Philip Serwer

Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA

The complex double-stranded DNA bacteriophages assemble DNA-free protein shells (procapsids) that subsequently package DNA. In the case of several double-stranded DNA bacteriophages, including P22, packaging is associated with cutting of DNA from the concatemeric molecule that results from replication. The mature intravirion P22 DNA has both non-unique (circularly permuted) ends and a length that is determined by the procapsid. In all known cases, procapsids consist of an outer coat protein, an interior scaffolding protein that assists in the assembly of the coat protein shell, and a ring of 12 identical portal protein subunits through which the DNA is presumed to enter the procapsid. To investigate the role of the portal protein in cutting permuted DNA from concatemers, we have characterized P22 portal protein mutants. The effects of several single amino acid changes in the P22 portal protein on the length of the DNA packaged, the density to which DNA is condensed within the virion, and the outer radius of the capsid have been determined. The results obtained with one mutant (NT5/1a) indicate no change (+/- 0.5%) in the radius of the capsid, but mature DNA that is 4.7% longer and a packing density that is commensurately higher than those of wild-type P22. Thus, the portal protein is part of the gauge that regulates the length and packaging density of DNA in bacteriophage P22. We argue that these findings make models for DNA packaging less likely in which the packing density is a property solely of the coat protein shell or of the DNA itself.

The complete sequence of marine bacteriophage VpV262 infecting vibrio parahaemolyticus indicates that an ancestral component of a T7 viral supergroup is widespread in the marine environment

The 46,012-bp sequence of the marine bacteriophage VpV262 infecting the bacterium Vibrio parahaemolyticus is reported. The VpV262 sequence reveals that it is a distant relative of marine Roseophage SIO1, and an even more distant relative of coliphage T7. VpV262 and SIO1 appear to represent a widespread marine phage group that lacks an RNA polymerase gene and is ancestral to the T7-like phages. We propose that this group together with the T7-like phages be designated as the T7 supergroup. The ancestral head structure gene module for the T7 supergroup was reconstructed by using sensitive biased Psi-blast searches supplemented by statistical support derived from gene order. In the early and replicative segments, these phages have participated in extensive interchange with the viral gene pool. VpV262 carries a different replicative module than SIO1 and the T7-like phages.

Propagating the missing bacteriophages: a large bacteriophage in a new class

The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly). As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter), tail (486 × 26 nm), corkscrew-like tail fibers (187 × 10 nm) and genome (221 Kb) that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305φ8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage), has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305φ8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305φ8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305φ8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion.

Exclusion of spheres by agarose gels during agarose gel electrophoresis: dependence on the sphere's radius and the gel's concentration

Agarose gel electrophoresis of spheres (radius = R) has been used to determine the effective radius (PE) of the pores of an agarose gel (percentage of agarose in a gel = A). The value of PE at a given A was taken to be the R of the largest sphere that enters the gel. When log PE is plotted as a function of log A, the results can be represented by: PE = 118A-0.74 for 0.2 less than or equal to A less than or equal to 4.0 (PE in nm). However, the data suggest significant nonlinearity in this plot, the magnitude of the exponent of the PE vs A relationship increasing by about 20% as A increases from 0.2 to 4.0. From these data, PEs as big as 1500 nm and as small as 36 nm can be achieved with agarose gels formed with unmodified, unadulterated agarose and usable for electrophoresis.

The relationship of agarose gel structure to the sieving of spheres during agarose gel electrophoresis.

