Cryo-reconstructions of P22 polyheads suggest that phage assembly is nucleated by trimeric interactions among coat proteins

Bacteriophage P22 forms an isometric capsid during normal assembly, yet when the coat protein (CP) is altered at a single site, helical structures (polyheads) also form. The structures of three distinct polyheads obtained from F170L and F170A variants were determined by cryo-reconstruction methods. An understanding of the structures of aberrant assemblies such as polyheads helps to explain how amino acid substitutions affect the CP, and these results can now be put into the context of CP pseudo-atomic models. F170L CP forms two types of polyhead and each has the CP organized as hexons (oligomers of six CPs). These hexons have a skewed structure similar to that in procapsids (precursor capsids formed prior to dsDNA packaging), yet their organization differs completely in polyheads and procapsids. F170A CP forms only one type of polyhead, and though this has hexons organized similarly to hexons in F170L polyheads, the hexons are isometric structures like those found in mature virions. The hexon organization in all three polyheads suggests that nucleation of procapsid assembly occurs via a trimer of CP monomers, and this drives formation of a T = 7, isometric particle. These variants also form procapsids, but they mature quite differently: F170A expands spontaneously at room temperature, whereas F170L requires more energy. The P22 CP structure along with scaffolding protein interactions appear to dictate curvature and geometry in assembled structures and residue 170 significantly influences both assembly and maturation.     

Structure of T4 polyheads: II. A pathway of polyhead transformations as a model for T4 capsid maturation

We have studied the aberrant tubular polyheads of bacteriophages T4D and T2L as a model system for capsid maturation. Six different types of polyhead surface lattice morphology, and the corresponding protein compositions are reported and discussed. Using in vitro systems to induce transformations between particular polyhead types, we have deduced that the structural classes represent successive points in a transitional pathway. In the first step, coarse polyheads (analogous to the prohead τ-particle) are proteolytically cleaved by a phagecoded protease, a fragment of the gene 21 product. This cleavage of P23 to P23¹ induces a co-operative lattice transformation in the protein of the surface shell, to a conformation equivalent to that of T2L giant phage capsids. These polyheads (derived either from T4 or T2L lysates) can accept further T4-coded proteins. In doing so, they pass through intermediate structural states, eventually reaching an end point whose unit cell morphology is indistinguishable from that of the giant T4 capsids. At least one protein (called soc (Ishii & Yanagida, 1975)) is bound stoichiometrically to P23¹ in the end-state conformation. The simulation of several aspects of capsid maturation (cleavage of P23 to P23¹, stabilization, and lattice expansion) in the polyhead pathway suggest that it parallels the major events of phage T-even capsid maturation, decoupled from any involvement of DNA packaging.     

Folding and capsomere morphology of the P23 surface shell of bacteriophage T4 polyheads from mutants in five different head genes

Mutants in five different “head formation” genes (20, 22, 24, 40, IPIII)² of bacteriophage T4 produce polyheads. “Coarse” polyheads, which contain uncleaved P23, constitute over 90% of these tubular particles in fresh lysates. Using optical diffraction and filtration, we show that the pseudo-hexagonal net and the capsomere morphology are common to all coarse polyheads, regardless of genetic origin or polyhead diameter. Micropolymorphism is exhibited in each genetic class with respect to the cylindrical folding of the hexagonal net. We find that the frequency distribution of the diameters and pitch angles is significantly different for polyheads made by mutants affecting either of the major prohead core proteins (IPIII and P22). In every case, the foldings differ from the unique folding characteristic of giant phage capsid, suggesting that the assembly error responsible for producing polyheads instead of proheads involves a misdirection in arranging the P23 shell. By analysing the properties common to the various structures which may be formed out of this net (single-layered polyheads, multi-layered polyheads, proheads), we find that the P23 molecules possess form-determining specificity in terms of an intrinsic curvature of the capsomere bonding. These observations are discussed within the context of form determination of the phage prohead (τ-particle) and of its subsequent conservative maturation to the head of the infective wild-type phage.     

