Variation of the permeability of bacteriophage T4: analysis by use of a protein-specific probe for the T4 interior

The permeability of bacteriophage T4 and the change in T4 permeability caused by mutation to osmotic shock resistance are investigated here by quantification of the kinetics with which both a DNA-specific probe (ethidium) and a protein-specific probe [1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid, or bis-ANS)] bind to T4. In the case of an osmotic shock-resistant mutant, T40s41, both ethidium and bis-ANS bind with first order kinetics. The first-order rate constant (k*) for both bis-ANS and ethidium is a function of anion type and concentration. Adenosine triphosphate, phosphate, bisulfite, sulfate, and acetate anions all reduce k* below the k* observed when chloride is the only anion. When chloride is the only anion at 25 degrees C, k* values for binding to T40s41 are orders of magnitude above k* values for binding to wild-type T4 (T4wt). At 25 degrees C, k* for T4wt is too small to measure but k* for T4wt increases at 50-55 degrees C to values approaching those measured for T40s41, without inactivating T4wt, when chloride is the only anion; during heating, T4wt is stabilized by both ethidium and bis-ANS. Binding to T4wt is reversible at 50-55 degrees C, but not at 25 degrees C. Equilibrium binding of bis-ANS to T40s41 reveals 112 +/- 24 sites per T4 capsid. Equilibrium binding of ethidium to T40s41 reveals both high- and low-affinity sites previously observed in the packaged DNA of other bacteriophages. The ATP-induced decrease in k* is not accompanied by a decrease in equilibrium binding. The following hypotheses are presented to explain the above data: (a) All detected bis-ANS binding sites on T4 are interior to the outer surface of T4. (b) The value of k* for both bis-ANS and ethidium is controlled at the port(s) of passage through the outer shell of the T4 capsid. (c) The anions present control k* values at the port(s) of entry, probably by controlling the size of this port. The effects on k* of phosphate explain the otherwise paradoxical observation [P. J. McCall and V. A. Bloomfield (1976) Biopolymers 15, 2323-2336] that in a phosphate buffer the permeabilities of T4wt and T40s41 are the same.     

DNA packaging in vitro by an isolated bacteriophage T7 procapsid.

The results of previous in vivo studies indicate that a DNA-free procapsid (capsid I) packages bacteriophage T7 DNA during infection of Escherichia coli. It was shown here that capsid I, isolated by electrophoresis in metrizamide density gradients, packaged DNA and formed infectious phage particles when incubated in vitro with extracts deficient in capsid proteins.     

Assembly of bacteriophage T7. Dimensions of the bacteriophage and its capsids.

The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering. Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10(-16) ml. The outer radius determined for T7 phage in solution is approximately 30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy. Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV). In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approximately 5% smaller than the envelope of T7 phage. The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained. The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10(-17) ml. The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between, DNA strands. The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- less than 2.2 A. A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV. This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV.     

Detection and characterization of agarose-binding, capsid-like particles produced during assembly of a bacteriophage T7 procapsid.

It has previously been shown that: (i) during infection of its host, the DNA bacteriophage T7 assembles a DNA-free procapsid (capsid I), a capsid with an envelope differing physically and chemically from the capsid of the mature bacteriophage, and (ii) capsid I converts to a capsid (capsid II) with a bacteriophage-like envelope as it packages DNA. Lysates of phage T7-infected Escherichia coli contained a particle (AG particle) which copurified with capsid II during buoyant density sedimentation, velocity sedimentation, and solid support-free electrophoresis, but was distinguished from capsid II by its apparent diversity during electrophoresis in agarose gels. Treatment of AG particles with trypsin converted most of them to particles that comigrated with trypsin-treated capsid II during electrophoresis in agarose gels. Irreversible binding of AG particles to agarose gels was shown to contribute to the apparent diversity of AG particles during agarose gel electrophoresis. The results of quantitation of AG particles and of capsid I and capsid II in lysates of a nonpermissive host infected with T7 amber mutants suggested that, in site of their capsid II-like properties, most AG particles were produced during assembly of capsid I and not during DNA packaging. The presence of AG particles in T7 lysates explains contradictions in previous data concerning the pathway of T7 assembly.     

Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels.