To understand the organization of fibers in an agarose gel, digitized electron micrographs are used here to determine the frequency distribution of interfiber distance (2Pc) in thin sections of agarose gels. For a preparation of underivatized agarose, a 1.5% gel has a Pc distribution that is indistinguishable from the Pc distribution of a computer-generated, random-fiber gel; the log of the occurrence frequency (F) decreases linearly as a function of Pc. As the agarose concentration decreases below 1.5%, the semilogarithmic F versus Pc plot becomes progressively less linear. Two straight lines represent the data; the plot is steeper at the lower Pc values. As the percentage of agarose increases above 1.5%, the semilogarithmic F versus Pc plot becomes steeper at the higher Pc values. This change in the shape of semilogarithmic F versus Pc plots is possibly explained by the existence in agarose gels of two zones, one whose Pc distribution is more sensitive to the average agarose concentration than the other. To compare the structure of agarose gels to their sieving during electrophoresis, the root mean square value of Pc (Pc) is compared to the sieving-based radius of the effective pore (PE; Griess et. al. (16)) for both underivatized agarose and a derivatized agarose that has a smaller PE at any given agarose percentage. For 0.8-2.0% gels of either underivatized or derivatized agarose, PE/Pc is a constant within experimental error. Deviations from this constant are observed at lower gel percentages. This relationship of PE to Pc constrains theoretical descriptions of the motion of spheres in fibrous networks.

Models of bacteriophage DNA packaging motors

An ATP-dependent motor drives a DNA genome into a bacteriophage capsid during morphogenesis of double-stranded DNA bacteriophages both in vivo and in vitro. The DNA molecule enters the capsid through a channel in the center of a symmetric protein ring called a connector. Mechanisms in two classes have been proposed for this motor: (1) An ATP-driven rotating connector pulls a DNA molecule via serial power strokes. (2) The connector rectifies DNA motion that is either thermal, biased thermal, or oscillating electrical field-induced (motor-ratchet hypothesis). Mechanisms in the first class have previously been proposed to explain the detailed structure of DNA packaging motors. The present study demonstrates that the motor-ratchet hypothesis also explains the current data, including data in the following categories: biochemical genetics, energetics, structure, and packaging dynamics.

Arrangement of double-stranded DNA packaged in bacteriophage capsids. An alternative model.

Toroidal winding of double-stranded DNA in the protein capsids of bacteriophages has been proposed previously. An alternative model for the packaging and arrangement of DNA in bacteriophage capsids is presented here. By introducing sharp folds, the alternative model avoids toroidal winding and its accompanying difficulties. This alternative model is in agreement with the current data obtained with several different bacteriophages.

Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7

Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used single-particle electron cryomicroscopy to study the multilayer structure of the portal vertex of the bacteriophage T7 procapsid, the recipient of T7 DNA in packaging. A focused asymmetric reconstruction method was developed and applied to selectively resolve neighboring pairs of symmetry-mismatched layers of the portal vertex. However, structural features in all layers of the multilayer portal vertex could not be resolved simultaneously. Our results imply that layers with mismatched symmetries can join together in several different relative orientations, and that orientations at different interfaces assort independently to produce structural isomers, a process that we call combinatorial assembly isomerism. This isomerism explains rotational smearing in previously reported asymmetric reconstructions of the portal vertex of T7 and other bacteriophages. Combinatorial assembly isomerism may represent a new regime of structural biology in which globally varying structures assemble from a common set of components. Our reconstructions collectively validate previously proposed symmetries, compositions, and sequential order of T7 portal vertex layers, resolving in tandem the 5-fold gene product 10 (gp10) shell, 12-fold gp8 portal ring, and an internal core stack consisting of 12-fold gp14 adaptor ring, 8-fold bowl-shaped gp15, and 4-fold gp16 tip. We also found a small tilt of the core stack relative to the icosahedral fivefold axis and propose that this tilt assists DNA spooling without tangling during packaging.

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.

Improvements in procedures for electrophoresis in dilute agarose gels

Abstract Procedures have been developed for performing electrophoresis in agarose gels with agarose concentrations as low as 0.035%. Using these procedures, agarose gel electrophoresis of the following has been performed: (a) bacteriophage T7 missing its tail fibers; no detectable sieving of this spherical particle (radius = 30.1 nm) occurred below 0.075% agarose, (b) duplex DNAs with molecular weights between 26.5 × 106 and 110 × 106.