the solute. In dilute solution, where solute-solute interaction can be neglected, the ultrasound velocity is a linear function of concentration and the value for A is a constant depending on the bulk elastic properties of the solute and its hydration shell (Sarvazyan, 1983a; Sarvazyan and Kharakoz, 1977). Figure 3 shows a concentration dependence of ultrasound velocity in a suspension of phage T4B. The ultrasound velocity increment is independent of the concentration of phage over
/
/. 7
2
3
. 4
. 5
. 6
CONCENTRATION (,&ml)
Fig. 3. The difference between ultrasonic velocities in the bacteriophage T4B suspension and in the solvent as a function of bacteriophage concentration.
the investigated range of 0.3 to 6 mg/ml. Also the shape of the temperature dependence of A as shown in Figs. 4 and 5, which will be discussed below, remained the same even when the phage concentration was varied. This indicates that over the investigated range of temperatures and concentrations there are no artefacts introduced by possible aggregation of the phage particles. Figure 4 presents the effects of varying the temperature on the acoustic parameter A for native and osmotically shocked T4B phage, respectively. The temperature dependence of ultrasound velocity in an aqueous solution of a substance having no conformational transitions should be a monotonically decreasing line because an increase in temperature increases both the compressibility of the water of hydration and the intermolecular compressibilities of macromolecules and their complexes (Sarvazyan and Kharakoz, 1977; Sarvazyan, 198313).The monotonic increase in compressibility results in a corresponding monotonic decrease in the velocity of the ultrasonic waves. However, Fig. 4 shows that there is an additional transition which occurs between 35 and 45°C and results in an increase in the acoustic velocity increment. There are two physical processes which could be responsible for this observed increase of ultrasound velocity. These are (1) an increase in the amount of the hydration water due to rearrangement of the structural elements of the phage resulting in an increase in the accessible surface for solvation; and/or (2) structural transitions in the phage decreasing the compressibility of some of its elements. These rearrangements and/or transitions must be reflecting some normal physiological process since it covers the range of temperatures
Temperature induced structural changes in T4B and T7 bacteriophages 0 G. V. SHILNIKOV et al.
over which the bacteriophage is maximally infective. It is therefore of interest to attempt to identify which of the structural components comprising the bacteriophage could be responsible for this presumed transition. Osmotically shocked phage particles do not contain any DNA and yet the magnitude of the temperature dependent transition is almost identical with that of the native bacteriophage. This transition must therefore be occurring within the protein components and not within the DNA. The magnitude of the transition (roughly a 10% increase in acoustic velocity increment) indicates that it must either involve a large structural element such as a head capsule or a tail piece or else be associated with a phenomenon which is known to cause a large increase of hydration such as the production or uncovering of a large number of charged groups (Sarvazyan, 1983a; 1983b). Conformational changes within a small structural element such as the long tail fibers (which only comprise about 3% of the phage structure) as they move from the up to the down position would not be large enough to account for a change in velocity of this magnitude. The role of the tail piece in the observed transition was revealed by the investigation of the temperature dependence of ultrasound velocity in suspensions of T7 bacteriophage. The difference in the structure of T7 and T4B bacteriophages is mainly in the dimensions of the tail piece which is extremely short in T7. Figure 5 presents the effect of varying the temperature on the acoustic parameter A for T7 bacteriophage. Despite its short tail piece it also exhibited a distinct transition in the temperature range 35 to 40°C. The presence of such a transition in the same temperature range as that shown by the T4B bacteriophage again implies that .
?
0,35c
a X ti
0,3oc
! PI
0,25C
20
30
40
5’
TEMPERA!NRE(W) Fig. 5. Temperature dependence of the relative specific concentrational increment of ultrasound velocity (A) for a suspension of bacteriophage T7. The measurement errors are smaller than the size of the data points.
