Bacteriophage T4 baseplate components. I. Binding and location of the folic acid.

Two different proteins with high affinities for the pteridine ring of folic acid have been used to determine the location of this portion of the folate molecule in the tail plate of T4D and other T-even bacteriophage particles. The two proteins used were (i) antibody specific for folic acid and (ii) the folate-binding protein from bovine milk. Both proteins were examined for their effect on various intact and incomplete phage particles. Intact T2H was weakly inactivated by the antiserum but not by the milk protein. No other intact T-even phage, including T4D, was affected by these two proteins. When incomplete T4D particles were exposed in an in vitro morphogenesis system, it was found that neither of the two proteins affected either the addition of the long tail fibers to fiberless particles or the addition of tail cores to tail plates. On the other hand, these two proteins specifically blocked the addition of T4D gene 11 product to the bottom of T4D baseplates. After the addition of the gene 11 protein, these two reagents did not inhibit the further addition of the gene 12 protein to the baseplate. It can be concluded that the phage folic acid is a tightly bound baseplate constituent and that the pteridine portion of the folic acid is largely covered by the gene 11 protein.     

Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein.

Dihydrofolate reductase plays a dual role in bacteriophage T4, first, as an enzyme of thymidylate metabolism, and second, as a protein component of the tail baseplate. Antibody to the purified enzyme has been used to study its synthesis and intracellular turnover. The antibody specifically precipitates one protein from T4D-infected cell extracts. This has been identified as dihydrofolate reductase, although the polypeptide molecular weight (22,000) is lower than that earlier determined for this enzyme. The protein comigrates on gels with pY, a genetically undefined protein component of the baseplate. However, it is not pY, for pY is synthesized late in infection, whereas virtually no dihydrofolate reductase synthesis occurs later than 10 min after infection at 37 degrees C. Dihydrofolate reductase, once formed, is neither degraded nor converted to proteins of higher or lower molecular weight. Thus, it is probably incorporated into virions at the same molecular weight as that of the soluble enzyme. 125I-radiolabeled antibody binds to the wedge substructure of the baseplate, and this binding is blocked by preincubation with purified T4 dihydrofolate reductase. Thus, the enzyme protein seems to be a component of the wedge.     

Bacteriophage T4 baseplate components. III. Location and properties of the bacteriophage structural thymidylate synthetase.

Two T4D thymidylate synthetase (td) temperature-sensitive mutants have been isolated and characterized. Both mutants produce heat-labile phage particles. This observation supports the view that this viral-induced protein is a phage structural component. Further, antiserum to td has been shown to block a specific step in tail plate morphogenesis. The results indicated that the td protein is largely covered by the T4D tail plate gene 11 protein. Since the phageinduced dihydrofolate reductase (dfr) also is partially covered by the gene 11 protein, it appears that td was adjacent to the tail plate dfr. This location has been confirmed by constructing a T4D mutant which is dfrtstdts and showing that these two tail plate constituents interact and give altered physical properties to the phage particles produced. A structural relationship for the tail plate folate, dfr, and td has been reported.     

Bacteriophage T4 tail length is controlled by its baseplate

Purified T4 baseplate, when treated with high concentrations of pancreatic RNase, are inactive in $\text{in}\text{vitro}$ complementation assays. Their ability to initiate tail tube assembly is not altered; but the most probably length of the tube-baseplate formed is only 800A, compared to 1000A, the normal tube length, when untreated baseplates are used. Thus, baseplates help to regulate tube length, possibly by a template mechanism. Several minor baseplate proteins which may be involved in determining the length, including gp54, are missing from RNase - treated base-plates. These effects may be due to an unidentified protease contaminant of the RNase, since they are inhibited by phenylmethane sulfonyl fluoride.     

Ligase-Defective Bacteriophage T4 II. Physiological Studies

The timing of the suppression of gene 30 (deoxyribonucleic acid ligase) mutations by rII mutations was studied by temperature shift-down experiments with a temperature-sensitive rII mutation. The rII function must remain inactivated for about 5 to 8 min at 37 C for suppression to occur, thus making suppression an early function. This result is in agreement with the timing of expression of other rII functions. A gene 30 defect can also be overcome by replacing the Na(+) cation in the growth medium with the Mg(2+) cation, a result similar to the relief of the lethality of rII mutations in lambda lysogens. Prior infection with bacteriophages T3 or T7, which produce their own deoxyribonucleic acid ligases, can also partially overcome the lethality of gene 30 mutations.     