Determinants of bacteriophage P22 polyhead formation: the role of coat protein flexibility in conformational switching

We have investigated determinants of polyhead formation in bacteriophage P22 in order to understand the molecular mechanism by which coat protein assembly goes astray. Polyhead assembly is caused by amino acid substitutions in coat protein at position 170, which is located in the β‐hinge. In vivo scaffolding protein does not correct polyhead assembly by F170A or F170K coat proteins, but does for F170L. All F170 variants bind scaffolding protein more weakly than wild‐type as observed by affinity chromatography with scaffolding protein‐agarose and scaffolding protein shell re‐entry experiments. Electron cryo‐microscopy and three‐dimensional image reconstructions of F170A and F170K empty procapsid shells showed that there is a decreased flexibility of the coat subunits relative to wild‐type. This was confirmed by limited proteolysis and protein sequencing, which showed increased protection of the A‐domain. Our data support the conclusion that the decrease in flexibility of the A‐domain leads to crowding of the subunits at the centre of the pentons, thereby favouring the hexon configuration during assembly. Thus, correct coat protein interactions with scaffolding protein and maintenance of sufficient coat protein flexibility are crucial for proper P22 assembly. The coat protein β‐hinge region is the major determinant for both features.     

Maturation of the head of bacteriophage T4. II. Head-related, aberrant tau-particles

Three somewhat different types of particle accumulate in cells infected with a phage carrying a mutation in gene 21 (in addition to the tubular variant (polyhead) of the head). The major type is the so-called τ-particle. These particles are very fragile, associated with the cell membrane, and have a sedimentation coefficient of about 420 S. They possess no DNA if isolated, and contain predominantly the precursor proteins P23, P24, P22 and the internal protein IP III, in addition to protein P20 and several proteins of unknown genetic origin.The remainder of the particles are partially or completely filled with DNA. The ratio of τ-particles to these partially or completely filled particles depends upon the particular mutant (in gene 21) phage used. In cells infected with a phage carrying the amber mutation (N90) in gene 21, about 10% of the precursor head protein P23 is cleaved to P231, and correspondingly about 10% of the particles are partially or completely filled with DNA. In cells infected with the temperature-sensitive mutant (N8) in gene 21, about 1% of the particles are fully or partially filled, and correspondingly about 1% of the P23 is cleaved to P231. In either case, the DNA-associated particles contain predominantly the cleaved proteins P231 and IP III1, and have none of the P22 and IP III found in τ-particles. This observation, and the correlation of the amount of partially or completely filled particles with the extent of the cleavage of P23 in the lysates, strongly suggest that cleavage of the head proteins is required for DNA packaging to occur.The τ-particles have properties similar to the so-called prohead I particles which we have isolated as intermediates in wild-type head assembly (preceding paper). However, temperature shift-down experiments, using several different phage carrying temperature-sensitive mutations in gene 21, indicate that the bulk of the τ-particles cannot be used for normal phage production.     

Head morphologies in bacteriophage T4 head and internal protein mutant infections.

Escherichia coli infected with phage T4 mutants defective in synthesis of the three major internal proteins found in the phage head, IPI-, IPII-, IPIII-, or IP degrees (lacking all three) were examined in the electron microscope for head formation. Infection with IPI- or IPII- does not appear to induce increased aberrant head formation, whereas IPII- or IP degrees infections result in production of polyheads and viable phage. Multiple mutants of the early head formation genes 20, 21, 22, 23, 24, 31, 40 and IP degrees were constructed. Combination with IP degrees increases polyhead formation when head formation is not blocked at a more defective stage but results in a qualitative shift to lump formation in association with gene 22 mutants. Thin-sectioning studies show morphologically similar cores in amber 21 and 21am IP degrees tau particles. These morphological observations, genetic evidence for interaction between ts mutants in gene 22 and the IP mutants, and analysis of the protein composition of tau particles further support the idea that p22 and the internal proteins form an unstable assembly core necessary for an early stage of head formation (M. K. Showe and L. W. Black, 1973).     

Chaperonin-mediated folding of bacteriophage T4 major capsid protein. II. Production of gene product 23 deletion mutants

Folding of bacteriophage T4 major capsid protein, gene product 23 (534 a.a.), is aided by two proteins: E. coli GroEL chaperonin and viral gp31 co-chaperonin. In the present work a set of mutants with extensive deletions inside gene 23 using controlled digestion with Bal31 nuclease has been constructed. Proteins with deletions were co-expressed from plasmid vectors with phage gp31 co-chaperonin. Deletions from 8 to 33 a.a. in the N-terminal region of the gp23 molecule covering the protein proteolytic cleavage site during capsid maturation have no influence on the mutants' ability to produce in E. coli cells proteins which form regular structures—polyheads. Deletions in other regions of the polypeptide chain (187-203 and 367-476 a.a.) disturb the correct folding and subsequent assembly of gp23 into polyheads.     