Agarose gel electrophoresis of the following was performed in 0.05 M sodium phosphate-0.001 M MgCl2 (pH 7.4): (i) bacteriophage T7; (ii) a T7 precursor capsid (capsid I), isolated from T7-infected Escherichia coli, which has a thicker and less angular envelope than bacteriophage T7; (iii) a second capsid (capsid II), isolated from T7-infected E. coli, which has a bacteriophage-like envelope; and (iv) capsids (capsid IV) produced by temperature shock of bacteriophage T7. Bacteriophage T7 and all of the above capsids migrated towards the anode. In a 0.9% agarose gel, capsid I had an electrophoretic mobility of 9.1 +/- 0.4 X 10(-5) cm2/V.s; bacteriophage T7 migrated 0.31 +/- 0.02 times as fast as capsid I. The mobilities of different preparations of capsid II varied in such gels: the fastest-migrating capsid II preparation was 0.51 +/- 0.03 times as fast as capsid I and the slowest was 0.37 +/- 0.02 times as fast as capsid I. Capsid IV with and without the phage tail migrated 0.29 +/- 0.02 and 0.42 +/- 0.02 times as fast as capsid I. The results of the extrapolation of bacteriophage and capsid mobilities to 0% agarose concentration indicated that the above differences in mobility are caused by differences in average surface charge density. To increase the accuracy of mobility comparisons and to increase the number of samples that could be simultaneously analyzed, multisample horizontal slab gels were used. Treatment with the ionic detergent sodium dodecyl sulfate converted capsid I to a capsid that migated in the capsid II region during electrophoresis through agarose gels. In the electron microscope, most of the envelopes of these latter capsids resembled the capsid II envelope, but some envelope regions were thicker than the capsid II envelope.     

Comparison of the physical properties and assembly pathways of the related bacteriophages T7, T3 and phi 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.     

Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro.

Bacteriophage T7 packages its double-stranded DNA genome in a preformed protein capsid (procapsid). The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the mature 40-kilobase pair genome. Mature genomes are cleaved from the concatemer during packaging. In the present study, fluorescence microscopy is used to observe T7 concatemeric DNA packaging at the level of a single (microscopic) event. Metabolism-dependent cleavage to form several fragments is observed when T7 concatemers are incubated in an extract of T7-infected Escherichia coli (in vitro). The following observations indicate that the fragment-producing metabolic event is DNA packaging: 1) most fragments have the hydrodynamic radius (R(H)) of bacteriophage particles (+/-3%) when R(H) is determined by analysis of Brownian motion; 2) the fragments also have the fluorescence intensity (I) of bacteriophage particles (+/-6%); 3) as a fragment forms, a progressive decrease occurs in both R(H) and I. The decrease in I follows a pattern expected for intracapsid steric restriction of 4',6-diamidino-2-phenylindole (DAPI) binding to packaged DNA. The observed in vitro packaging of a concatemer's genomes always occurs in a synchronized cluster. Therefore, the following hypothesis is proposed: the observed packaging of concatemer-associated T7 genomes is cooperative.     

Binding of ethidium to bacteriophage T7 and T7 deletion mutants

Equilibrium binding of ethidium, quantitated by fluorescence enhancement, to DNA packaged in bacteriophage T7 and T7 deletion mutants has been compared with the binding of this dye to DNA released from its capsid (free DNA). During achievement of apparent equilibrium binding, no change in bacteriophage T7 structure occurred, by the criterion of agarose gel electrophoresis. However, excessive incubation with ethidium bromide caused detectable changes in bacteriophage structure, a possible explanation of disagreements in similar studies previously performed with T-even bacteriophages. Scatchard plots for packaged DNA had a curvature greater than the previously demonstrated [Bresloff, J. L. & Crothers, D. M. (1981) Biochemistry 20 , 3547–3553] curvature for free DNA. By treating plots for packaged DNA as though they were biphasic, it was found that binding to most sites occurred with an apparent association constant (K_ap) 3.3–4.3 times lower than the K_ap of free DNA. The number of these sites increased significantly as the density of packaged DNA was decreased by use of the deletion mutants. Values of ΔH° for these sites were negative and equal to the ΔH° for free DNA; values of ΔS° were positive and about half the ΔS° for free DNA. A second class of sites, roughly 1.2% of the total, had a significantly higher K_ap and more negative ΔH° than those of the majority of sites.     

Effect of rifampicin on the infectivity of RNA bacteriophage f2.

RNA bacteriophage f2, treated in vitro with rifampicin, loses infectivity dramatically. Rifampicin interacts with phage RNA, binding to a few specific sites. Inhibition of phage RNA infectivity occurs at 10-100 times lower molar excess of rifampicin than inhibition of infectivity of intact phage particles. Thus the phage capsid acts as a barrier, diminishing interaction of the drug with phage RNA.     

A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Stability and in vitro DNA packaging of bacteriophages: effects of dextrans, sugars, and polyols.