Bacteriophage T4 tail assembly: proteins of the sheath, core and baseplate

Structural intermediates in phage tail formation have been isolated by sucrose gradient centrifugation from cells infected with mutants blocked at various stages in tail assembly. The polypeptide chains of these structures containing ¹⁴C-labeled amino acids have been analyzed by sodium dodecyl sulfate—acrylamide gel electrophoresis, enabling us to identify the proteins forming the various morphological components of the tail. Comparison of sheathed tails with corebaseplates shows that the contractile sheath is composed of a single species of subunit, the product of gene 18 (mol.wt 80,000). The site for head attachment terminating the tail is composed of the product of gene 15 (mol.wt 35,000). Comparison of core-baseplates with free baseplates shows that the tail core is composed of a single species of subunit, the product of gene 19 (mol.wt 21,000).Free baseplates are composed of at least twelve species of proteins: the products of genes 6, 7, 8, 9, 10, 11, 12 and 29, and four genetically unidentified species.The incomplete tails which accumulate in cells infected with mutants defective in genes 9, 11 and 12, which specify proteins on the outside of the baseplate, have also been characterized. Tails from 9^? lysates lack only P9. Tails from 11^? lysates lack both Pll and P12. Tails from 12^? infection lack only P12. Incorporation of P12 into the baseplate requires the function of gene 57, which is also required for tail fiber assembly. P57 thus appears to take part in the maturation of three different phage structural proteins.The sequential nature of the protein interactions in tail formation is discussed in terms of the regulation of morphogenesis at the level of assembly.     

Structure and location of gene product 8 in the bacteriophage T4 baseplate

Many bacteriophages, such as T4, T7, RB49, and phi29, have complex, sometimes multilayered, tails that facilitate an almost 100% success rate for the viral particles to infect host cells. In bacteriophage T4, there is a baseplate, which is a multiprotein assembly, at the distal end of the contractile tail. The baseplate communicates to the tail that the phage fibers have attached to the host cell, thereby initiating the infection process. Gene product 8 (gp8), whose amino acid sequence consists of 334 residues, is one of at least 16 different structural proteins that constitute the T4 baseplate and is the sixth baseplate protein whose structure has been determined. A 2.0A resolution X-ray structure of gp8 shows that the two-domain protein forms a dimer, in which each monomer consists of a three-layered beta-sandwich with two loops, each containing an alpha-helix at the opposite sides of the sandwich. The crystals of gp8 were produced in the presence of concentrated chloride and bromide ions, resulting in at least 11 halide-binding sites per monomer. Five halide sites, situated at the N termini of alpha-helices, have a protein environment observed in other halide-containing protein crystal structures. The computer programs EMfit and SITUS were used to determine the positions of six gp8 dimers within the 12A resolution cryo-electron microscopy image reconstruction of the baseplate-tail tube complex. The gp8 dimers were found to be located in the upper part of the baseplate outer rim. About 20% of the gp8 surface is involved in contacts with other baseplate proteins, presumed to be gp6, gp7, and gp10. With the structure determination of gp8, a total of 53% of the volume of the baseplate has now been interpreted in terms of its atomic structure.     

Bacteriophage Tail Components: II. Dihydrofolate Reductase in T4D Bacteriophage

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.     

The conformation of T4 bacteriophage dihydrofolate reductase from circular dichroism

The secondary and tertiary structure of T4 bacteriophage dihydrofolate reductase is investigated by vacuum ultraviolet circular dichroism (CD) spectroscopy and probability analysis of the primary amino acid sequence. The far ultraviolet CD spectrum of the enzyme in the range of 260-178 nm is analyzed by the generalized inverse and variable selection methods developed by our laboratory. Variable selection yields an average content of 26% alpha-helix, 21% antiparallel beta-sheet, 10% parallel beta-sheet, 20% beta-turns, and 32% "other" structures within the T4 protein. The characteristic peaks of the CD spectrum indicate that the enzyme has a lot of antiparallel beta-sheet, which is typical of the alpha + beta tertiary class of globular proteins. The secondary structure of the protein is also analyzed by using four statistical methods on the amino acid sequence. Although the secondary structures predicted by each individual statistical method vary to a considerable extent, the fractions of each structure jointly predicted by a majority of the methods are in excellent agreement with our CD analysis. The alternating arrangement for some segments of alpha-helix and beta-sheet predicted from primary structure to be within the enzyme is characteristic of proteins containing parallel beta-sheet. This supports our conclusion that the protein contains both parallel and antiparallel beta-sheet structures, but finding both types of beta-sheet also means that the protein may have the variation on alpha/beta tertiary structure recently found in EcoRI endonuclease and thymidylate synthase. These observations, in conjunction with other physical properties of the T4 reductase, suggest that the enzyme perhaps shares an evolution in common with the dihydrofolate reductases derived from type I R-plasmids rather than with the host-cell protein.     

