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.     

Cleavage of head and tail proteins during bacteriophage T5 assembly: selective host involvement in the cleavage of a tail protein

Electrophoresis studies showed that at least three phage-specified proteins undergo proteolytic cleavage during the development of bacteriophage T5. One of these proteins has a molecular weight of about 135,000 and the product of this cleavage reaction is a minor component of the T5 tail, having a molecular weight of about 128,000. All of the tail-defective T5 mutants studied in this report failed to induce this cleavage reaction under restrictive conditions. This reaction also failed to occur in Escherichia coli groEA639 and groEA36 infected with wild type T5. Examination of lysates of infected groE cells in the electron microscope revealed the presence of filled and empty heads as well as tubular head structures, but no tails were detected. The filled heads were able to combine with separately prepared T5 tails in vitro to form infectious phage particles. Therefore, propagation of T5 in these groE mutants is prevented primarily by a specific block in tail assembly. A T5 mutant, T5?6, was isolated, which has the capacity to propagate in these groE hosts. The gene locus in T5?6 was mapped.The second T5 protein which is cleaved has a molecular weight of 50,000 and is related to head morphogenesis. Treatment of infected cells with l-canavanine (50 μg/ml) inhibited cleavage of this polypeptide. Only small quantities of the major head protein (32,000 mol. wt) were produced in these treated cells. Treatment with canavanine lead to production of tubular heads. The major protein component of partially purified tubular heads has a molecular weight of 50,000. Cells infected with T5 amber H30b, a mutant defective in head gene D20, does not produce the 50,000 and 32,000 molecular weight proteins. These findings suggest that the 50,000 molecular weight protein undergoes cleavage to form the major head polypeptide. A third T5 protein is cleaved to form a minor head component with a molecular weight of 43,000 and its cleavage is linked to that involving the major head protein.     

Role of the Host Cell in Bacteriophage Morphogenesis: Effects of a Bacterial Mutation on T4 Head Assembly

In certain bacterial mutants, called groE, T4 phage head assembly is blocked specifically, implying that the host plays a direct role in head assembly. The block occurs early in the assembly process at the level of action of T4 gene 31.     

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.     

Head-tail connector of bacteriophage lambda

The head-tail connector of phage λ, a protein knob inside the head shell to which the tail attaches, is composed primarily of head protein gpB ⁴ and its cleaved form gpB¹. All of the gpB and gpB¹ in the virion is located in the connector. gpFII, the protein that is thought to form the site on the head to which the tail binds, is also located in the connector. Head proteins gpE, gpD, X1 and X2 are not components of the connector. These assignments were made by disrupting virions with guanidine hydrochloride, in such a way that heads and tails separate with the connectors attached to the tails, and determining which head proteins co-purify with the tails.We find that lysates from a λE^? infection contain a high proportion of tails with connectors attached. (Gene E codes for the major component of the head shell.) Connectors are also present on tails from a λE^?C^? infection, arguing that gpE, gpC, and their processed forms, X1 and X2, are all unnecessary for assembly of biologically competent connectors. The gpB in the connectors on E^? and E^?C^? tails is in the uncleaved form. Connectors are not seen on tails from infections by λE^?B^?, λE^?FII^?, or λE^? in a groE^? host.     

Assembly of bacteriophage lambda heads: Protein processing and its genetic control in petit λ assembly

Petit bacteriophage λ is a hollow λ head precursor which is found in λ-infected lysates, including lysates of phage λ carrying mutations in head genes. Wild-type petit λ has a protein composition similar to heads, except that it is missing pD ⁴, a major component of heads. About 95% of the mass of petit λ is pE, the major structural protein of heads, and in addition it has proteins pB, h3, X1, and X2. Tryptic fingerprint analysis shows that h3 is a proteolytic cleavage product of pB, and previous experiments have shown that X1 and X2 are protein fusion products, closely related to each other and containing amino acid sequences of both pC and pE. Petit lambdas derived from infection by phages defective in genes A or D are indistinguishable from wild-type petit λ. B⁻, C⁻, or groE⁻ defective petit lambdas show differences from wild-type in protein composition and in extent of protein processing. On the basis of the properties of mutant petit lambdas it is concluded that: (1) the protein processing reactions (cleavage of pB; fusion of pC with pE) occur on the petit λ structure; (2) cleavage of pB requires the functioning of genes C and groE but not A or D; (3) fusion of pC and pE requires gene groE but not A, B or D; (4) pNu3 participates directly in petit λ assembly but is lost from the structure by the time assembly is complete.Physical studies of petit λ show that wild-type, A⁻, B⁻ and D⁻ petit lambdas sediment at 150 S, while C⁻ and groE⁻ petit lambdas sediment at 190 S. Purified petit λ of either class has an ultraviolet absorption spectrum characteristic of pure protein.     

Morphogenesis of bacteriophage lambda deletion mutants. I. Abnormal head-related structures produced in normal Escherichia coli.

