Irwin J. Bendet

Functions and properties related to the tail fibers of bacteriophage T4

Abstract It is shown that adsorbability of T4 is regularly correlated with the extended state of the tail fibers, suggesting that in T4 fiber extension is a necessary condition for adsorption. Furthermore the extension and retraction of fibers is correlated with the dual sedimentation of T4 observed during ultracentrifugation. For T4, 38, which requires tryptophan for adsorption, electron microscopy shows the tail fibers to be extended in the presence of tryptophan and retracted in its absence. Phages with retracted fibers (no tryptophan) show a faster sedimentation than those with extended fibers (with tryptophan). In the absence of tryptophan T4, 38 does not show fiber extension even at pH 7, a condition sufficient for nontryptophan-requiring T4, whose fibers are retracted at pH 5. A conditional lethal mutation in gene 37 of phage T4D results in phages which lack tail fibers entirely. These phages no longer adsorb. In the ultracentrifuge, only the fast form can be observed whether at pH 7 or 5. Phages with extended fibers are more rapidly inactivated by ultrasonic waves than phages with retracted fibers. Measurements on electron micrographs show that the head size of T4 is invariant. A model of the functioning fiber apparatus is proposed and discussed.

The hydration of viruses.

Publisher Summary The size and shape of virus nucleoprotein particles can be evaluated unambiguously only when the degree of hydration is known. This chapter discusses several methods that are available for the determination of the hydration of protein crystals. These include the gravimetric analysis of crystals before and after drying, density determinations of crystals, analyses of secondary solute concentrations in equilibrium with crystals, X-ray diffraction analyses of crystal lattice dimensions in the hydrated and in the unhydrated state, and optical analyses of crystals. Only the X-ray diffraction and the optical methods actually have been applied to the crystals of virus nucleoproteins. Most methods of investigating hydration, particularly hydration of particles in solution, fall into three categories. The first category provides information about the total displacement of the hydrated particle. This category includes the direct displacement method of Lauffer and Taylor and methods involving various combinations of sedimentation, diffusion, rotational diffusion, and viscosity. The second category evaluates the effective specific volume of the hydrated particle such as sedimentation in media of different densities. The third category evaluates directly the volume or the mass of solvate bound by the particle. This category includes the methods that presumably determine the amount of solvent not free to dissolve a secondary solute such as vapor pressure determinations, freezing point depressions, boiling point elevations, and the distribution of secondary solute among phases or across membranes. Direct gravimetric or chemical analyses and X-ray diffraction analyses of crystals also fall within this classification.

The size of T3 DNA

The nucleic acid of the bacteriophage T3 has been studied with the electron microscope in order to determine whether it exists either as a single unit or as several pieces of equal or varying size. The nucleic acid strands, obtained by mild alkali treatment of the virus, were oriented on collodion films and shadowed perpendicular to their length. None of the strands exhibited any branching, being continuous from one end to the other, and their diameters were found to be relatively constant. Measurement of seven such strands revealed a mean length of 14·0 ± 0·6 μ . The relatively good agreement of this value with that arrived at from theoretical calculations can be interpreted to mean that all of the T3 DNA is in one piece.

Dual sedimentation of T2 bacteriophage of Escherichia coli

Abstract For T2 bacteriophage, diffusion coefficients of 3.25·10 −8 cm 2 /sec and 2.63·10 −8 cm 2 /sec were obtained in pH 5 and pH 7 buffers, respectively. The transition limits for dual sedimentation encompass approximately one-half pH units, with a central value at about pH 6.1. Sedimenting the virus from a medium of one pH directly into a second medium of greater density and different pH has indicated that the sedimentation rate change is quite sharp and complete within a few minutes after passing through the interface. s 5 / D 5 differs significantly from s 7 / D 7 but not enough to represent one-to-one aggregation. It is, therefore, assumed that the molecular weight is the same at pH 7 and pH 5. It follows from this assumption and the experimental results that f s 7 / f s 5 is significantly greater than f D 7 / f D 5 . A model has been found which can account for all of these facts. It is assumed that at pH 5 the particle is tadpole-shaped but that at pH 7 it projects thin filaments laterally from the tail. Such filaments have been demonstrated with the electron microscope. Calculations of friction coefficients made for complex ellipsoidal models approximating the actual models discussed above yield results consistent with the experimental observations, if it is assumed that the particles are oriented randomly during diffusion but sediment in the direction of their longest axis.

Some biological and physicochemical properties of blue-green algal virus LPP-1☆

Adsorption and one-step growth experiments performed with blue-green algal virus (BGAV) LPP-1 under normal conditions revealed a slow adsorption rate, in comparison with bacteriophage systems, followed by a 7-hour latent period. The burst size was approximately 100 virus particles per cell. The molecular weight of the virus obtained from sedimentation and diffusion measurements was 51 +/- 3 x 10(6) daltons, and the hydration was calculated to be 0.37 g of H2O per gram of virus. Phosphorus analysis indicated a DNA content of 40% w/w, while amino acid analysis revealed all the common amino acids to be present. At reduced magnesium concentrations, the virus breaks apart, liberating its DNA, which then can be separated from the protein by sedimentation in a CsCl density gradient. A protein component was obtained as ghosts. Neutralization experiments with BGAV antiserum established no relationship to bacteriophages T1, T2, or T3.

