Attachment of tail fibers in bacteriophage T4 assembly. Identification of the baseplate protein to which tail fibers attach

A phage-neutralizing rabbit antiserum collected after immunization with tail-fiberless bacteriophage T4 particles was adsorbed with complete T4 phage. The resulting adsorbed serum inhibited tail fiber attachment in vitro. To identify the antigens against which this inhibitory activity was directed, blocking experiments were carried out with the adsorbed serum. Isolated complete baseplates and mutant-infected-cell extracts lacking known baseplate gene products but containing gene 9 product showed similar high levels of blocking activity. By contrast, both tail-fiberless particles lacking gene 9 product and infected-cell extracts made with gene 9 mutants showed 30-fold to 100-fold lower blocking activity. These results strongly support the conclusion that gene 9 product is the baseplate protein to which tail fibers attach.     

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 Tail Components V. Complementation of T4D Gene 28?-Infected Bacterial Extracts with Pteroyl Hexaglutamate

Synthetic pteroyl hexaglutamate (9 x 10(-6) M) stimulated the formation of new T4D particles in vitro in extracts of Escherichia coli B infected with T4D gene 28(-). The stimulation was specific for this form of folic acid since neither pteroyl pentaglutamate nor pteroyl heptaglutamate stimulated phage formation. T4D formation in vitro in E. coli B extracts prepared after infection with 11 other phage mutants known to be involved in phage tail plate formation (5(-), 6(-), 7(-), 8(-), 10(-), 25(-), 26(-), 27(-), 29(-), 51(-), 53(-)) was not stimulated by the addition of pteroyl hexaglutamate. It can be concluded that the T4D gene 28 product is involved in the formation of the phage tail plate pteroyl hexaglutamate.     

Effect of Host Cell Wall Material on the Adsorbability of Cofactor-requiring T4

The adsorbability of T4 on host cells was determined as a function of time after their liberation from infected cells. Freshly liberated (nascent) particles are readily adsorbed but lose their adsorbability with a half-time of about 2 days at 5 C, but only about 20 min at 37 C. They can be made adsorbable again with an alpha-amino acid cofactor like l-tryptophan, and this state of adsorbability can be stabilized by cell wall material from Escherichia coli. Such stabilized particles lose their adsorbability at a rate similar to that at which nascent particles lose theirs. Most freshly liberated particles are observed by means of electron microscopy to have "debris" attached to their baseplates and to have most of their six, long tail fibers free, whereas "old" particles that have lost their adsorbability appear relatively "clean" with most of their tail fibers wrapped around their sheaths. Nascent particles have densities that are lower than those of old particles. The material responsible for nascent adsorbability seems to be a fragment of the host's cell wall, for nascent adsorbability is destroyed by lysozyme. Furthermore, nascent T4 particles liberated from host cells with radioactively labeled walls carry the label in density gradients but lose it as they lose adsorbability. In addition, only a small proportion of particles liberated from infected spheroplasts are nascently adsorbable, whereas most particles liberated from intact cells are adsorbable.     

Hydrodynamics of macromolecular complexes. III. Bacterial viruses

We have calculated the translational and rotational frictional coefficients of structures related to T2 and T4 bacteriophage, using the theoretical framework developed in the preceding two papers. The structures considered were models for tail-fiberless phage, and for whole phage with fibers in the extended and retracted portions. We also computed and compared with the experiment the changes in translational frictional coefficient produced by successive addition of 1–6 fibers to the fiberless particle. Agreement with experimental results is markedly improved over previous theoretical efforts, especially with respect to the effect of tail-fiber extension. Some significant discrepancies remain, however, in the comparison of fiber-retracted and fiberless phage.     

Crystalline T4 bacteriophage

Concentrated suspensions of T4 phage crystallize spontaneously in the absence of tryptophan. The crystals, in one plane, contain repeated bilayers of phage in head to head and tail to tail contact, probably with tail fibers retracted. In the plane of each layer there is hexagonal packing of the phage.     

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.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates I. Identification of T4D Gene 28 Product in the Tail Plug

The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28^ts phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28^ts and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28^ts and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28^ts particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28^ts particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28^ts revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.     

Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.     

Tryptophan synthetase in Euglena gracilis strain G

The five enzyme activities in the synthesis of l-tryptophan have been obtained in extracts of Euglena gracilis. One of these, tryptophan synthetase, has been studied in detail. The general catalytic properties of tryptophan synthetase, including the range of reactions catalyzed and its substrate and cofactor affinities, are similar to those reported for other organisms. The Euglena enzyme has two properties never previously observed for tryptophan synthetase. First, the rate of catalysis of the conversion of indole-glycerol phosphate to l-tryptophan remained at its maximal value and was unaffected by the ionic environment up to 0.3 m KCl. In contrast, the conversion of indole to tryptophan showed a sharp maximum at 0.08 m KCl. Second, the enzyme is a component of a complex that includes every enzyme in the pathway committed to tryptophan biosynthesis with the exception of anthranilate synthetase, the regulatory enzyme.     

