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 length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail.Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1¹, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII¹ is present in reduced amounts as compared to wild type. The precursor to B1¹ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved.The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).     

Mechanism of head assembly and DNA encapsulation in Salmonella phage p22. I. Genes, proteins, structures and DNA maturation

The functions of ten known late genes are required for the intracellular assembly of infectious particles of the temperate Salmonella phage P22. The defective phenotypes of mutants in these genes have been characterized with respect to DNA metabolism and the appearance of phage-related structures in lysates of infected cells. In addition, proteins specified by eight of the ten late genes were identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; all but two are found in the mature phage particle. We do not find cleavage of these proteins during morphogenesis.The mutants fall into two classes with respect to DNA maturation; cells infected with mutants of genes 5, 8, 1, 2 and 3 accumulate DNA as a rapidly sedimenting complex containing strands longer than mature phage length. 5^? and 8^? lysates contain few phage-related structures. Gene 5 specifies the major head structural protein; gene 8 specifies the major protein found in infected lysates but not in mature particles. 1^?, 2^? and 3^? lysates accumulate a single distinctive class of particle (“proheads”), which are spherical and not full of DNA, but which contain some internal material. Gene 1 protein is in the mature particle, gene 2 protein is not.Cells infected with mutants of the remaining five genes (10, 26, 16, 20 and 9) accumulate mature length DNA. 10^? and 26^? lysates accumulate empty phage heads, but examination of freshly lysed cells shows that many were initially full heads. These heads can be converted to viable phage by in vitro complementation in concentrated extracts. 16^? and 20^? lysates accumulate phage particles that appear normal but are non-infectious, and which cannot be rescued in vitro.From the mutant phenotypes we conclude that an intact prohead structure is required to mature the virus DNA (i.e. to cut the overlength DNA concatemer to the mature length). Apparently this cutting occurs as part of the encapsulation event.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway

We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis.In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26.Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3^? infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads.Detailed examination of 8^? lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well.All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.     

Morphogenesis of the tail of bacteriophage lambda. III. Morphogenetic pathway.

Precursors of the tail of bacteriophage λ have been detected by measurements of in vitro complementation activities and serum blocking activity in sucrose gradients of lysates defective in tail genes.On the basis of these measurements, a pathway for the assembly of the λ tail is proposed:The morphogenesis of the λ tail starts from the tail fiber (product of gene J) located at the distal end of the tail, and proceeds to the proximal end. Gene J by itself produces a 15 S structure with serum blocking activity but without any detectable in vitro complementation activity, which may be the least advanced precursor of the λ tail or an abortive product. Functions of genes J, I, K, L are required for the formation of a 15 S precursor that has in vitro complementation activities with J⁻, I⁻, K⁻ and L⁻ lysates and serum blocking activity. If the products of genes G and H act on the latter 15 S precursor, a 25 S precursor is made, but this precursor seems either to be in equilibrium with the 15 S precursor or to degrade easily into the 15 S precursor. Gene M has a function of stabilizing the 25 S precursor. After the action of gene M product, the 25 S precursor is ready to serve as a nucleus on which the product of gene V (the major tail protein) assembles. However, gene U product is also necessary at this step for the correct assembly of the major tail protein on the 25 S precursor. Without gene U product the assembly of the major tail protein does not stop at the correct length and a polytail is formed instead of a morphologically normal tail. Finally, gene Z product acts on the morphologically normal tail and makes it a biologically active tail. Without the action of gene Z product, the defective tail binds to a head and forms a phage-like particle which is only very weakly infectious. (The position of gene T in the pathway is not determined, because no sus mutant is available in gene T.)Two abnormal, less efficient pathways are also present in vitro. (1) If gene U product acts on a polytail in an U⁻ lysate, the polytail finally binds to a head and forms a phage particle with an extra long tail which is infectious to a small extent. (2) The function of gene K seems to be bypassed to some extent: K⁻ lysates accumulate particles which sediment as fast as normal phage and which are complemented by other tail⁻ lysates.     

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.     

Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). II. The propagation of phage lambda

In the accompanying paper (Sternberg, 1973) the properties of three independently isolated strains of Escherichia coli with groE mutations (NS-1, NS-2 and NS-3) have been characterized. In this report the ability of these strains to propagate phage λ is examined in greater detail. In the temperature -sensitive groE strain NS-1, all early phage functions tested (curing, infective center formation, DNA synthesis and early messenger RNA synthesis) are expressed normally. In addition, two late phage functions (late mRNA synthesis and tail formation) are also expressed normally, and a third, phage-induced cell lysis, is expressed with only a slight delay. Based upon head-tail in vitro complementation assays, however, λ fails to make any functional heads at elevated temperatures (41 °C) in this host. Electron microscopic studies of strain NS-1 defective lysates indicate that aberrant head-like forms, including tubular forms and “monsters,” are made.Mutants of λ, designated λEP, which are able to grow in the three groE strains, have been isolated. An analysis of these mutants indicates that at least some carry a mutation in λ head gene E and these make reduced levels of active gene E protein in groE hosts.A further study of all known λ head genes indicates that it is the interaction between the gene E protein and the proteins specified by head genes B and C that is adversely affected by the groE mutation. Presumably, the relative level of gene E protein is too high in groE strains for proper head formation. The λEP mutation compensates for this effect by reducing the level of this protein, and so restoring a balance.     