Late in the morphogenesis of bacteriophage lambda, DNA condenses into the nascent head and is cut from a concatemeric replicative intermediate by a nucleolytic function, Ter, acting at specific sites, called cos. As a result of this process, heads of lambda deletion mutants contain less DNA than those of the wild-type phage. It has been reported that phage with very large deletions (22% of the genome or more) grow poorly but that normal growth can be restored by the non-specific addition of DNA to the genome. This finding implies that DNA content may exert a physical effect on some stage of head assembly.We have investigated the effects of two long deletions, b221 and tdel33, on head assembly. Bacteria infected with the mutants were lysed with non-ionic detergent under conditions favoring stabilization of labile structures containing condensed DNA. It has proved possible to isolate two aberrant head-related structures produced by the deletion mutants. One of these (“overfilled heads”) contains DNA which is longer than the deletion mutant genome and is about the same size as that found in wild-type heads. These structures appear to be unable to attach tails. The second type of structure (“incompletely filled heads”) contains a short piece of DNA, 40% of the length of the mutant genome. The incompletely filled heads are found both with and without attached tails. Both of these abnormal structures are initially attached to the replicating DNA but are released by treatment with DNAase. The nature of these abnormal structures indicates that very small genomes affect a late stage of head morphogenesis, after the DNA is complexed with a capsid of normal size. The results presented suggest that underfilling of the capsid interferes with the ability of the Ter function to properly cleave cos.     

DNA packaging steps in bacteriophage lambda head assembly

Petit λ is an empty spherical shell of protein which appears wherever λ grows. If phage DNA and petit λ are added to a cell-free extract of induced lysogenic bacteria, then phage particles are formed that contain the DNA and protein from the petit λ. Petit λ is transformed, without dissociation, into a phage head by addition of DNA and more phage proteins.The products of ten genes, nine phage and one host, are required for λ head assembly. Among these, the products of four phage genes, E, B, C, and Nu3 and of the host gene groE are involved in the synthesis of petit λ, consequently these proteins are dispensable for head assembly in extracts to which petit λ has been added. The products of genes A and D allow DNA to combine with petit λ to form a head that has normal morphology. In an extract, DNA can react with A product and petit λ to become partially DNAase-resistant, as if an unstable DNA-filled intermediate were formed. ATP and spermidine are needed at this stage. This intermediate is subsequently stabilized by addition of D product. The data suggest a pathway for head 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.     

Structural studies of bacteriophage lambda heads and proheads by small angle X-ray diffraction.

We have examined a series of lambda proheads and mature structures by small angle X-ray diffraction. This technique yields spherically averaged density distributions and some information about surface organization of particles in solution.We find that gpE ² of proheads and heads forms shells with one of two radii; A^?, B^?, groE^?, and Nu3^? proheads have shells of radius 246 Å, while mature heads, urea-treated A^? proheads and C^? proheads have a radius of 300 Å. The expansion of proheads to mature heads is accompanied by a corresponding decrease in the thickness of the shell. groE^? proheads contain a core. This core is lost spontaneously from the structure and is only observed if the structures are fixed with glutaraldehyde prior to examination by X-ray diffraction or electron microscopy.C^? proheads expand to mature head size spontaneously. A preparation of C^? proheads which was fixed with glutaraldehyde at an early stage of the purification had the smaller, prohead radius. Unfixed particles from this preparation expanded to the mature head size after further purification and standing in the cold for several days. This result suggests that gpC may be involved in regulating head expansion.The radii of the protein shells of mature heads are identical for a series of phages that contain between 78% and 105% of the wild-type complement of DNA, and this radius is the same as that of proheads expanded in the absence of DNA. These results with phage lambda indicate that assembly of a double shell structure composed of coat and scaffolding protein, followed by expansion to a larger shell containing only coat protein is a general feature of the morphogenesis of dsDNA phages.     

Studies on the In Vitro Assembly of Bacteriophage φ80 and φ80-Lambda Hybrids

Suppressor-sensitive (sus) mutants of bacteriophage 80 defective in late functions were classified, by means of in vitro assembly tests, into two complementation groups: head donors and tail donors. Each group of mutants was subdivided, by means of two-factor crosses, into six cistrons. Deletion mapping revealed clustering of tail and also of head cistrons. The two clusters were located in the left arm of vegetative 80 (the tail specifying cluster being distal). In vitro cross complementation between 80 and lambda sus mutants revealed that whereas lambda heads could quite efficiently bind 80 tails to form viable phage, the union of 80 heads and lambda tails was very much less efficient. Deletion mapping of the 80 sus mutants, using both 80 and i⁸⁰h^λ deletion lysogens indicated congruent gross gene arrangement in the two related bacteriophages.     

