We describe a method for the rapid partial purification of intermediate structures of phage lambda tail assembly, using formaldehyde-fixed Escherichia coli cells to precipitate tail-related structures. The purification depends on the specific interaction between the E. coli lambda receptor protein and lambda tail protein gpJ. Protein compositions of tail assembly intermediates were analyzed to determine when in the assembly sequence the minor tail protein gpH is cleaved. gpH joins the tail precursor structure early in the pathway, during assembly of the initiator (a structure that becomes the tail tip). However, gpH is not cleaved until after initiator assembly is complete and after the tail shaft has polymerized onto the initiator. These results suggest that each gpH molecule is extended along the length of the tail. Our results also appear to eliminate an ambiguity in the tail assembly pathway determined by earlier experiments: we argue that gene G acts between genes H and M.

We have studied the phenotype of a heat-sensitive mutation that defines λ tail gene Induction and growth at the nonpermissive temperature of a λTts40 prophage results in production of morphologically normal but biologically inactive tails, which can be activated in vitro by lysates supplying the products of genes T, U, and Z. Thus, gene T acts between genes V and U in the tail assembly pathway or immediately after the completion of tail shaft polymerization. λTts40 lysates also contain λ substantial number of morphologically normal phage particles which are deficient in cleavage of the minor tail protein gpH. This result is consistent with the fact that 9PH cleavage normally occurs later in the assembly pathway than the time found for gene T action. Purified λTts40 virions that were produced at the permissive temperature are normal with respect to gpH cleavage. However, they are more sensitive to heat inactivation than are wild-type virions, suggesting that the T gene product is in the virion. At the permissive temperature, the mutant protein made by λTts40 is active in vivo even if it has been synthesized at the nonpermissive temperature.

Correct assembly of the heads of bacteriophages lambda and T4 requires the function of the groE gene of the Escherichia coli host. We have isolated a transducing derivative of lambda, called lambda gt-Ec.groE, that carries a functional copy of the groE gene. Unlike wild-type lambda, this phage is able to form plaques on hosts with a mutant groE gene. We have isolated an amber mutation in the groE gene carried by the phage, and this has made it possible to identify the groE product as a protein of molecular weight 65,000. In the phage, the groE gene is under the control of an early phage promoter.

The effect of multiplicity of infection was studied in Escherichia coli with λ phage, using phage endolysin as an example of a late gene product. A very sensitive endolysin assay method was used so that the initiation time of endolysin synthesis could be more accurately determined. It was observed that high multiplicity of infection (1) increases the rate of lysogenization, (2) progressively delays lysis time, and (3) significantly delays and reduces the synthesis of endolysin in λcIII+-infected cells. The extent of delay and reduction in endolysin synthesis increases with increasing multiplicity. In contrast, λcIII67cII68-infected cells show no delay in endolysin synthesis at high multiplicity of infection when compared with the λcIII+cII+-infected cells. The results suggest that (1) the expression of cIII and cII genes is multiplicity dependent, (2) high multiplicity of infection enhances the expression of the cIII and cII genes, and (3) the expression of the cIII and cII genes interferes with the expression of the late genes. A model to explain how the expression of the cIII and cII genes interferes with the expression of the late genes is proposed.