J. Mol. Biol. (1965) 12, 287-289
Optical Rotatory Dispersion of Bacteriophages The optical rotatory dispersion of a variety of bacteriophages with different morphologies and different contents of DNA has been measured. The bacteriophages studied were: T2, T4B and T40 (shock-resistant strain), T6, T5, T7, ,\ (phages attacking Escherichia coli), bacteriophage a (phage of Bacillus megatherium) and B3 (phage of Pseudomonas aeruginosa). In addition, the optical rotatory dispersion of shocked T2 phage, T2 ghosts, and T2 and T4 DNA complexes with amines (spermidine and spermine) was determined. The optical rotation was measured with a Cary model 60 spectropolarimeter in the wavelength region from 350 miL to 220 or 210 tiu». (The optical rotatory dispersion was not measured above 350 miL because a much higher concentration of bacteriophage would be needed. Therefore, we have no data suitable for calculation of Drude constants.) Solutions of phage in water at neutral pH and 0·1 to 0·5 ionic strength buffers were used; their absorbance at 260 miL in a l-cm cell was 1 or less. A cell of l-cm path length (3 ml. vol.) was used for the rotation measurements; the results were reported as specific rotation [a] in degrees/decimeter per g/ml. of DNA in solution. The concentration of DNA was estimated from an extinction coefficient for DNA of A~ro mil = 200 (Samejima & Yang, 1964) and a 10% scattering correction to the measured absorbance. The 10% scattering factor was arrived at by absorption measurements on T4 and T6 bacteriophage on a Cary model 14 spectrophotometer with a Cary model 1462 scattered transmission attachment. The salient features of the measurements are: (1) Each phage has a characteristic optical rotatory dispersion curve, by which it can be easily distinguished; even very closely related phages (such as the T-even phages) show significantly different curves. (2) The optical rotatory dispersion curves fall into three general types. Type I:
T2, T4B, T40, T6, ,\
T5, T7, B3
Type III: a, T4 DNA, T2 DNA, shocked T2 phage. A comparison of these three types is shown in Fig. 1, where the specific rotation curves of T2, T7 and a are presented. (3) A correlation can be found between the specific rotation values at 290 miL and the DNA content of each phage. Thus if a plot is made of [ah90 versus the percentage by weight of DNA in each phage, we obtain the graph shown in Fig. 2. One sees that the correlation is very good except for T2, T4 and T6. However, if a correction is made for the rotation due to the glucosylation of the 5.hydroxymethyl cytosine of the T-even phage family, it is found that the corrected points lie on the line (open circles). (The corrections made assumed that the deviation Ll[a]290 for T2 bacteriophage from the line is due to the 70% glucosylation of the 5-hydroxymethylcytosine in the phage DNA. Then T4 and T6 were proportionately corrected using the fact that T4 is 100% glucosylated and T6 is 147% glucosylated (Stent, 1963).) 287
l\1. F. l\lAE STBE AN D 1. TINO CO, .TR .
o E -0
FIG. 1. Optical rotatory disp ersion curves for T2. T7 and a bacteriophages.
% D NA in bac teri o phage
FIG . 2. P lot of values of [a]290 m .. ver8U8 the we ight per cen t of DNA in the phages. The filled c ircles are the values m easured for T2, T4B, T40 and T6. The values corrected for p ercentage glucosylation are shown by the empty circles la beled T2*, T4*B, T4*o and T6*. The weights per cent were determined or obtained from the following sources: Stent, 1963 (T2, T4, T6) ; Aurisicchio, Frontali & Graziosi, 1962 (a phage); Freifeld er (private communication) an d Davison & Freifeld er, 1962 (T7, T5); and MacHattie & T homas, 1964 (,\ phage) . The values wer e com. puted either from the rep orted m olecular weights of t h e phages and t hei r DNA's or t he r ep ort ed volumes and the relative d ensities from CsCI d en sit .y-gradient determinations. The weight per cent calcu lations are approximate and were done on ly to obtain a possible correlation between [ah 90 an d a mo lecular parameter of the phage.
LETTERS TO THE EDITOR
(4) The optical rotatory dispersion of osmotically shocked T2 phage is essentially that of free T2 DNA in solution. For complexes of T4 DNA and polyamines, it is essentially that of free T4 DNA in solution, except for a decrease in the magnitude of rotation caused by precipitation of aggregated DNA. (5) Measurements of T2 ghosts show that the protein coat does not contribute appreciably to the optical rotation of the phage in the wavelength region above 210 tau, The main influence on the optical rotatory dispersion of a polymer is the interaction among the polymer units. Therefore we suggest that the differences in optical rotation for the different phages mirror conformation differences of the DNA inside the phage. The correlation of specific rotation with DNA content of the phage is consistent with this hypothesis. A high DNA concentration in the phage should lead to a conformation of the DNA different from its conformation in solution. This explanation is not entirely satisfactory, because extrapolation of the line in Fig. 2 to zero DNA content (free DNA in solutiont] leads to an [a.]290 higher than any measured in solution. Samejima & Yang (1965) give values which range from about 1500 to 2500 for DNA's of different composition. However, other explanations of the results are possible. Specific interactions of the DNA with the phage polyamines or protein coat could change the rotation. The experiments done on shocked phage or mixtures of DNA and polyamines cannot rule this out. Also the effective pH and ionic strength inside the phage (which can be quite different from that in solution) could influence the DNA rotation. Whatever interpretation for the optical rotatory dispersion curves is finally established, it is clear that such measurements can be very useful in the study of bacteriophages. A small amount of material (3 ml. of A 2 6 0 = 1) is sufficient to allow identification of the type and amount of phage present. Furthermore, the optical rotatory dispersion is apparently very sensitive to the state of the DNA inside the phage and is therefore particularly suited for studies of this aspect of phage structure. We are indebted to Dr D. Freifelder of the Donner Laboratories at the University of California, Berkeley, for the supply of bacteriophages T5, T6, T7, ex, B3 and A. T4B was obtained from Dr M. Gellert of the National Institutes of Health, Bethesda, Md. and T40 (shock-resistant mutant) from Dr J. Hearst, Chemistry Department, University of California, Berkeley. The T2 (r+ strain) was grown from an original strain obtained from Dr P. Kahn at Yale University, New Haven, Conn. One of us (M.F.M.) is sponsored by a U.S. Public Health postdoctoral fellowship, number 5-F2-Al·13, 230-02. The work was supported in part by Public Health Service research grant GM 10840. Chemistry Department University of California Berkeley, California, U.S.A.
MARcos F. MAESTRE IGNACIO TINOOO, JR.
Received 16 January 1965 REFERENCES Aurisicchio, S., Frontali, C. & Oraziosi, F. (1962). Nuovo Oimento Supp, 25, no. 1, 35. Davison, P. F. & Friefelder, D. (1962). J. Mol. Biol. 5, 635. MacHattie, L. A. & Thomas, Jr., C. A. (1964). Science, 144, 1142. Samejima, T. & Yang, J. T. (1964). Biochemistry, 3, 613. Samejima, T. & Yang, J. T. (1965). J. Bioi. Ohern. in the press. Stent, G. S. (1963). Molecular Biology oj Bacterial Vir~e8. San Francisco, California: W. H. Freeman & Company. 19