Construction and characterization of hybrids containing genes coding for proteins of the central part of bacteriophage T4 baseplate.

The central part (hub or plug) of bacteriophage T4 baseplate consist of several proteins which are present in only few copies per phage particle. The presence of these minor baseplate components was inferred from the genetic data but only some of them were identified as distinct proteins species by biochemical analysis. We have constructed a number of plasmids containing segments of bacteriophage T4 genome coding for baseplate proteins. The following genes were cloned into expression vectors: 54, 48, 29, 28, 27, 51, 26 and 25. The presence of a particular gene product was confirmed by in vivo complementation test. On the basis of these results we could more precisely localize the position of a particular gene on T4 phage genetic map. The hybrids contain sets of genes which make aggregation impossible, so bacteria harbouring these plasmids are convenient starting point for the purification of baseplate proteins.     

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.     

Expression of bacteriophage T4 minor baseplate proteins in the bacteriophage T7 promoter/RNA polymerase expression system.

The central part of bacteriophage T4 baseplate is built of several proteins which are present in only a few copies per phage particle. Only some of these minor baseplate components have been identified previously as distinct protein species by biochemical analysis. We have used the bacteriophage T7 RNA polymerase expression system to identify and overexpress the minor baseplate proteins. The products of genes 25, 26 and 51 were identified on the autoradiographs after selective labelling with [35]S methionine. The overexpression of gene 25 and 51 products was high enough to make possible undertaking their purification and studies of their properties.     

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.     

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.     

The Baseplate Wedges of Bacteriophage T4 Spontaneously Assemble into Hubless Baseplate-Like Structure In Vitro

The baseplate of phage T4 is an important model system in viral supramolecular assembly. The baseplate consists of six wedges surrounding the central hub. We report the first successful attempt at complete wedge assembly using an in vitro approach based on recombinant proteins. The cells expressing the individual wedge proteins were mixed in a combinatorial manner and then lysed. Using this approach, we could both reliably isolate the complete wedge along with a series of intermediate complexes as well as determine the exact sequence of assembly. The individual proteins and intermediate complexes at each step of the wedge assembly were successfully purified and characterized by sedimentation velocity and electron microscopy. Although our results mostly confirmed the hypothesized sequential wedge assembly pathway as established using phage mutants, interestingly, we also detected some protein interactions not following the specified order. It was found that association of gene product 53 to the immediate precursor complex induces spontaneous association of the wedges to form a six-fold star-shaped baseplate-like structure in the absence of the hub. The formation of the baseplate-like structure was facilitated by the addition of gene product 25. The complete wedge in the star-shaped supramolecular complex has a structure similar to the baseplate in the expanded “star” conformation found after infection. Based on the results of the present and previous studies, we assume that the strict order of wedge assembly is due to the induced conformational change caused by every new binding event. The significance of a 40-S star-shaped baseplate structure, which was previously reported and was also found in this study, is discussed in the light of a new paradigm for T4 baseplate assembly involving the star-shaped wedge ring and the central hub. Importantly, the methods described in this article suggest a novel methodology for future structural characterization of supramolecular protein assemblies.     

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.     

Structure of the central hub of bacteriophage Mu baseplate determined by X-ray crystallography of gp44

Bacteriophage Mu is a double-stranded DNA phage that consists of an icosahedral head, a contractile tail with baseplate and six tail fibers, similar to the well-studied T-even phages. The baseplate of bacteriophage Mu, which recognizes and attaches to a host cell during infection, consists of at least eight different proteins. The baseplate protein, gp44, is essential for bacteriophage Mu assembly and the generation of viable phages. To investigate the role of gp44 in baseplate assembly and infection, the crystal structure of gp44 was determined at 2.1A resolution by the multiple isomorphous replacement method. The overall structure of the gp44 trimer is similar to that of the T4 phage gp27 trimer, which forms the central hub of the T4 baseplate, although these proteins share very little primary sequence homology. Based on these data, we confirm that gp44 exists as a trimer exhibiting a hub-like structure with an inner diameter of 25A through which DNA can presumably pass during infection. The molecular surface of the gp44 trimer that abuts the host cell membrane is positively charged, and it is likely that Mu phage interacts with the membrane through electrostatic interactions mediated by gp44.     