25°C
515
40°C
Fig. 6. Electron micrographic images of bacteriophage T4B before and after the observed temperature transition.
this phenomenon plays an important role in host cell recognition and/or infection. If the observed temperature-induced transition is caused by a rearrangement of the phage elements which expose new molecular surfaces to the solvent and thus increases hydration, then it could also result in a substantial change in the shape of the phage. A series of
electron micrographic investigations were therefore performed on phage T4B fixed with 2% formaldehyde in the presence and absence of tryptophan over the temperature range 20-40°C. Figure 6 shows that within the limitations of the negative staining technique, there were no significant changes either in the dimensions of the phage or in its morphology. These observations confirm the measurements of Greve and Blok ( 1973; 1975) who found that the coefficient of rotational diffusion determined by electric birefringence remained constant over the temperature range 20-50°C for a mutant of phage T4B which had no long tail fibers. Thus, if any temperature-dependent structural transitions are occurring within the head or tail piece of the bacteriophage, they are not detectable by electron microscopy and they have little effect on its hydrodynamic behaviour. Greve and Blok (1973; 1975) also investigated the temperature dependence of the coefficient of rotational diffusion for T4B phage both in the absence of tryptophan (i.e. with the long tail fibers in the up position) and in the presence of tryptophan (i.e. with the long tail fibers in the down position). They observed a large change in the presence of tryptophan over the temperature range 30-40°C which they attributed to lowering of the long tail fibers. However, they also observed a smaller change over the same temperature range in the absence of tryptophan which they attributed to an
516
Ultrasound in Medicine and Biology
June 1986, Volume 12, Number 6
Table 1. Values of the sedimentation constants for bacteriophage T4B normalized to 20°C and the viscosity of water Position of long tail libres Temperature This article Results of Greve and Blok (1973; 1975)
down
up 20°C 1017 + 20 s 1023 f 12 S
40°C 950 + 20 s
20°C 856 f 20 S 818 + 11 s
40°C 806 + 20 s
S = Svedberg units
error in their measurement system but which (in the light of our results) may have been a real effect. Acoustic results have shown that the temperature induced transition could not be explained just in terms of the lowering of the long tail fibers as proposed by Greve and Blok and we therefore performed sedimentation analysis experiments to link the acoustic and hydrodynamic results. We have measured sedimentation constants of bacteriophage T4B at 20 and 40°C both in the presence and in absence of tryptophan, that is with the long tail fibers in their up and down position, respectively. Table 1 is a compilation of our own and Greve and Blok’s (1973; 1975) data on the hydrodynamic properties of bacteriophage T4B. All the results are normalised to 20°C and to the viscosity of water. In all of the sedimentation analysis experiments only one peak was observed which indicated that our samples were homogeneous and we did not have aggregation. Table 1 shows that the constants of sedimentation for T4B phage in the absence of tryptophan measured at 20 and 40°C differed from each other by a value greater than the measurement error even though the long tail fibers were still in their up position. This indicates that the transition detected by acoustic velocity measurements in the range 35-45°C (which reflects changes in the hydration and/or intrinsic compressibility of the phage) is associated with a detectable change in the hydrodynamic properties of the phage. It is possible that this same thermally-induced change in hydrodynamic properties of T4B in the absence of tryptophan was also detected by Greve and Blok ( 1973; 1975) but was interpreted by them as an experimental error. An important conclusion which can be made from our sedimentation data shown in Table 1 is that the temperature transitions seems to be independent of the position of the long tail fibers. This conclusion is made from the consideration of the values of the ratio of sedimentation constants of the phage with the long tail fibers in up position at two temperatures and the same ratio for the down position: JS0.w(UP 20°C) = I o7 5-203 (UP 40°C) *
and S20,w(down 20°C) = 1.06. S20,w(down 40°C) These two ratios coincide well within the experimental error. Thus, our high precision ultrasonic velocity measurements have enabled us to detect a change in the state of hydration and/or the intrinsic compressibility of both T4B and T7 bacteriophages within the temperature range 35-40°C. This change is not associated with the DNA or the tail piece of the phage since osmotically shocked phages or short-tailed phages yield similar results. This change can also be detected whether the long tail fibers are in their up or down positions. By elimination, we deduce that this change is most probably associated with some unidentified structural rearrangement within the protein of the head capsule which may be important in maximising the infectivity of the phage to its host cell. Acknowledgments-The authors are grateful to Drs 0. N. Ozolin and S. G. Kamzolova for culturing the phage particles. A. R. Williams wishes to thank the Royal Society for selecting him to participate in the Inter-Academy Exchange Program, and the Academy of Sciences, USSR, and the Institute of Biological Physics in Pushchino for making his visit possible and productive.
