Assembly-associated structural changes of bacteriophage T7 capsids. Detection by use of a protein-specific probe.

To detect changes in capsid structure that occur when a preassembled bacteriophage T7 capsid both packages and cleaves to mature-size longer (concatameric) DNA, the kinetics and thermodynamics are determined here for the binding of the protein-specific probe, 1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid (bis-ANS), to bacteriophage T7, a T7 DNA deletion (8.4%) mutant, and a DNA-free T7 capsid (metrizamide low density capsid II) known to be a DNA packaging intermediate that has a permeability barrier not present in a related capsid (metrizamide high density capsid II). Initially, some binding to either bacteriophage or metrizamide low density capsid II occurs too rapidly to quantify (phase 1, duration < 10 s). Subsequent binding (phase 2) occurs with first-order kinetics. Only the phase 1 binding occurs for metrizamide high density capsid II. These observations, together with both the kinetics of the quenching by ethidium of bound bis-ANS fluorescence and the nature of bis-ANS-induced protein alterations, are explained by the hypothesis that the phase 2 binding occurs at internal sites. The number of these internal sites increases as the density of the packaged DNA decreases. The accompanying change in structure is potentially the signal for initiating cleavage of a concatemer. Evidence for the following was also obtained: (a) a previously undetected packaging-associated change in the conformation of the major protein of the outer capsid shell and (b) partitioning by a permeability barrier of the interior of the T7 capsid.     

Thermal analysis of bacteriophage T4.

The process of bacteriophage T4 morphogenesis was studied using a heat leakage scanning calorimeter. Thermograms of defective mutant 49 (am NG727) in permissive and non-permissive cells of $\text{Escherichia coli}$ showed a difference in thermal properties between packaged and non-packaged DNA molecules. $\text{In vivo}$, non-packaged DNA carried out their thermal transition at 85°C, the same temperature as that of T4 DNA melting measured in the standard saline citrate buffer, while the packaged DNA gave a sharper peak at 87°C due to some interaction with the head shell structure. Empty head shells showed a sharp heat absorption peak at 89°C both $\text{in vivo}$ and $\text{in vitro}$, indicating the high degree of cooperativity in their conformational changes.     

Marker rescue of the adsorption properties of bacteriophage T 4 by bacteriophage T 2

Rescue of adsorption properties from UV-irradiated T4 by T2 as a helper phage, revealed progeny phage with intermediate properties. Fourteen independent progeny phages, plating onE. coli B/2, were plated on several indicator strains and their adsorption properties were also studied with specific T4 antibodies. Two of these, plating onE. coli KS/4, were not inactivated by the T4 antiserum, and were T2h without apparent T4 properties. The other 12 progeny phages did not plate on KS/4, and were inactivated, but at a slower rate than the parental T4. Their mean efficiency of plating onE. coli B/2 (0.83) was significantly lower than that of the parental T4. The efficiency of plating was positively correlated with the velocity of inactivation by T4 antiserum. The observations were explained by assuming that the progeny phages were recombinants of T4 and T2 loci for adsorption sites. Plating of these 12 progeny phages on several indicator strains showed that they were allrII mutants and all, except one, wererI mutants too. In addition, two weretu andh ⁴, respectively. The condition for the appearance of multiple mutants might be a complementation by T2 of UV-damaged functions, which otherwise fail to induce the completion of the lytic cycle in monocomplexes of extracellularly irradiated T4.     

Functional analysis of the bacteriophage T4 DNA-packaging ATPase motor

Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp255) preceded by four hydrophobic residues (251MIYI254), which are predicted to form a beta-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the beta-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.     

Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins

We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection. which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids: these have been shown to contain a special type of T4 replication origin.     

Late effect of bacteriophage T4D on the permeability barrier of Escherichia coli.

Cold centrifugation of lysis-inhibited Escherichia coli B infected with wild-type T4D results in extensive lysis beginning around 20 min after infection at 37 degrees C. Infection with an e mutant, which fails to make lysozyme, prevents lysis, but does not prevent a marked loss of K+ and Mg3+. The t gene product, thought to disrupt the cytoplasmic membrane in natural lysis, is not required for this handling-induced cation loss or lysis. Three lines of evidence argue that late protein synthesis is required to develop this potential for cation loss; the potential does not develop in infections by: (i) mutants defective in DNA synthesis, (ii) mutants defective in gene 55, and (iii) wild-type T4 when chloramphenicol is added at 6 min after infection. All late mutants examined, which are blocked in the major pathways of morphogenesis, do not prevent development of the potential. The evidence argues for a new, late effect of T4 infection on the cytoplasmic membrane.     