Attempts were made to increase the efficiency of infectious particle formation during the in vitro assembly of bacteriophage T7 from procapsids and DNA. It was found that dextrans and some smaller, related compounds (sucrose and sorbitol) increase this efficiency by a factor of 8 to 50. Dextrans also inhibited elevated temperature-induced emptying of DNA from bacteriophages T7, P22, and T4, suggesting that the stimulation of assembly is caused, at least in part, by the stabilization of packaged DNA in capsids. The data indicated that the sugars and polyols can slow DNA emptying from bacteriophages at elevated temperature whether they permeate the bacteriophage capsid or not. In contrast, the data suggested that permeation of some particle, probably a capsid, results in inhibition of in vitro T7 assembly.     

Interaction of a DNA-binding protein, the gene product of D5 of bacteriophage T5, with double-stranded DNA. Analysis by metrizamide gradient centrifugation

Interactions of DNA and the gene product D5 (gpD5) of bacteriophage T5, a DNA-binding protein that binds preferentially and cooperatively to double-stranded DNA, were analyzed by metrizamide gradient centrifugation. Conditions were set so that DNA and DNA protein complex sedimented to apparent equilibrium positions. DNA has a buoyant density of 1.12 g/cm3, and DNA saturated with gpD5 has a buoyant density of 1.17 g/cm3. These values are independent of DNA size and base composition in the range studied. At gpD5 concentration below the saturation value in a low ionic strength buffer, DNA distribution is bimodal, indicating cooperative binding of gpD5 to DNA. However, in the presence of 10 mM MgCl2, the binding process becomes distributive, with the buoyant density increasing linearly with the amount of gpD5 added until the saturation. From these data, one molecule of gpD5 is calculated to cover 40 base pairs at saturation. The technique as described has general applicability to the study of any interaction between DNA and dNA-binding proteins that bind in sufficient amount to cause detectable changes in buoyant density.     

A small molecule inhibits and misdirects assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of -28.0 +/- 2.0 kJ/mol (-6.7 +/- 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was -24.5 +/- 0.9 kJ/mol (-5.9 +/- 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular "wedge" that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.     

Bacteriophage P22 in vitro DNA packaging monitored by agarose gel electrophoresis: rate of DNA entry into capsids.

Bacteriophage P22, like other double-stranded DNA bacteriophages, packages DNA in a preassembled, DNA-free procapsid. The P22 procapsid and P22 bacteriophage have been electrophoretically characterized; the procapsid has a negative average electrical surface charge density (sigma) higher in magnitude than the negative sigma of the mature bacteriophage. Dextrans, sucrose, and maltose were shown to have a dramatic stimulatory effect on the in vitro packaging of DNA by the P22 procapsid. However, sedoheptulose, smaller sugars, and smaller polyols did not stimulate in vitro P22 DNA packaging. These and other data suggest that an osmotic pressure difference across some particle, probably a capsid, stimulates P22 DNA packaging. After in vitro packaging was optimized by including dextran 40 in extracts, the entry kinetics of DNA into P22 capsids were measured. Packaged DNA was detected by: (i) DNA-specific staining of intact capsids after fractionation by agarose gel electrophoresis and (ii) agarose gel electrophoresis of DNase-resistant DNA after release of DNase-resistant DNA from capsids. It was found that the first DNA was packaged by 1.5 min after the start of incubation. The data further suggest that either P22 capsids with DNA partially packaged in vitro are too unstable to be detected by the above procedures or entry of DNA into the capsid occurs in less than 0.25 min.     

Conformational Changes Leading to T7 DNA Delivery upon Interaction with the Bacterial Receptor

The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity. Bacteriophage T7 belongs to the Podoviridae family and has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Here we characterize the kinetics and structure of bacteriophage T7 DNA delivery process. We show that T7 recognizes lipopolysaccharides (LPS) from Escherichia coli rough strains through the fibers. Rough LPS acts as the main phage receptor and drives DNA ejection in vitro. The structural characterization of the phage tail after ejection using cryo-electron microscopy (cryo-EM) and single particle reconstruction methods revealed the major conformational changes needed for DNA delivery at low resolution. Interaction with the receptor causes fiber tilting and opening of the internal tail channel by untwisting the nozzle domain, allowing release of DNA and probably of the internal head proteins.     

Fibrous projections from the core of a bacteriophage T7 procapsid

A cylindrical core previously demonstrated in a bacteriophage T7 procapsid (capsid I) has been further examined by electron microscopy. Fibrous extensions of the core have been observed; these fibers appear to connect the core to the capsid I envelope. After infection of a nonpermissive host with bacteriophage T7 amber mutant in any gene coding for a core protein, the resulting lysates contained more noncapsid assemblies of capsid envelope protien than did wild-type lysates; these assemblies had a mass two to at least 500 times greater than the mass of capsid I. This suggests that the internal core and fibers assist the assembly of subunits in the envelope of capsid I.     

