J.R. Christensen

Functional characterization of the genes of bacteriophage T1

Abstract Amber mutants representing the 18 known genes of coliphage T1 Michalke 1967 were examined for DNA synthesis, head or tail production, and cell lysis in a nonpermissive host. Mutants in genes 1 or 2 are nearly completely defective in phage DNA synthesis. However, they are still capable of rapidly shutting off host DNA synthesis. This shutoff is caused by a phage-induced protein, since it is inhibited by chloramphenicol. Gene 1 or gene 2 mutants also affect lysis kinetics of the infected cells to permit only a delayed and incomplete lysis. The gene 4 mutant causes a premature arrest of phage DNA synthesis, but lyses the host normally. Mutants in all other genes are normal in DNA synthesis and lysis, but are defective in some stage of maturation. An in vitro complementation assay and electron microscopy of lysates from the nonpermissive host were used to ascertain that the mutants of gene 3 and genes 5 to 11 inclusive are producers of heads, but not tails. Presumably, these genes are involved in tail biosynthesis. Conversely, gene 4 and genes 12 to 18 inclusive are involved in head biosynthesis because mutants in these genes produce only tails.

Genetic crosses between restricted and unrestricted phage T1 in lysogenic and nonlysogenic hosts

Abstract When Shigella dysenteriae is lysogenized by the phage P1, it becomes immune to infection with “ordinary” T1 phage (“restricted T1” or “rT1”). A host-controlled variant of T1 (“unrestricted T1” or “uT1”) can multiply in the lysogenic S. dysenteriae. Three-factor crosses were done between suitably marked strains of rT1 and uT1 in both lysogenic and nonlysogenic S. dysenteriae. When lysogenic cells are infected with both rT1 and uT1, one finds some recombinants among the progeny, but few, if any phage bearing the genotype of the restricted parent. The proportion of recombinants in the total yield is about one-eighth to one-twelfth that in control crosses (in nonlysogenic hosts) and is independent of the order of addition of either parent and of the time elapsing between the addition of each parent. We infer, then, that (1) the genome of rT1 enters the lysogenic cells; (2) it is neither replicated nor is it destroyed; and (3) it can “mate” with genomes of uT1 which may be present. The proportions of the various recombinant classes are unusual. Complementary classes are not equal; certain markers may be recovered together with a high frequency, whereas certain others, more closely linked, are seldom found together. Certain “double crossover” classes are more frequent than certain “single crossover” classes. These observations can be explained in terms of a “copy choice” mechanism of recombination by assuming that the rT1 genome contains at least two “bad spots,” the location of which can be determined approximately and which force a “switch” in replication from the restricted genome to the unrestricted genome.

The kinetics of reversible and irreversible attachment of bacteriophage T 1

Abstract Solutions are presented for differential equations representing the kinetics of reversible and irreversible attachment of bacteriophage to bacteria according to two different hypotheses: reversible attachment is an intermediate state in the infectious process (sequential theory); reversible attachment is noninfectious and competes with irreversible, infectious attachment (competitive theory). Curves computed from the respective solutions are compared. Experimentally, total attachment and irreversible attachment kinetics were followed during the attachment of phage T1 to Escherichia coli B(P1), to which T1 attaches normally, but in which T1 fails to produce progeny because of prophage interference. Consistent with the sequential theory, a shoulder was found under suitable conditions in the irreversible attachment curve. Consistent with the competitive theory, all attachment curves followed long enough showed a marked decrease in attachment rates after most of the phage had attached. The results on this point were not explainable on the basis of heterogeneity in the phage population. The curves for reversible attachment were more complex than could be explained by either theory. Differential equations are presented corresponding to the hypothesis (modified sequential theory) that attachment is a sequential process, but that a certain proportion of reversible attachment sites do not allow any subsequent irreversible step. Analytical solutions for these equations were not obtained, but curves were computed from them by numerical methods. By a trial and error process, it was possible to obtain curves that agreed quite well with experimental results. In some cases, the best fit could be obtained only by making the additional assumption that the phage population contained a small proportion of slowly attaching phages. The transition from reversibly bound to irreversibly bound state was directly observed by attaching briefly, at high bacterial densities, at 0°C, followed by dilution into attachment medium at 37°C. A large majority of the phage rapidly became irreversibly attached under these conditions. Stirring did not affect the result. From these results, it was concluded that reversible attachment is an intermediate state in the infectious process.

On the process of host-controlled modification of bacteriophage.