Assembly and disassembly of bacteriophage T4 polyheads

The assembly of the product of bacteriophage T4 gene 23 (gp23), the uncleaved form of the main shell protein, has been studied. Assembly and disassembly follow the predictions for entropy-driven processes; assembly is strongly favored by conditions of high salt concentrations and high temperatures, whereas low salt and low temperatures promote disassembly. In the absence of the scaffolding core proteins in vitro, only polyheads, the tubular variant of the prohead, are produced. Kinetic studies show that the rate of polyhead dissociation depends on the concentration of associated protein, not on the number and length of the particles. Comparable to crystal formation, assembly of gp23 occurs above a critical concentration, which is dependent on salt concentration, pH and temperature. These characteristics are common to most self-assembling systems. The oligomeric states of gp23 have been investigated by analytical ultracentrifugation, which indicated the existence, at very low salt concentration and low temperature, of an equilibrium between monomers and higher oligomers, culminating in the hexamer. At pH 9.0 polyheads are completely dissociated into their monomeric gp23 subunits. Our data suggest that the hexamer is a true intermediate of polyhead assembly.     

Studies related to the head-maturation pathway of bacteriophages T4 and T2: II. Nuclear disruption,protein synthesis and particle formation with the mutant 43−·30−·46−

We describe the aberrant phage multiplication of the triple conditional lethal mutant 43^?(polymerase)· 30^?(ligase)·46^?(exonuclease) of bacteriophage T4D in which phage DNA replication is arrested but some late protein synthesis occurs (33). The nuclear disruption is indistinguishable from wild type. Forty-five empty small and empty large particles are assembled per cell when the multiplicity of infection (m.o.i.) is 100. This number corresponds closely to the 38 phage equivalents of cleaved major head protein determined biochemically. By reducing the m.o.i. the number of observable particles decreases, reaching 1–5 per cell at an m.o.i. of 5(+5). The total synthesis of phage related proteins is not significantly dependant on the m.o.i. The synthesis of late proteins is about 10% of that of wild type at high m.o.i. and decreases with the m.o.i. The different early and late proteins do not show the same relative proportions as in wild type and respond differently to an increased m.o.i. These and other results are discussed with respect to the role of phage DNA in prehead assembly and head maturation.     

W-reactivation of phage lambda in X-irradiated mutants ofEscherichia coli K-12

Summary The survival of UV irradiated phage lambda was increased on X-irradiatedE. coli K-12 host cells over that on unirradiated cells. The frequency of c mutants among the surviving phages was to a similar extent increased by the X-ray exposure of the host cells as by UV light. This W-reactivation of phage lambda occurred inuvrA, polA, andrecB mutants besides the wild type at about equal X-ray doses, however, at a reduced reactivation efficiency compared with the wild type. W-reactivation was undetectable inrecA mutants. While maximal UV induced W-reactivation occurred 30 min after irradiation, the maximal X-ray induced reactivation was found immediately after irradiation. Chloramphenicol (100 µg/ml) and nitrofurantoin (50 µg/ml) inhibited W-reactivation of phage lambda if added before irradiation of the host cells, indicating the necessity of protein synthesis for W-reactivation.     

meoA is the structural gene for outer membrane protein c of Escherichia coli K12

Summary The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Mel resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptorcomplex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c^*. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.     

Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.     

Absence of a protein constituent and occurrence of an oversized DNA genome in a high density mutant particle of phage P22

Summary Mutants of P22 phage with abnormal density in CsCl solution (P22ndc phage) were analyzed in detail for this report. Two dimensional polyacrylamide gel electrophoresis revealed that wild-type P22ndc ⁺ phage virions contained a new protein (gpU) in addition to nine already identified proteins, while P22ndc lacked gpU. The molecular weight of gpU was essentially the same as that of gp5 (45 500), and one mature virion of phage P22ndc ¹ contained as many as 30–50 molecules of gpU. As P22ndc is a plaque-forming phage, gpU cannot be essential for the growth and assembly of P22 phage. Both genetical and biochemical analysis of the phage DNA in the virion revealed that P22ndc phage contained 2%–4% longer DNA than wild type P22ndc ⁺. A model is presented to account for the formation of P22ndc phage.     