Host participation in bacteriophage lambda head assembly

Mutants of Escherichia coli, called groE, specifically block assembly of bacteriophage λ heads. When groE bacteria are infected by wild type λ, phage adsorption, DNA injection and replication, tail assembly, and cell lysis are all normal. No active heads are formed, however, and head related “monsters” are seen in lysates. These monsters are similar to the structures seen on infection of wild-type cells by phage defective in genes B or C.We have isolated mutants of λ which can overcome the block in groE hosts and have mapped these mutants. All groE mutations can be compensated for by mutation of phage gene E (hence the name groE). Gene E codes for the major structural subunit of the phage head. Some groE mutants, called groEB, can be compensated by mutation in either gene E or in gene B. Gene B is another head gene.During normal head assembly the protein encoded by phage head gene B or C appears to be converted to a lower molecular weight form, h3, which is found in phage. The appearance of h3 protein in fast sedimenting head related structures requires the host groE function.We suggest that the proteins encoded by phage genes E, B and C, and the bacterial component defined by groE mutations act together at an early stage in head assembly.     

Bacteriophage T4 head morphogenesis: host DNA enzymes affect frequency of petite forms

Petite T4 phage particles have a shorter head than normal T4 phage and contain less DNA. They are not viable in single infections but are able to complement each other in multiply infected cells. Such particles normally make up 1 to 3% of T4 lysates. We show here that lysates of T4 grown on Escherichia coli H560 (end-A^?, pol-A^?) contain 33% of such petite particles. These particles are identical in physical and biological properties to those described previously, only their high frequency is abnormal. The frequency of petite particles in lysates grown on H560 is controlled by the presence or absence of the gene for DNA polymerase I (pol-A1) and apparently also a gene for endonuclease I (end-A). The involvement of these host DNA enzymes with T4 head morphology and DNA content indicates that DNA is directly involved in head morphogenesis. Such an involvement is incompatible with models of T4 head morphogenesis in which dimensionally stable, preformed empty heads are precursors of filled heads. The processing or repair of DNA apparently helps decide whether the assembly of T4 head subunits produces normal or petite heads.     

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.     

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.     

Escherichia coli HflK and HflC can individually inhibit the HflB (FtsH)-mediated proteolysis of λCII in vitro

λCII is the key protein that influences the lysis/lysogeny decision of λ by activating several phage promoters. The effect of CII is modulated by a number of phage and host proteins including Escherichia coli HflK and HflC. These membrane proteins copurify as a tightly bound complex ‘HflKC’ that inhibits the HflB (FtsH)-mediated proteolysis of CII both in vitro and in vivo. Individual purification of HflK and HflC has not been possible so far, since each requires the presence of the other for proper folding. We report the first purification of HflK and HflC separately as active and functional proteins and show that each can interact with HflB on its own and each inhibits the proteolysis of CII. They also inhibit the proteolysis of E. coli σ³² by HflB. We show that at low concentrations each protein is dimeric, based on which we propose a scheme for the mutual interactions of HflB, HflK and HflC in a supramolecular HflBKC protease complex.     

Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22

Escherichia coli recA protein directs the inactivation of the repressor of Salmonella typhimurium phage P22 in vitro. As is true for repressor of the E. coli phage λ, inactivation of P22 repressor is accompanied by proteolytic cleavage of the repressor into two detectable fragments.We have investigated the kinetics of inactivation of the λ and P22 repressors in vitro. The fraction of λ repressor inactivated per unit time decreases as its concentration in the reaction is increased. However, high concentrations of λ repressor do not inhibit the inactivation of P22 repressor. Thus, it does not appear that the inactivation system is saturated by λ repressor, but rather that λ repressor is a less efficient substrate at higher concentrations.     

CPAF: A Chlamydial Protease in Search of an Authentic Substrate

Bacteria in the genus Chlamydia are major human pathogens that cause an intracellular infection. A chlamydial protease, CPAF, has been proposed as an important virulence factor that cleaves or degrades at least 16 host proteins, thereby altering multiple cellular processes. We examined 11 published CPAF substrates and found that there was no detectable proteolysis when CPAF activity was inhibited during cell processing. We show that the reported proteolysis of these putative CPAF substrates was due to enzymatic activity in cell lysates rather than in intact cells. Nevertheless, Chlamydia-infected cells displayed Chlamydia-host interactions, such as Golgi reorganization, apoptosis resistance, and host cytoskeletal remodeling, that have been attributed to CPAF-dependent proteolysis of host proteins. Our findings suggest that other mechanisms may be responsible for these Chlamydia-host interactions, and raise concerns about all published CPAF substrates and the proposed roles of CPAF in chlamydial pathogenesis.     