Genetic analysis of bacteriophage T4 transducing bacteriophages.

Mutations in the genes for nuclear disruption (ndd), endonuclease IV (denB), and the D1 region of the T4 genome are essential for converting bacteriophage T4 into a generalized transducing phage. These mutations gave rise to a very low frequency of transduction, about 10(-8) per infected bacterium. The addition of an rII mutation raised the transduction frequency about 20-fold. An additional 100-fold increase in the transduction frequency was observed with mutations in genes 42, 56, and alc. High-frequency generalized transduction by T4 results from the cumulative effect of these mutations.     

N6-methyldeoxyadenosine 5'-triphosphate as a probe of the fidelity mechanisms of bacteriophage T4 DNA polymerase

The incorporation of m6dATP by T4 DNA polymerase has been investigated. Unlike Escherichia coli DNA polymerase I (Engel, J.D., and von Hippel, P.H. (1978) J. Biol. Chem, 253, 935-939), the T4 enzyme discriminates at the insertion step against the methylated triphosphate as compared to the normal substrate (dATP). The apparent Km values measured in two ways agree with the overall 7-fold discrimination measured in double label experiments. The apparent Vmax values measured for net DNA synthesis are the same, while those measured for nucleotide turnover show that the rate for m6dATP is 2-fold greater than for dATP itself. The T4 enzyme results are consistent with the generally held theory that fidelity at the insertion step of DNA polymerization is determined by the relative free energies of primer-enzyme-triphosphate ternary complexes formed by competing, alternative substrate dNTPs. These results are also consistent with the view that these free energies chiefly depend on formation of satisfactory hydrogen bonds between the bases of the template and triphosphate.     

Determination of mutation rates in bacteriophage T4 by unneighborly base pairs: Genetic analysis

Earlier studies showed that the 2-aminopurine-induced mutation rate at a particular base pair can be influenced by the base adjacent to, or one additional base-pair removed from, the measured site (Koch, 1971). The present study extends to 0.3 map unit (about 30 base pairs) the distance at which a single base-pair substitution can exert such an effect. A particular base-pair substitution (defined as a ts mutation in the rIIA gene of bacteriophage T4) reduces the spontaneous, 2-aminopurine-induced and nitrous acid-induced reversions of an rIIA amber mutation approximately threefold. The ts mutation also reduces the 2-aminopurine-induced conversion of the corresponding ochre codon to amber (UAA → UAG) about twofold and to opal (UAA → UGA) about eightfold. The 2-aminopurine-induced reversion of the ochre codon to a glutamine codon (UAA → CAA), however, is not affected. Control experiments demonstrate that these observed reductions in mutation frequency do not result from unacceptable pathways of reversion in the presence of the ts allele.     

An electron microscopic analysis of pathways for bacteriophage T4 DNA recombination

The interaction between ribosomes of Bacillus stearothermophilus and the RNA genomes of R17 and Qβ bacteriophage has been studied. Whereas Escherichia coli ribosomes can initiate the synthesis of all three RNA phage-specific proteins in vitro, ribosomes of B. stearothermophilus were previously shown to recognize only the A (or maturation) protein initiation site of f2 or R17 RNA. Under these same conditions, a Qβ region is bound and protected from nuclease digestion. Qβ RNA, however, does not direct the synthesis of any formylmethionyl dipeptide in the presence of B. stearothermophilus ribosomes, nor does the binding of either this Qβ region or the R17 A protein initiation site to these ribosomes show the same fMet-tRNA requirement for recognition of initiator regions as that previously established with E. coli ribosomes. Analysis of a 38-nucleotide sequence in the protected Qβ region reveals no AUG or GUG initiator codon. These observations suggest that messenger RNA may be recognized and bound by B. stearothermophilus ribosomes quite independently of polypeptide chain initiation.Binding experiments using R17 RNA and mixtures of components from B. stearothermophilus and E. coli ribosomes confirm the conclusion drawn by Lodish (1970a) that specificity in the selection of authentic phage initiator regions by the two species resides in the ribosomal subunit(s). However, anomalous attachment of B. stearothermophilus ribosomes to R17 RNA, which is observed upon lowering the incubation temperature of the binding reaction, is clearly a property of the initiation factor fraction. The results are discussed with respect to current ideas on the role of ribosomes and initiation factors in determining the specificity of polypeptide chain initiation.     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.