Nucleic acid economy in bacteria infected with bacteriophage T2

1. During the first 10 minutes of viral growth following infection of E. coli by phage T2 in broth, a pool of DNA is built up that contains phosphorus later to be incorporated into phage. This pool receives phosphorus from, but does not contain, the bacterial DNA. 2. After 10 minutes, DNA synthesis and phage maturation keep pace in such a way that the amount of precursor DNA increases moderately for a time and then remains constant. 3. The pool so described is defined in terms of the kinetics of transport of phosphorus from its origins in the culture medium, the bacterial DNA, and the DNA of the parental phage, to the viral progeny. The most interesting parameter of this system is the size of the precursor pool, which measures 10^–9 to 2 x 10^–9 µg. DNA-P (50 to 100 phage particle equivalents) per bacterium. 4. Neither the precursor nor the intracellular phage population exchanges phosphorus with the phosphate in the medium. More interestingly, the phosphorus in mature phage does not exchange with phosphorus in the precursor, showing that maturation is an irreversible process. 5. Maturation is also a remarkably efficient process. About 90 per cent of labeled phosphorus introduced early into the precursor pool is later incorporated into phage. 6. Viral DNA is synthesized at the rate of about 1.5 x 10^–10 µg. DNA-P (7 or 8 phage particles) per bacterium per minute. This is somewhat faster than bacterial DNA is formed, but considerably slower than RNA is formed, in uninfected bacteria. 7. The transport of phosphorus from medium to viral precursor DNA takes an average of 8 or 9 minutes, and from precursor to phage an additional 7 or 8 minutes. 8. Metabolically active RNA has been detected in infected bacteria.     

Absence of the psbH gene product destabilizes the Photosystem II complex and prevents association of the Photosystem II-X protein in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

PS II-H is a small hydrophobic protein that is universally present in the PS II core complex of cyanobacteria and plants. The role of PS II-H was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The psbH disruptant could grow photoautotrophically; however, its growth was much slower than that of the wild type cell. Chromatography enabled the isolation of active oxygen-evolving PS II complexes from both the mutant and the wild type. The mutant yielded a relatively large amount of inactive PS II complex that lacked the following extrinsic proteins: the 33-kDa protein, the 12-kDa protein, and cytochrome c ₅₅₀ . There were differences between the psbH disruptant and the wild type in terms of the oxygen evolution activities of the cells, thylakoids, and PS II complexes. At high concentrations of 2,6-DCBQ, the activity was much lower in the mutant than in the wild type. Gel filtration chromatography of the PS II complexes showed that both active and inactive PS II complexes isolated from the mutant were mostly in the monomeric form, while the active PS II complex from the wild type was in the dimeric form. The polypeptide composition of both active and inactive PS II complexes from the mutant showed the absence of another small polypeptide, PS II-X. These results suggest that the PS II-H protein is essential for stable assembly of native dimeric PS II complex containing PS II-X.     

Regulation of the intracellular concentration of T4 induced tRNA

Summary We have studied the biosynthesis of T4 induced tRNA's upon infection of E. coli B ^E cells in low phosphate (l.p.) medium (10^-4 M PO ₄ ^--- ). Under our experimental conditions the onset of phage DNA synthesis occurs at about 15 min after infection, while the first intracellular phage appears one hour later. Amounts of newly synthesized DNA and phage burst size are equivalent to the values obtained in standard (M9) medium (10^-1 M PO ₄ ^--- ). We present evidence that the synthesis of mature tRNA's and of at least one dimeric precursor drastically declines 20 min after infection. In addition we show that T4 induced tRNA molecules are stable and that the triphosphate nucleoside precursor pool does not change significantly during infection. Therefore we conclude that T4 induced tRNA molecules behave similarly to other early gene products.     

Structure of the scaffold in bacteriophage lambda preheads removal of the scaffold leads to a change of the prehead shell

Small angle X-ray scattering was performed on unprocessed and processed preheads, intermediates in the morphogenesis of bacteriophage λ heads. Unprocessed preheads possess an internal structure (scaffold), necessary for efficient assembly of closed shells. Processed preheads, formed after removal of the scaffold, are able to pack and cut the viral DNA in vitro. Our data show that the scaffold fills out the inside of the shell in an almost (but not completely) homogeneous fashion; structures of the scaffold with the bulk of the mass in a small core inside the shell can be excluded. Unprocessed preheads are larger than processed ones. A change in shell architecture takes place upon transition from unprocessed to processed prehead; the shell becomes roughened up. Shrinking of the shell as well as roughening up can be triggered by accidental partial degradation of the scaffold. The lattice constant of type A polyheads is in agreement with the lattice constant derived from our icosahedral models of the shell, indicating a close relationship between processed preheads and type A polyheads. This observation, together with the type of subunit clustering found, leads us to propose a simple model for the interaction of prehead shell and protein pD, which stabilizes phage DNA after packaging.     