Solution structure of bacteriophage T4D and icosahedral capsid geometry visualized in freeze-fractured, deep-etched replicas

The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80 degrees angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q number of 21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.     

Production of Substituted l-Tryptophans by Fermentation

Claviceps purpurea has been shown to produce extracellular l-tryptophan from indole in stirred fermentors. The substrate specificity of this conversion was investigated by using substituted indoles, anthranilic acid, and 4-chloro-anthranilic acid. Addition of 2-, 4-, 5-, 6-, and 7-methyl indole or 6-chloroindole to C. purpurea C1M produced the corresponding substituted l-tryptophan. In contrast, addition of l-methyl, 6-trifluoromethyl, 6-nitro-, or 4-benzyloxy-substituted indoles, or anthranilic and 4-chloroanthranilic acids did not produce detectable amounts of the corresponding tryptophan.     

The function of tail fibers in triggering baseplate expansion of bacteriophage T4

Normal particles of bacteriophage T4 have six long tail fibers attached to a hexagonal baseplate. T4 particles having various complements of tail fibers were prepared by in vitro addition of fibers to fiberless particles, and the infectivity of the particles was determined. Particles having fewer than six fibers (partially fibered) were found to have a decreased probability of infection. Partially fibered particles having T4 fibers were completed by addition of T6 fibers, and the infectivity was determined on a host that lacked the T6 tail fiber receptor. Attachment of the additional fibers increased the infectivity even though the T6 fibers could not bind to the host cell. The infectivity of particles having mixtures of T4 and T6 fibers was determined on cells having only one type of receptor. The results indicated that particles bound by only three fibers have a low probability of infection. The effect of thermolabile baseplate mutations was also examined. Studies of partially fibered particles and particles with mixtures of fibers indicated that particles with altered baseplates have a less stringent requirement for binding of the tail fibers for infection.     

Product induction in Pseudomonas acidovorans of a permease system which transports L-tryptophan

Growth of Pseudomonas acidovorans in the presence of l-tryptophan resulted in the appearance of a tryptophan transport system which was extremely sensitive to sodium azide or 2,4-dinitrophenol. Asparagine-grown cells possessed no detectable tryptophan "permease" activity. Substitution of l-kynurenine for l-tryptophan in the growth medium also induced the tryptophan permease activity, along with tryptophan oxygenase and kynurenine formamidase. This is the first reported example of the product induction of a permease activity. Irrespective of whether Pseudomonas cells are grown in the presence of d- or l-tryptophan, the resulting induced tryptophan permease activity is specific for the l-isomer. In addition, the radioactive compounds l-leucine, l-phenylalanine, or dl-5-hydroxytryptophan are not transported. When dl-5-fluorotryptophan is a component of the inducing medium (with l-tryptophan), induction of tryptophan permease activity, as well as tryptophan oxygenase, is inhibited. In the permease assay system, using normally induced cells, the fluoroanalogue inhibited strikingly tryptophan transport. Therefore, this analogue may inhibit induction by blocking inducer transport into the cell. When added to the l-tryptophan-inducing medium, dl-7-azatryptophan markedly enhanced induction of tryptophan oxygenase, but the level of tryptophan permease activity was not further elevated. The mechanism of this analogue is unclear at present. Invariant tryptophan permease activity levels are found in cells grown with 5 or 15 mml-tryptophan or 5 mml-kynurenine, whereas the respective tryptophan oxygenase levels are greatly different. Together with other results, these results indicate that the synthesis of tryptophan permease activity is not coordinate with that of tryptophan oxygenase. Tryptophan transport is strongly inhibited by l-formylkynurenine and by l-kynurenine. These two metabolites were prepared in radioactive form, and they are actively transported following bacterial growth on l-tryptophan or l-kynurenine. Preliminary results suggest the tryptophan permease activity may be distinct from the permease(s) activity for l-formylkynurenine and l-kynurenine. Kynurenine, then, is capable of inducing tryptophan permease and kynurenine permease activities.     

Bacteriophage T4 baseplate components. II. Binding and location of bacteriophage-induced dihydrofolate reductase.

The location of T4D phage-induced dihydrofolate reductase (dfr) has been determined in intact and incomplete phage particles. It has been found that phage mutants inducing a temperature-sensitive dfr (dfrts) procude heat-labile phage particles. The structural dfr produced by these ts mutants was shown to assume different configurations depending on the temperature at which the phage is assembled. Morphogenesis of incomplete phage particles lacking the gene 11 protein on their baseplates was found to be inhibited by reagents binding to dfr, such as antibodies to dfr. Further, cofactor molecules for dfr, such as reduced nicotinamide adenine dinucleotide phosphate and reduced nicotinamide adenine dinucleotide, also inhibited the step in morphogenesis involving the addition of gene 11 product. On the other hand, inhibitors of dfr, such as adenosine dephosphoribose, stimulated the addition of the gene 11 protein. It has been concluded that the phage-induced dfr is a baseplate component which is partially covered by the gene 11 protein. The properties of phage particles produced after infection of the nonpermissive host with the one known T4D mutant containing a nonsense mutation in its dfr gene suggested that these progeny particles contained a partial polypeptide, which was large enough to serve as a structural element.     