Abstract The modification of restricted phage T1 (rT1) to the unrestricted form (uT1), and the analogous modification of phage P2 (rP2 to uP2) can be brought about by the presence in the same nonlysogenic host cell, of the vegetative phase of the phage P1. In P1-lysogenic host cells, joint infection with P1 and rT1 increases only slightly the yield of uT1 as compared to infection with rT1 alone. It is postulated that the rare event that causes P1-lysogenic hosts to become yielders when infected with rT1 or rP2, is prophage activation and that a subsequent interaction between vegetative P1 and the restricted phage causes some of the latter to become modified.

A new effect of the rex gene of phage λ: Premature lysis after infection by phage T1

Abstract When phage T1 infects bacteria lysogenic for lambda, lysis is premature and burst sizes are small. This has been shown to be an effect of the lambda rex gene.

Genes of coliphage T1 whose products promote general recombination.

The RecE bacterial recombination pathway, expressed in strains carrying a sbcA mutation, can substitute for the function of gene 4 of phage T1. RecE will also substitute for the function of a newly discovered phage gene, 3.5. Like mutants in gene 4, gene 3.5 mutants have a DA (DNA synthesis arrest) phenotype under nonpermissive conditions. In addition to their effects on DNA synthesis, mutations in genes 3.5 and 4 profoundly depress recombination in T1. Inhibition of DNA synthesis by nalidixic acid does not effect the frequency of recombinants among the small population of progeny phage that are produced. Isolation of T1 mutants dependent on the RecE function has yielded additional mutants specifically in genes 3.5 and 4. Together, these results are interpreted to mean that these two phage genes encode components of a general recombination system, referred to as T1 Grn. During replication of T1 in conventional hosts the essential function of this system is to provide for the formation, via recombination, of concatameric DNA molecules, which are the substrates for the packaging of DNA into T1 heads.

The synthesis of coliphage T1 DNA: requirement for host dna genes involved in elongation.

Abstract Available Escherichia coli mutants with temperature-sensitive mutations in genes essential for DNA replication have been used to investigate the host protein involvement in the DNA synthesis of bacteriophage T1. This study has shown that T1 is independent of the E. coli initiation gene products dna A, dna C, dna I, dna P, and dna T. The gene products of the pol C, dna G, and dna Z loci, know to be involved in the elongation events of replication, are required for a productive T1 infection. T1 was found to be independent of the functioning of the dna B gene product in the four dna B mutants tested.

The fate of genes from restricted T1 in lysogenic Shigella dysenteriae, Sh(P1)☆

Abstract When rT1 infects Sh(P1), the release of modified uT1 by the rare yielder cells is delayed in a portion of these cells for at least 1 hour. In the nonyielding cells the Hr gene persists in an inactive, nonmultiplying state for some time, but after several hours of bacterial multiplication its presence cannot be demonstrated. Even in Sh(P1) cells jointly infected by rT1 and uT1, the genes from rT1 neither function nor replicate, unless they are rescued by recombination with uT1. Two strains of rT1, which have become restricted independently, fail to cooperate in the infection of Sh(P1).

The synthesis of coliphage T1 DNA: Studies on the roles of T1 genes 1, 2, and 4

Abstract We have used T1 with is mutations in genes 1, 2, and 4 in temperature-shift experiments in order to explore further the roles of the products of these genes in the replicative cycle for T1 DNA. The products of genes 1 and 2 are required throughout infection; cessation of synthesis after a shift to a nonpermissive temperature was quite rapid with all of the mutants. Gene 4 product, in contrast, becomes progressively dispensible as the infection proceeds; this process starts promptly after infection under permissive conditions. DNA concatamers appearing early under permissive conditions are not capable of being packaged into viable phage without further DNA synthesis. However, concomitant DNA synthesis is not a direct requirement for DNA maturation. Certain possible explanations for the DNA synthesis arrest (DA) phenotype have been ruled out: DNA synthesized under nonpermissive conditions for gene 4 is not subject to degradation; concomitant activity of the gene 4 product is not required for the continued synthesis of either monomeric or concatamric DNA molecules.

Cooperative infection of P1-lysogenic bacteria by restricted phage T1☆

Abstract Restricted phage T1 exhibits cooperation in the infection of P1-lysogenic bacteria at high multiplicities. Above a multiplicity of 5, essentially all the progeny phage become modified. Genetic crosses done under the conditions of cooperative infection show unusual features: (1) the recombinant frequency is strongly dependent on the multiplicity; (2) above a multiplicity of 10, the recombinant frequency is three to four times that found in control crosses; (3) complementary classes of recombinants are found in unequal numbers; (4) single bursts often contain a single genotype of progeny; this can be a recombinant genotype. Certain strains of T1 show erratic cooperation and are poorly modified. The existence of a nonreplicating but genetically recombining state of the genome is proposed.