Ultraviolet Dichroism of fd Bacteriophage

The internal structure of the bacterial virus fd was investigated by ultraviolet dichroism of virus solutions oriented by flow through a small capillary tube. The dichroism was found to be positive for wavelengths longer than 262 mmicro and shorter than 239 mmicro, and negative for the intermediate wavelengths. The magnitude of the effect was at all times small, with dichroic ratios of 1.22 and 0.83 at 280 mmicro and 250 mmicro, respectively. the intuitive interpretation that this was the result of the addition of negative DNA dichroism and positive protein dichroism was confirmed by the application of a simple theory which allowed the calculation from protein and DNA absorption data of a dichroism curve closely approximating the experimental one. The parameters arrived at by this procedure indicate a semiangle of 25 degrees +/-5 degrees for a cone described by the normals to the DNA base planes inside the virus. The protein absorbers tryptophan and probably tyrosine were found to be oriented on the average relatively parallel to the longitudinal axis of the virus.

The protein subunit of cucumber virus 4; degradation of viruses by succinylation.

Abstract The protein of cucumber virus 4 was dissociated into subunits by succinylation. The subunit molecular weight was found to be 19,000 by sedimentation-diffusion and 21,500 by osmotic pressure. The intact virus was degraded into subunits by succinylation as well. Tobacco mosaic virus, Tipula iridescent virus, and T2 bacteriophage were also degraded to slower-sedimenting components by treatment with succinic anhydride.

Biophysical Characterization of Bacteriophage Nucleic Acid

Publisher Summary The chapter discusses the biophysical characterization of bacteriophage nucleic acid. While bacteriophages have molecular weights of millions to hundreds of millions, nevertheless, in a few instances, their chemical composition is known to a considerable extent. In the main, their principal chemical constituents are protein and deoxyribonucleic acid (DNA), with the latter accounting for up to one-half of the particle weight. For the T-even bacteriophages, one generally alludes to three classes of proteins, characterized fortuitously perhaps, by their morphological localizations: (1) a “head” protein, which comprises the membrane surrounding the nucleic acid of the head, and is left behind as a “ghost” when bacteriophages are subjected to osmotic shock and their DNA is released; (2) “tail” proteins, which are involved in the adsorption and penetration phases of infection; and (3) a non-sedimentahle protein, commonly referred to as “internal” protein. The DNA of the T-even bacteriophages is unique since along with the usual constituent bases adenine, thymine, and guanine it contains 5hydroxymethy lcytosine instead of cytosine. Apparently a small amount of 6-methylaminopurine also occurs in T2 DNA, while it has been reported that 5-hydroxymethyl uracil has been isolated from the DNA of the bacteriophage SP8. The physical dimensions and the organization of the nucleic acid of bacteriophage is very much the result of many different areas of biophysical investigation, among which may be mentioned density gradient equilibrium sedimentation, autoradiography, electron microscopy, birefringence studies, and X-ray diffraction. The chapter discusses these techniques in the light of what information each has contributed to the elucidation of the physical state of the nucleic acid within bacteriophage.

Orientation of the RNA in tobacco mosaic virus

Abstract The configuration of the ribonucleic acid within tobacco mosaic virus was studied by comparing the ultraviolet dichroism of flow-oriented virus and repolymerized, nucleic acid free tobacco mosaic virus protein. The dichroism of the RNA within tobacco mosaic virus, obtained by subtracting the protein contribution from the dichroic spectra of the virus, has been interpreted as being due to a preferred orientation of the planes of the purine and pyrimidine bases of the RNA “approximately” parallel to the longitudinal axis of the virus. The dichroism of the protein suggests a preferred orientation of the aromatic amino acids, especially tryptophan, “approximately” parallel to the longitudinal axis. The dichroic ratio of tobacco mosaic virus was found to be influenced by the asymmetry of the particles in the flowing solution and by the geometry of the flow cell. With linearly aggregated particles at pH 5.15 and an optical flow cell of square cross-section, the dichroic ratios for tobacco mosaic virus in this study were appreciably higher than those previously obtained by others.

Subunits of T4 head structures

Abstract Capsids of bacteriophage T4 have been dissociated in three different solvents: (1) 6 M guanidine hydrochloride, (2) 6 M guanidine hydrochloride plus 0.1 M β-mercaptoethanol, and (3) 67% acetic acid. Sedimentation equilibrium experiments done in the presence of mercaptoethanol have shown that the capsid subunits can be divided into two classes with molecular weights of approximately 11,000 and 46,000. In the absence of mercaptoethanol, a component of molecular weight 78,000 has been found, indicating that disulfide bonds may be important in stabilizing the phage head structure.