Structural transformations accompanying the assembly of bacteriophage P22 portal protein rings in vitro

The Salmonella typhimurium bacteriophage P22 assembles an icosahedral capsid precursor called a procapsid. The oligomeric portal protein ring, located at one vertex, comprises the conduit for DNA entry and exit. In conjunction with the DNA packaging enzymes, the portal ring is an integral component of a nanoscale machine that pumps DNA into the phage head. Although the portal vertex is assembled with high fidelity, the mechanism by which a single portal complex is incorporated during procapsid assembly remains unknown. The assembly of bacteriophage P22 portal rings has been characterized in vitro using a recombinant, His-tagged protein. Although the portal protein remained primarily unassembled within the cell, once purified, the highly soluble monomer assembled into rings at room temperature at high concentrations with a half time of approximately 1 h. Circular dichroic analysis of the monomers and rings indicated that the protein gained alpha-helicity upon polymerization. Thermal denaturation studies suggested that the rings contained an ordered domain that was not present in the unassembled monomer. A combination of 4,4'-dianilino-1,1'-binapthyl-5,5'-disulfonic acid (bis-ANS) binding fluorescence studies and limited proteolysis revealed that the N-terminal portion of the unassembled subunit is meta-stable and is susceptible to structural perturbation by bis-ANS. In conjunction with previously obtained data on the behavior of the P22 portal protein, we propose an assembly model for P22 portal rings that involves a meta-stable monomeric subunit.     

Recognition of chemically damaged DNA by the gene 32 protein from bacteriophage T4

We have used fluorescence spectroscopy to investigate the binding of gene 32 protein from bacteriophage T4 to DNA which has been chemically modified with carcinogens or antitumor drugs. This protein exhibits a high specificity for single-stranded nucleic acids and binds more efficiently to DNA modified either with cis-diaminodichloroplatinum(II) or with aminofluorene derivatives than to native DNA. This increased affinity is related to the formation of locally unpaired regions which are strong binding sites for the single-strand binding protein. In contrast, gene 32 protein has the same affinity for native DNA, DNA containing methylated purines and DNA that has reacted with trans-diaminodichloroplatinum(II) or with chlorodiethylenetriaminoplatinum(II) chloride. These types of damage do not induce a sufficient structural change to allow gene 32 protein binding. Depurination of DNA does not create binding sites for the T4 gene 32 protein but nicked apurinic sites are strong ligands for the protein. This T4 single-strand binding protein does not exhibit a significantly increased affinity for nicked DNA as compared with native DNA. These results are discussed with respect to the recognition of DNA damage by proteins involved in DNA repair and to the possible role of single-strand binding proteins in DNA repair mechanisms.     

Parent-to-Progeny Transfer and Recombination of T4rII Bacteriophage

Transfer of parental, light (not substituted with 5-bromodeoxyuridine) (32)P-deoxyribonucleic acid (DNA) from rII(-) mutants of T4 bacteriophage to heavy (5-bromodeoxyuridine-substituted) progeny in Escherichia coli B was less homogeneous than in wild phages. The net transfer was 5 to 20% of the value for wild T4 phage, and the parental contribution per progeny DNA molecule amounted to 7 to 100% of the genome. Three classes could be distinguished, based on the density distribution of parental label in CsCl analysis of the progeny phages. "Far recombined" phages contain parental material only in semiconservatively replicated subunits covalently attached to progeny DNA, amounting to 5 to 10% parental contribution per genome. "Intermediate recombinants" contain, aside from conventional recombinant DNA, parental DNA banding at the original, light density. This DNA may be unattached to heavy progeny DNA or attached by weak bonds which are very sensitive to shearing during the extraction procedure. The parental contribution is 10 to 50% per progeny DNA molecule in this class. "Conservative" phages band close to the parental, light density in CsCl; their DNA is purely light. When the parental phage is labeled with both (3)H-leucine (capsid) and (32)P (DNA), the specific activity of (3)H/(32)P in the "conservative progeny" is 10 to 40% of that in the parental, showing that at least some of the (32)P in this area belongs to phages with parental DNA as the sole DNA component inside an unlabeled capsid, i.e., parental DNA which has been injected into the host and matured in a new capsid without replication or recombination. This phenomenon occurs to about the same extent in both single and multiple infection.