Control of the synthesis of phage p22 scaffolding protein is coupled to capsid assembly

The assembly of the precursor shells of bacteriophage P22 entails the co-polymerization of gene 5 coat protein with gene 8 scaffolding protein into double shell structures. During DNA encapsidation, the inner shell of scaffolding molecules dissociates and exits from the prohead. These molecules then recycle, catalyzing the assembly of newly synthesized coat protein to form new proheads (King and Casjens, 1974).Although gene 5 and gene 8 are adjacent on the phage chromosome, we find that the synthesis of the two proteins is differentially regulated. In productively infected cells, scaffolding protein is synthesized at a low rate relative to the coat protein. In contrast, cells that are infected with mutants blocked in DNA packaging and accumulate precursor shells synthesize scaffolding protein at a much higher rate. If a mutation is introduced into the coat protein gene, however, preventing shell assembly, the rate of scaffolding protein synthesis decreases to less than the wild-type rate.The experiments are consistent with models in which either continued synthesis of scaffolding protein depends upon co-polymerization with coat subunits, or soluble scaffolding subunits (but not assembled subunits) depress their own further synthesis. The finding that amber fragments of the scaffolding protein are synthesized at a very low rate is inconsistent with the second model. There is evidence, however, that fragments of the protein may have regulatory activity.The regulatory circuit couples scaffolding protein synthesis to morphogenesis. Gene dosage experiments show that regulation results in the maintenance of coat and scaffolding subunits in the proper ratio for shell assembly.     

The influence of growth conditions on the synthesis of molybdenum cofactor in Proteus mirabilis

Cell-free extracts of Proteus mirabilis were able to reconstitute NADPH-dependent assimilatory nitrate reductase in crude extracts of the Neurospora crassa mutant strain nit-1, lacking molybdenum cofactor. Molybdenum cofactor was formed in the cytoplasm of the bacterium even in the presence of oxygen during growth though under these conditions no molybdo enzymes are formed. As a consequence no cofactor could be released by acid treatment from membranes of cells grown aerobically. The amount of cofactor released from membranes of cells grown anaerobically under various conditions was proportional to the amount of molybdo enzymes formed. During growth in the presence of tungstate a cofactor, which lacks molybdenum, was found in the cytoplasm. For detection of this so-called demolybdo cofactor the presence of molybdate during reconstitution was essential. Moreover, the cytoplasmic cofactor pool in cells grown in the presence of tungstate appeared to be two to three times higher than in cells grown under similar conditions without tungstate. After anaerobic growth in the presence of tungstate, the inactive demolybdo reductases were shown to contain partly no cofactor and partly a demolybdo cofactor. The P. mirabilis chlorate resistant mutant S 556 did not contain molybdenum cofactor. In two other chl-mutants the cofactor activity was the same as in the wild type.     

The properties of hybrids formed between the P-group plasmid RP1 and various plasmids from Pseudomonas aeruginosa

Summary Ribosomal DNA content has been determined in several adult and larval tissues of Drosophila melanogaster. Underreplication of rRNA genes was observed in polytenic salivary glands of larvae. On the contrary, polytenic/polyploid ovaries showed no decrease in rDNA. It is concluded that polyteny is not necessarily associated with underreplication of rDNA. No other tissue examined displayed any change in rDNa redundancy. Third-instar-larvae showed a decrease in rDNA amount which might be partly accounted for by underreplication of rDNA in salivary glands. No such decrease was seen in pupae. Bobbed genotypes were essentially similar to wild type in all tissues except salivary glands. In this case, it was found that the extent of underreplication is less in bobbed as compared to wild genotypes.Ribosomal DNA activity was examined in various tissues of Drosophila melanogaster. The rates of rRNA synthesis vary greatly between various tissues. It is concluded that a control at the level of gene activity operates as differences in the amount of precursor rRNA synthesized can be observed both in flies of varying rDNA contents as well as in various tissues of the same genotype.