REFERENCES Arisaka F., Engel J. and Klump H. (198 1) Contraction and dissociation of the bacteriophage T4 tail sheath induced by heat and urea. In Bacteriophage Assembly, pp. 365-379. A. R. Liss, N.Y. Boer J., Beck-Boot J. and Greve J. (1981) The influence of bacteriophage T4D short tail fibers on long tail fiber extension. Virology 110, 344-348. Boontje W., Greve J. and Blok J. (1978) Transient electric birefringence of T-even bacteriophages. IV. T2LO and T6 with extended tail fibers. Biopolymers 17, 2689-2702. Bradley D. E. ( 1967) Ultrastructure of the bacteriophages and bacteriocins. Bact. Rev. 31,230-3 14. Fidy J., Mauss G., Pataki K., Chamron J. and Ronto Gy. (1983) Flourescence label studies of the phase transition of T7. Biophys. Struct. Mech. 10, 109-l 19. Gendon U. S., Poglazov B. F. and Tichonenko T. J. (1975) Nuclear Acids and Virion Proteins, 98- 150. Nauka, Moscow. Greve J. and Blok J. (1973) Transient electric birefringence of Teven bacteriophages. I. T4B in the absence of tryptophan and fiberless T4 particles. Biopolymers 12, 2607-2622. Greve J. and Blok J. (1975) Transient electric birefringence of T-
Temperature induced structural changes in T4B and T7 bacteriophages 0 G. V. even bacteriophages. II. T4B in the presence of tryptophan and T4D. Biopolymers 14, 139- 154. Heriot R. M. and Barlow J. L. (1957) The protein coats or “ghosts” of coliphage T2. Gen. Physiol. 40, 809-825. King J. ( 1968) Assembly of the tail of bacteriophage T4. J. Mol. Biol. 32,23 l-262. Pouta H., RahwsdorfH. J., Pai S. H., Hi&i-Kauffmann M., Herrlich P. and Schweiger M. (1974) Control of gene expression in bacteriophage T7. Mol. Gen. Genet. 134,281-287. Rolbin J. A., Swergun D. J., Feigin L. A., Gaspar Sh. and Ronto D. ( 1982) Bacteriophage T7 structure according to the data of smallangle X-ray scattering. Docl. Acad. Nauk USSR 225, 1497-1500. Sarvazyan A. P. (1982) Development of methods of precise ultrasonic measurements in small volumes of liquids. Ultrasonics 20, 15l154. Sarvazyan A. P. (1983a) Ultrasonic velocimetry of biological substances. Molekulyarnaya Biologiya 17, 739-750. Sarvazyan A. P. (1983b) Propagation of ultrasound in solutions of biological substances. In Ultrasound Interactions in Biology and
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195-202. Plenum Press, New York. Sarvazyan A. P. and Kharakoz D. P. (1977) Acoustic investigation of the conformational changes of protein in water solutions. In Molecular and Cellular Biophysics (Edited by G. M. Frank), pp. 93- 106. Nauka, Moscow. Simon L. D. and Anderson T. F. ( 1967) The Infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope. Virology 32, 279-297. Stent G. S. and Wollman E. L. (1952) On the two-step nature of bacteriophage adsorption. Biochim. et Biophys. Ada 8,260-269. Studier F. W. (1972) Bacteriophage T7-genetic and biochemical analysis of this simple phage gives information about basic genetic processes. Science 176,367. Thomas G. J. (1976) Raman spectroscopy and virus research. Appl. Spectroscopy 30,483-494.
Tichonenko T. J., Koudelka L. and Borshpolec S. J. (1963) Concentration and purification of bacteriophages by column chromotography. Microbiology 22, 723-726.