Maturation of the head of bacteriophage T4. I. DNA packaging events

Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P231 (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P231, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells.The prohead II particles are in turn converted into particles which sediment with about 550 S after DNase treatment (prohead III). During this conversion about 50% of normal DNA complement becomes packaged in a DNase-resistant form, and roughly 50% of the core proteins P22 and IP III are cleaved. In lysates the prohead III particles are attached to the replicative DNA. The prohead III particle appears to be the immediate precursor of the full mature head (1100 S). Cleavage of protein P22 to small polypeptides and conversion of IP III IP III1 are completed at this time. No precursor proteins are found in the full heads. Studies with various mutant phage showed that the prohead II to III conversion is blocked by mutations in genes 16 and 17 and that the conversion of the prohead III particles to the mature heads is blocked by mutations in gene 49. Cleavage of the head proteins, however, occurs normally in these mutant-infected cells. We conclude that the cleavage of the major component of the viral shell, P23, into P231 precedes the DNA packaging event, whereas cleavage of the core proteins P22 and IP III appears to be intimately linked to the DNA packaging event. Models relating the cleavage processes to DNA encapsulation are discussed.     

Assembly of Both the Head and Tail of Bacteriophage Mu Is Blocked in Escherichia coli groEL and groES Mutants

Like several other Escherichia coli bacteriophages, transposable phage Mu does not develop normally in groE hosts (M. Pato, M. Banerjee, L. Desmet, and A. Toussaint, J. Bacteriol. 169:5504–5509, 1987). We show here that lysates obtained upon induction of groE Mu lysogens contain free inactive tails and empty heads. GroEL and GroES are thus essential for the correct assembly of both Mu heads and Mu tails. Evidence is presented that groE mutations inhibit processing of the phage head protein gpH as well as the formation of a 25S complex suspected to be an early Mu head assembly intermediate.     

Mutational Analysis of the Pseudomonas aeruginosa Myovirus ?KZ Morphogenetic Protease gp175

Pseudomonas aeruginosa myovirus ϕKZ has a 270-kb genome within a T=27 icosahedral capsid that contains a large, unusual, and structurally well-defined protein cylindrical inner body (IB) spanning its interior. Proteolysis forms a pivotal stage in ϕKZ head and IB morphogenesis, with the protease gp175 cleaving at least 19 of 49 different head proteins, including the major capsid protein and five major structural IB proteins. Here we show that the purified mature form of gp175 is active and cleaves purified IB structural proteins gp93 and gp89. Expression vector synthesis and purification of the zymogen/precursor yielded an active, mature-length protease, showing independent C-terminal gp175 self-cleavage autoactivation. Mutation of either the predicted catalytic serine or histidine inactivated mature gp175, supporting its classification as a serine protease and representing the first such direct biochemical demonstration with purified protease and substrate proteins for any phage protease. These mutations also blocked self-cleavage of the precursor while allowing intermolecular gp175 processing. To confirm the cleavage specificity of gp175, we mutated three cleavage sites in gp93, which blocked proteolysis at these sites. The N-terminal propeptide of gp93 was shown to undergo more extensive proteolysis than previously identified. We found that proteolysis in gp93 progressed from the N to C terminus, while blocking cleavage sites slowed but did not eliminate downstream proteolysis. These findings were shown by informatics to be relevant to the head morphogenesis of numbers of other related IB-containing giant phages as well as to T4 and herpesviruses, which have homologous proteases.     

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.     

Studies on the structure, protein composition and aseembly of the neck of bacteriophage T4

We have developed an osmotic shock procedure which disconnects the tail from the head of intact bacteriophage T4, leaving the neck region attached to the tail. Purification of these necked tails permitted detailed structural observations of the neck and the collar/whisker complex attached to it, as well as comparison by gel electrophoresis with tails lacking the neck. Five or six neck proteins were found: N1 (M_r = 52,000; 39 copies/phage) is the product of the wac³ gene (Pwac), forms both the collar and six whiskers as a multimeric fibrous protein, and probably assembles onto phage after head to tail joining; N2 (M_r= 35,000; 5 to 6 copies/phage), N3 (M_r= 33,000; 17 copies/phage) identified here as P13, and N6 (M_r= 28,000; 10 to 11 copies/phage) are all assembled in heads prior to tail joining; N4 (M_r= 32,000; 6 to 9 copies/phage) is unusual in that it is present in wac or wac⁺ phage and necked tails but is absent from purified heads; N5 (M_r =29,000) is probably P14 and like N4 is not found in heads. However, while we find one to two copies of N5 per necked tail, we have not observed it in phage.An aberrant neck structure called the extension assembles on the distal end of the tail connector late (after 33 min, 30 °C) in head-defective, mutant-infected cells. The extension contains five of the six neck proteins (N2 is absent), and blocks head to tail joining in vitro. Mutations in genes 13 and 14, and the double mutant 49:Wac block extension assembly.Other results show that the wac mutant E727J is an amber lesion, and that Pwac can assemble on collarless, wac phage in vitro.