Bacteriophage lambda FII gene protein: role in head assembly

The in vitro completion of bacteriophage lambda F_II^? heads to form phage can be used as an assay for the λ F_II gene protein. F_II protein activity is released from highly purified phage particles or phage heads by treatment with heat or denaturing agents. F_II protein was purified from isolated phage particles and from an extract of E^? infected cells in which it is not bound to any large structures. No differences in molecular weight (11,500), isoelectric point (4.75), electrophoretic mobility, or purification properties could be demonstrated between the F_II proteins from the two sources. Thus the polypeptide does not seem to be modified during assembly.Phage φ80 is closely related to λ. φ80 heads will join to φ80 tails in vitro but will not join to λ tails, though λ heads will join to either type of tail. Mixing experiments between F_II^? heads, tails, and F_II protein from λ or φ80 show that the specificity of head-tail joining is correlated with the source of the F_II protein and not with the source of the other head proteins. Thus, F_II protein is apparently responsible for this specificity of head-tail joining.     

The lambda head-tail joining reaction: purification, properties and structure of biologically active heads and tails

Procedures were developed to obtain biologically active lambda heads and tails at high purity with 20 to 40% recovery. Free heads, free tails and phage particles differ markedly in stability. Phage are stable in solutions containing Mg²⁺ but tails are not. The protein subunits which form the shaft of the tail dissociate in the presence of Mg²⁺ and form multisubunit spherical structures. EDTA protects free tails against inactivation but disrupts heads and phage particles. The four carbon diamine, putrescine, stabilizes heads against inactivation; the three and five carbon diamines are less effective. Electron micrographs reveal a new “knob” structure at the distal end of the tail fiber of phage and of free tails. Tails released from EDTA-disrupted phage possess a “head-tail connector”, a structure not present on the tail before its joining with a head.     

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.     

Involvement of a Tryptophan Residue in the Assembly of Bacteriophages φ80 and Lambda

The assembly of infective particles of bacteriophages lambda and phi80 from heads and tails was found to be inhibited by l-tryptophan and some of its analogues, most notably tryptamine. Both the rate of assembly and the final yield of phage were inhibited. The amino acid l-phenylalanine had a slight inhibitory effect, whereas all other amino acids found in proteins were ineffective. Evidence was presented to show that the binding of heads to tails was the affected process in the assay for assembly of infective units. The plaque-forming ability of preassembled phage was not affected by these inhibitors. Results of three different types of experiments suggest that inhibition is due to interaction of inhibitors with the head substructure. The assembly reaction is highly dependent on pH, ionic strength, and the presence of detergents.     

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.     

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).     

Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk “Viili”

“Viili,” a fermented milk product, has a firm but viscous consistency. It is produced with traditional mesophilic mixed-strain starters, which have various stabilities in dairy practice. Thirteen morphologically different types of phages were found in 90 viili samples studied by electron microscopy. Ten of the phage types had isometric heads with long, noncontractile tails, two had elongated heads with long, noncontractile tails, and one had a unique, very long elongated head with a short tail. Further morphological differences were found in the tail size and in the presence or absence of a collar, a baseplate, and a tail fiber. To find hosts for the industrially significant phages, we examined the sensitivities of 500 bacterial isolates from starters of the viili. Seven of the phages attacked Streptococcus cremoris strains, three attacked S. lactis subsp. diacetylactis strains, and four attacked Leuconostoc cremoris strains. Some phages differed only in their host specificity. Hosts were not found for 4 of the 13 morphological types of phages.     

Genome Sequence and Characteristics of Lrm1, a Prophage from Industrial Lactobacillus rhamnosus Strain M1

Prophage Lrm1 was induced with mitomycin C from an industrial Lactobacillus rhamnosus starter culture, M1. Electron microscopy of the lysate revealed relatively few intact bacteriophage particles among empty heads and disassociated tails. The defective Siphoviridae phage had an isometric head of approximately 55 nm and noncontractile tail of about 275 nm with a small baseplate. In repeated attempts, the prophage could not be cured from L. rhamnosus M1, nor could a sensitive host be identified. Sequencing of the phage Lrm1 DNA revealed a genome of 39,989 bp and a G+C content of 45.5%. A similar genomic organization and mosaic pattern of identities align Lrm1 among the closely related Lactobacillus casei temperate phages A2, ΦAT3, and LcaI and with L. rhamnosus virulent phage Lu-Nu. Of the 54 open reading frames (ORFs) identified, all but 8 shared homology with other phages of this group. Five unknown ORFs were identified that had no homologies in the databases nor predicted functions. Notably, Lrm1 encodes a putative endonuclease and a putative DNA methylase with homology to a methylase in Lactococcus lactis phage Tuc2009. Possibly, the DNA methylase, endonuclease, or other Lrm1 genes provide a function crucial to L. rhamnosus M1 survival, resulting in the stability of the defective prophage in its lysogenic state. The presence of a defective prophage in an industrial strain could provide superinfection immunity to the host but could also contribute DNA in recombination events to produce new phages potentially infective for the host strain in a large-scale fermentation environment.