Robert H. Watson

Comparison of the physical properties and assembly pathways of the related bacteriophages T7, T3 and II*

To understand constraints on the evolution of bacteriophage assembly, the structures, electrophoretic mobilities (mu) and assembly pathways of the related double-stranded DNA bacteriophages T7, T3 and phi II, have been compared. The characteristics of the following T7, T3 and phi II capsids in these assembly pathways have also been compared: (1) a DNA-free procapsid (capsid I) that packages DNA during assembly; (b) a DNA packaging-associated conversion product of capsid I (capsid II). The molecular weights of the T3 and phi II genomes were 25.2 X 10(6) and 25.9 (+/- 0.2) X 10(6) (26.44 X 10(6) for T7, as previously determined), as determined by agarose gel electrophoresis of intact genomes. The radii of T7, T3 and phi II bacteriophages were indistinguishable by sieving during agarose gel electrophoresis (+/- 4%) and measurement of the bacteriophage hydration (+/- 2%) (30.1 nm for T7, as previously determined). Assuming a T = 7 icosahedral lattice for the arrangement of the major capsid subunits (p10A) of T7, T3 and phi II best explains these data and data previously obtained for T7. At pH 7.4 and an ionic strength of 1.2, the solid-support-free mu values (mu 0 values) of T7, T3 and phi II bacteriophages, obtained by extrapolation of mu during agarose gel electrophoresis to an agarose concentration of 0 and correction for electro-osmosis, were -0.71, -0.91 and -1.17(X 10(-4) cm2V-1 s-1. The mu 0 values of T7, T3 and phi II capsids I were -1.51, -1.58 and -2.07(X 10(-4] cm2V-1 s-1. For the capsids II, these mu 0 values were -0.82, -1.07 and -1.37(X 10(-4] cm2V-1 s-1. The tails of all three bacteriophages were positively charged and the capsid envelopes (heads) were negatively charged. In all cases the procapsid had a negative mu 0 value larger in magnitude than the negative mu 0 value for bacteriophage or capsid II. A trypsin-sensitive region in capsid I-associated, but not capsid II-associated, T3 p10A was observed (previously observed for T7). The largest fragment of trypsinized capsid I-associated p10A had the same molecular weight in T7 and T3, although the T3 p10A is 18% more massive than the T7 p10A. It is suggested that the trypsin-resistant region of capsid I-associated p10A determines the radius of the bacteriophage capsid.

Conformation of DNA packaged in bacteriophage T7: Analysis by use of ultraviolet light-induced DNA-capsid cross-linking☆

The conformation of the linear, double-stranded, 39,936 kilobase-pair DNA packaged in the protein capsid of bacteriophage T7 is investigated here by use of short wavelength ultraviolet light-induced DNA-capsid cross-linking. To detect both DNA-capsid and DNA-DNA cross-links, DNA is expelled from the T7 capsid and the products of expulsion are analyzed by use of Nycodenz buoyant density centrifugation, followed by either pulsed field gel electrophoresis or invariant field gel electrophoresis. Short wavelength ultraviolet light is found to progressively induce both DNA-DNA and DNA-protein cross-links in intact bacteriophage T7, but not in T7 from which DNA had been expelled before exposure to ultraviolet light. Protein-protein cross-links are not induced. When DNA expelled from previously cross-linked T7 is cleaved with restriction endonuclease (1 to 3 sites cleaved), analysis of the resulting fragments reveals no regions on T7 DNA that are excluded from cross-linking to the capsid. However, the efficiency of cross-linking decreases as the distance from the left end (last end packaged) of the packaged DNA increases. Electron microscopy of negatively stained capsid-DNA complexes reveals no DNA-retaining structure other than the outer shell of the capsid. Together with previously reported data that indicate lack of protein-based specificity for ultraviolet light-induced cross-linking, these observations are interpreted by the assumptions that, within the limits of resolution of these experiments: (1) no region of packaged T7 DNA is excluded from contact with the outer shell of the T7 capsid; (2) the probability of contacting the outer shell decreases as the distance from the left end of packaged T7 DNA increases. Thus, T7 DNA packaging concentrates the last end packaged near the inner surface of the outer shell of the T7 capsid.