Multiple-Tailed T4 Bacteriophage.

Smith, Kendall O. (Baylor University College of Medicine, Houston, Tex.), and Melvin Trousdale. Multiple-tailed T4 bacteriophage. J. Bacteriol. 90:796-802. 1965.-T4 phage particles which appeared to have multiple-tails were observed. Experiments were designed to minimize the possibility that superimposed particles might account for this appearance. Double-tailed particles occurred at a frequency as high as 10%. Triple- and quadruple-tailed particles were extremely rare. All attempts to isolate pure lines of multiple-tailed phage have failed. Multiple-tailed phage particles were produced in highest frequency by Escherichia coli cells in the logarithmic growth phase which had been inoculated at a multiplicity of about 2.     

Control of synthesis of mRNA''s for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase.

A 30 degrees C, functional messengers for dCMP hydroxymethylase first appeared 3 to 6 min postinfection and reached their maximum levels at 12 min. Chloramphenicol, added before the phage, reduced the rate of mRNA accumulation. When the antibiotic was added 6 min postinfection, mRNA levels increased at their normal rate but there was no obvious repression of messenger accumulation. Delaying the addition of drug until 8 or 12 min had progressively less effect on the pattern of hydroxymethylase mRNA metabolism. When chloramphenicol was present from preinfection times or from 6 min postinfection, all hydroxymethylase mRNA's synthesized were stable; at later times, however, the ability of the drug to stabilize mRNA decreased with its ability to delay the turnoff of mRNA production. An overaccumulation of hydroxymethylase mRNA was also seen when phage-specific DNA synthesis was inhibited either by mutational lesion in an essential viral gene or by 5-fluorodeoxyuridine. By min 20 of a DNA-negative program, hydroxymethylase mRNA synthesis was repressed to the point where it no longer compensated for decay. However, a finite level of hydroxymethylase mRNA synthesis was maintained at later times of a DNA-negative infection. Such results indicate that replication of the phage chromosome is necessary but not sufficient for a complete turnoff of hydroxymethylase mRNA production. Functions controlled by the maturation-defective proteins (the products of genes 55 and 33) played only a minor role in the regulation of hydroxymethylase mRNA, metabolism. Thus, we favor the hypothesis that a complete turnoff of hydroxymethylase messenger production requires one or more new proteins as well as an interval of DNA replication. The absence of DNA synthesis had no particular effect upon dihydrofolate reductase messenger production. The preinfection addition of chloramphenicol likewise had little effect on dihydrofolate reductase messenger metabolism. These latter data imply that prior synthesis of a phage-coded protein synthesis may not be required for the turnoff of reductase messenger production.     

Intracellular location of nitrate reductase and nitrite reductase. II. Wheat roots

Approximately 15% of the total nitrite reductase of crude homogenates of wheat roots applied to sucrose gradients was separated with an organelle whose isopycnic density was about 1.22 g·cm⁻³. The activity recovered in the supernatant was thought to be particulate in origin, because similar ratios of activity of isoenzyme 1 and 2 of nitrite reductase were found in both particulate and supernatant fractions. The particle with nitrite reductase activity also contained glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triose phosphate isomerase and NADPH diaphorase. This root particle and whole chloroplasts from leaves had a similar isopycnic density as well as these enzymes, and thus the data suggest that the root particle may be a proplastid.

Nitrate reductase was found only in the supernatant and it was not associated with any of the root organelles.

Mitochondria from wheat roots had an equilibrium density of 1.18 g·cm₋₃ and contained both NAD and NADP glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triosephosphate isomerase and NADPH diaphorase but not nitrite reductase. Microbodies of wheat roots had an equilibrium density of about 1.20 g·cm⁻³ on the sucrose gradient and contained catalase and glycollate oxidase.     

Three-dimensional structure of bacteriophage T4 baseplate

The baseplate of bacteriophage T4 is a multiprotein molecular machine that controls host cell recognition, attachment, tail sheath contraction and viral DNA ejection. We report here the three-dimensional structure of the baseplate-tail tube complex determined to a resolution of 12 A by cryoelectron microscopy. The baseplate has a six-fold symmetric, dome-like structure approximately 520 A in diameter and approximately 270 A long, assembled around a central hub. A 940 A-long and 96 A-diameter tail tube, coaxial with the hub, is connected to the top of the baseplate. At the center of the dome is a needle-like structure that was previously identified as a cell puncturing device. We have identified the locations of six proteins with known atomic structures, and established the position and shape of several other baseplate proteins. The baseplate structure suggests a mechanism of baseplate triggering and structural transition during the initial stages of T4 infection.