Functional Role of the N-Terminal Domain of Bacteriophage T4-Gene Product 11

Bacteriophage T4 late gene product 11 (gp11), the three-dimensional structure of which has been solved by us to 2.0 A resolution, is a part of the virus' baseplate. The gp11 polypeptide chain consists of 219 amino acid residues and the functionally active protein is a three-domain homotrimer. In this work, we have studied the role of gp11 N-terminal domain in the formation of a functionally active trimer. Deletion variants of gp11 and monoclonal antibodies recognizing the native conformation of gp11 trimer have been selected. Long deletions up to a complete removal of the N-terminal domain, containing 64 residues, do not affect the gp11 trimerization, but considerably change the protein structure and lead to the loss of its ability to incorporate into the baseplate. However, the deletion of the first 17 N-terminal residues results in functionally active protein that can complete the 11(-)-defective phage particles in in vitro complementation assay. This region of the polypeptide chain is probably essential for gp11-gp10 stable complex formation at the early stages of phage baseplate assembly in vivo. A study of the gp10 deletion variants suggests that the central domain of gp10 trimer is responsible for the interaction with gp11.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates II. Folate Metabolism and Polyglutamate Cleavage Activity of Uninfected and Infected Escherichia coli Cells and Bacteriophage Particles

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28^ts or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28^ts production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28^ts was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29⁻, 26⁻, 27⁻, 51⁻, and 10⁻, but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28^ts. Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28^ts activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.     

The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in beta-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.     

Functional relationships and structural determinants of two bacteriophage T4 lysozymes: a soluble (gene e) and a baseplate-associated (gene 5) protein

Lysozymes have proved useful for analyzing the relation between protein structure and function and evolution. In bacteriophage T4, the major soluble lysozyme is the product of the e gene, gpe (gene product = gp). This lysozyme destroys the wall of its host, Escherichia coli, at the end of infection to release progeny particles. Phage T4 contains two additional lysozymes that facilitate penetration of the baseplates into host cell walls during adsorption. At least one of these, a 44-kD protein, is encoded by gene 5. We show here that a segment of the gp5 lysozyme amino acid sequence, deduced from the DNA sequence of gene 5, is remarkably similar to that of the T4 gene e lysozyme. Both T4 lysozymes are somewhat similar to the lysozyme of the Salmonella phage P22, but there is little significant DNA sequence homology among the two T4 lysozyme genes and the P22 lysozyme gene. We speculate that these lysozymes are adapted to differences in the composition of the cell walls of E. coli and S. typhimurium. The cloned gene 5 of the phage T4 directs synthesis of a 63-kD precursor protein that is approximately 19 kD larger than the gene 5 protein isolated from baseplates. Gp5 first associates with gp26 to form the central hub of this structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of P48 and P54 as components of bacteriophage T4 baseplates.

The involvement of two bacteriophage T4 gene products in the initiation of T4 tail tube and sheath polymerization on mature baseplates has been studied by radioautography of acrylamide gels of various partially completed tail structures. The products of genes 48 and 54 (P48[the nomenclature P48 refers to the protein product of bacteriophage T4 gene 48] and P54), which are known to be required for the synthesis of mature baseplates, have been shown to be structural components of the baseplate. These gene products have molecular weights of 42,000 and 33,000, respectively. The addition of P54 to the baseplate not only permits the polymerization of the core protein, P19, onto the baseplate, but also caused the disappearance of a polypeptide of molecular weight about 15,000 from the supernatant fraction of infected cells. Another gene product, P27, has been identified in the crude extracts of infected cells. This gene product, which is required for the synthesis of baseplate structures, has the same mobility as one of the unidentified structural polypeptides of the baseplate and is therefore probably also a baseplate component.     

Morphogenesis of the T4 tail and tail fibers

Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study.     

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.     