Solution Structure of Bacteriophage T4D and Icosahedral Capsid Geometry Visualized in Freeze-Fractured,Deep-Etched Replicas

Abstract

The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80° angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q number of 21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.     

Bacterial ice nucleation activity after T4 bacteriophage infection.

The changes in ice nucleation activity of transformed Ina+ Escherichia coli K12 after infection with T4D bacteriophage have been examined. Within 2 min after infection class A nucleation activity (measured at -4 degrees C) fell about 100-1000-fold whilst class B (measured at -5.5 degrees C) and class C (measured at -9 degrees C) nucleation activities increased 50-100-fold and then rapidly decreased. These changes also occurred after interaction with T4D ghost particles or T4D 11-/12- particles. Since ghost particles lack DNA and 11-/12- particles lack short tail fibres, the T4D particles appear to be exerting their effect by the attachment of the phage long tail fibres to the cell. The changes were not influenced by the addition of chloramphenicol.     

Entrapment of l-tryptophan producing Escherichia coli in different matrices: activity of immobilized cells

Cells of Escherichia coli induced for l-tryptophan synthase [l-serine hydro-lyase (adding indole-glycerol-phosphate), EC 4.2.1.20] have been assayed in DMF and DMSO aqueous solvents as reaction medium. Up to 20% DMF/water, cells retained 90% of their tryptophan synthase activity. Concentrations of 20 mM indole, which did not inhibit this reactivity, could be reached with 5% DMF/water. Four matrices were compared for cell immobilization: polyacrylamide, foam particles of bovine seum albumin, alginate and κ-carrageenan. The best activity was retained with the latter matrix, and the preparations thus obtained allowed high productivity of l-tryptophan. Various systems of production of l-tryptophan with κ-carrageenan and DMF/water were studied.     

Attachment of tail fibers in bacteriophage T4 assembly: role of the phage whiskers.

The collar and whiskers of bacteriophage T4 extend outward from the top of the tail and play a role in regulating retraction of the tail fibers (Conley &; Wood, 1975). The collar and whiskers also are required for efficient tail fiber attachment during phage assembly. The structural gene for the collar/whisker protein is called wac. In vitro, infected-cell extracts that contain tail fibers activate whiskerless (wac) tail fiberless particles and ordinary (wac⁺) tail fiberless particles at equal rates if the extracts contain the wac⁺ gene product. However, extracts that contain tail fibers but no wac⁺ gene product activate wac particles about ten times more slowly. In vivo, whiskers are not essential for plaque formation, but a wac mutation causes a delay in the appearance of intracellular phage and a fivefold decrease in the burst size of infectious particles.The effect of the whiskers on tail fiber attachment is due to an interaction between the whisker and the distal half of the tail fiber, similar if not identical to the interaction that controls tail fiber retraction in complete phage. The following observations support this view: a slow rate of in vitro tail fiber attachment similar to that described above is seen with wac⁺ particles when they are pretreated with anti-whisker serum, or when the tail fibers carry a mutational alteration in gp36, a structural protein in the distal half fiber near the central kink. Lack of whiskers does not affect the slow rate of attachment of proximal half fibers to the baseplate of fiberless particles, but lack of whiskers greatly decreases the rate at which particles with attached proximal half fibers are activated by addition of distal half fibers. Since whiskers normally are attached to the phage only after head—tail union (Coombs &; Eiserling, 1977; Terzaghi et al., 1978), these findings explain why tail fibers do not attach efficiently to the baseplates of free tails.     

Bacteriophage T4D receptors and the Escherichia coli cell wall structure: role of spherical particles and protein b of the cell wall in bacteriophage infection.

The nature of the interaction of bacteriophage T4D and the outer cell wall of its host, Escherichia coli B, has been investigated. Bacteria with altered or modified cell walls have been obtained by two different growth procedures: (i) growth in high osmolarity medium or (ii) growth in broth in the presence of divalent heavy metal ions. When these altered host cells were washed and subsequently added to regular growth medium, they interacted with added phage particles, but successful infection did not occur. Most of the phage particles released from these treated cells were observed to have full heads and an altered tail structure. The altered phage tails had contracted sheaths and unusual pieces of the bacterial cell wall attached to the distal portion of the exposed phage tail tube. Phage released from bacteria grown in the high osmolarity medium had attached cell wall pieces of two major types, these pieces being either 40 or 21 nm in diameter. The smaller-type cell wall pieces (21 nm) were formed by three spheres each measuring 7 nm in diameter. Phage particles released from cells previously exposed to the divalent metal ions had only one 7-nm cell wall sphere attached to the distal end of the tail tube. It was found that these 7-nm spheres (i) are normal components of the cell wall and are morphologically similar to endotoxin, (ii) are held in place on the cell wall by a component of the cell wall called protein b, and (iii) are most likely the site of penetration of the phage tail tube through which the phage DNA enters the host cell.