Capsid-DNA complexes in the DNA packaging pathway of bacteriophage T7: Characterization of the capsids bound to monomeric and concatemeric DNA

Abstract Complexes of capsids with monomeric bacteriophage T7 DNA and complexes of capsids with concatemeric bacteriophage T7 DNA have been isolated from lysates of T7-infected Escherichia coli and the capsids of these complexes have been characterized. In electron micrographs capsids bound to either monomeric DNA or concatemeric DNA have envelopes which are: (1) significantly less thick than the envelope of a DNA-free capsid (capsid I) isolated from T7-infected E. coli , (2) indistinguishable from the envelope of bacteriophage T7 and the envelope of a second DNA-free capsid (capsid II) isolated from T7-infected E. coli . By agarose gel electrophoresis (under nondenaturing conditions) and SDS-polyacrylamide gel electrophoresis the capsids released from the above capsid-DNA complexes are similar to capsid II and different from capsid I. The physical characteristics and radiolabeling kinetics of these capsid-DNA complexes suggest that they are DNA packaging intermediates; the results suggest a pathway for the packaging of DNA by bacteriophage T7.

Electrophoresis in density gradients of metrizamide

Abstract Linear density gradients of metrizamide have been used to prevent convection during electrophoresis in an apparatus originally designed for use with cylindrical gels. The procedures used are comparatively easy to perform. Two capsids of bacteriophage T7 previously shown to differ in average electrical surface charge density (σ) have been separated from each other by electrophoresis in gradients of metrizamide. These capsids were cleanly separated from both T7 DNA and a complex of a T7 capsid with T7 DNA. Thus, electrophoresis in density gradients of metrizamide is a technique for separating capsid-DNA complexes from DNA-free capsids and should be capable of separating other supramolecular complexes and organelles which differ in σ, some of which may be too large to be separated by electrophoresis in gels.

The structure of a bacteriophage T7 procapsid and its in vivo conversion product probed by digestion with trypsin.

Abstract In previous studies it was shown that a DNA-free bacteriophage T7 procapsid (capsid I) draws DNA to its interior during assembly and as it begins this packaging capsid I changes in structure to capsid II. To probe the structures of capsids I and II, they have been digested with trypsin. The most abundant protein in the envelopes of capsids I and II, P10, was cleaved when capsid I was digested, but was not cleaved when capsid II was digested. Cleavage of capsid I-associated P10 formed a major P10 fragment 18% reduced in molecular weight, but did not detectably affect the size of capsid I (determined from sieving during agarose gel electrophoresis) or appearance of capsid I in the electron microscope. The magnitude of the (negative) average electrical surface charge density of capsid I was increased by digestion with trypsin (determined by agarose gel electrophoresis). At least one minor P10 fragment remained associated with capsid I after digestion.

Formation of the right before the left mature DNA end during packaging-cleavage of bacteriophage T7 DNA concatemers

During bacteriophage T7 morphogenesis in a T7-infected cell, mature length T7 DNA molecules join end-to-end to form concatemers that are subsequently both packaged in the T7 capsid and cut to mature size. In the present study, the kinetics of the appearance in vivo of the mature right and left T7 DNA ends have been analyzed. To perform this analysis, the intercalating dye proflavine is used to interrupt DNA packaging. When used at 0.5 to 8.0 micrograms/ml, proflavine progressively inhibits events in the T7 DNA packaging pathway, without either altering protein synthesis or degrading intracellular T7 DNA. Restriction endonuclease kinetic analysis reveals that proflavine (8 micrograms/ml) completely blocks formation of the mature T7 DNA left end, but only partially blocks formation of the mature T7 DNA right end. Both these and other observations are explained by the hypothesis that, in the T7 DNA packaging pathway, events occur in the following sequence: (1) formation of a mature right end; (2) packaging of at least some of the genome; (3) formation of the mature left end.

Alterations of the bacteriophage T7 and T3 DNA packaging pathway in Escherichia coli mutant TSN B

Data previously obtained indicate that, during assembly of the related bacteriophages T7 and T3, a DNA-free procapsid (capsid I) is produced and that subsequently capsid I: (1) binds to a longer than mature (concatemeric) DNA and then becomes structurally altered to a particle isolated as a capsid (capsid II) physically resembling the mature bacteriophage capsid more than the procapsid (initiation phase of packaging), (2) draws DNA to its interior (entry phase of packaging), (3) participates in cutting the concatemeric DNA to mature size. It was found that, after infection of Escherichia coli mutant tsnB (selected for a deficiency in plating T7; M. Chamberlin [1974], J. Virol. 14, 509-516), T7 and T3 capsid I is assembled at a rate not significantly different from its rate of assembly in the wild-type host. However, the conversion of capsid I to capsid II was slowed in E. coli tsnB, suggesting that the tsnB mutation interferes with the initiation of DNA packaging. Although some T3 and T7 DNA enters capsids and is cut to mature size in the tsnB mutant, the data further suggest that the entry rate of DNA into capsid II is lower in the tsnB mutant than it is in an unaltered host. T7 capsid II-concatemeric DNA complexes accumulate during infection of the tsnB mutant. These observations suggest that use of the tsnB mutant as a host will simplify studies of bacteriophage T7 and T3 DNA packaging.