Isolation and characterization of precursors in bacteriophage T4 baseplate assembly. II. Purification of the protein products of genes 10 and 11 and the in vitro formation of the P(10/11) complex

Two bacteriophage T4 proteins which are precursors to the phage baseplate have been purified to homogeneity. These proteins, P10 and P11, are components of the P(10/11) complex, which is the first intermediate in the assembly of T4 baseplate 1/6th arms. Each protein was isolated from cells infected with a T4 amber mutant defective in the production of the other protein. Thus these purified proteins have never been assembled into the P(10/11) complex in vivo. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and the ability of these proteins to block the phage killing activity of specific antisera were used to monitor the purification steps. Sedimentation equilibrium experiments reveal a molecular weight of 188,000 g/mol for P10 and 60,000 g/mol for P11. These data together with the previously determined molecular weights of the gene 10 and gene 11 polypeptide chains (King & Mykolajewycz, 1973) and the in vivo assembled P(10/11) complex (Berget & King, 1978b) are consistent with P10 being a dimer of the product of gene 10, P11 being a dimer of the product of gene 11, and P(10/11) being a tetramer containing one of each of these dimers. Purified P10 and P11 are active in assembly because they complement 10- and 11- defective extracts, respectively, to form viable bacteriophage in vitro. Furthermore, these proteins assemble in vitro to form a protein structure identical to the P(10/11) complex formed in vivo as determined by non-denaturing gel electrophoresis. This P(10/11) complex formed in vitro complements 10-/11- defective extracts to form viable phage. The overall extent of this in vitro assembly reaction is not affected by NaCl to 1.5 M or 2% Triton X-100. The reaction is, however, prevented by the denaturing effects of urea and sodium dodecyl sulfate.     

Protein interactions in the assembly of the tail of bacteriophage T4

Protein interactions in the assembly of the baseplate have been investigated. The baseplate of the phage T4 tail consists of a hub and six wedges which surround the former. Both reversible and irreversible interactions were found. Reversible association includes gp5 and gp27 (gp: gene product) which form a complex in a pH-dependent manner and gp18 polymerization, i.e. the tail sheath formation depends on the ionic strength. These reversible interactions were followed by irreversible or tight binding which pulls the whole association reaction to complete the assembly. The wedge assembly is strictly ordered which means that if one of the seven wedge proteins is missing, the assembly proceeds to that point and the remaining molecules stay non-associated. The strictly sequential assembly pathway is suggested to be materialized by successive conformational change upon binding, which can be shown by proteolytic probe.     

Mutants of bacteriophage T4 that produce infective fibreless particles

In wild type bacteriophage T4 the long tail fibres serve both in the initial attachment of the phage to its host and in the triggering of tail contraction. A two-step model for the control of triggering suggests that particles lacking the product of gene 9, which are also structurally fibreless, might be infective. This is shown to be the case, even though such phage do not plate on restrictive strains of bacteria. However, starting from phage carrying an amber mutation in gene 9 it is easy to isolate additional mutations which, under restrictive conditions, permit fibreless plating (pfp mutations). Three such pfp mutations, having also a temperature-sensitive phenotype, have been isolated and shown to map in genes coding for structural components of the baseplate. The mode of action of these pfp mutations is not clear, though they certainly destabilize the baseplate, thereby making triggering easier. The pfp mutations are effective only when in combination with an amber mutation in gene 9 and not with amber mutations in tail fibre genes, establishing the essentially inhibitory nature of the control of triggering exercised by gene 9 product.     

The tail lysozyme complex of bacteriophage T4

The tail baseplate of bacteriophage T4 contains a structurally essential, three-domain protein encoded by gene 5 in which the middle domain possesses lysozyme activity. The gene 5 product (gp5) undergoes post-translational cleavage, allowing the resultant N-terminal domain (gp5*) to assemble into the baseplate as a trimer. The lysozyme activity of the undissociated cleaved gp5 is inhibited until infection has been initiated, when the C-terminal portion of the molecule is detached and the rest of the molecule dissociates into monomers. The 3D structure of the undissociated cleaved gp5, complexed with gp27 (another component of the baseplate), shows that it is a cell-puncturing device that functions to penetrate the outer cell membrane and to locally dissolve the periplasmic cell wall.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.