Erich W. Six

Bacteriophage P4: a satellite virus depending on a helper such as prophage P2

Abstract Bacteriophage P4 was isolated from cultures of Escherichia coli strain K-235, and two spontaneous mutants, P4 imp and P4 vir1, were obtained from P4 wild type. P4 was found to be a satellite virus, depending on a helper genome for the completion of its lytic life cycle. Genomes of phage P2 and of P2-related phages can serve as helpers. They are able to assist P4 either if present in the host as a prophage or if introduced by coinfection. P4 does not depend on a helper for lysogenizing its host. A specific P4 prophage site was recognized on the host chromosome. P4 depends on its helper for the expression of genes with late functions. This is indicated by the finding that the P4 virion reflects in certain of its properties, for example, host range and neutralizability, the genotype of its most recent helper. P4 production in cells lysogenic for a helper and infected with P4 proceeds without induction of the helper prophage and without lifting the (helper-specific) immunity of such cells. It is proposed that P4 produces a transactivating factor that triggers the expression of the helper genes that have to fulfill the late functions needed by P4. Some other findings concerning the propagation of P4 and properties of P4 lysogens are also reported.

The helper dependence of satellite bacteriophage P4: Which gene functions of bacteriophage P2 are needed by P4?☆

Abstract For its lytic growth cycle, bacteriophage P4 depends on its helper, the genome of bacteriophage P2, to provide all 18 known late gene functions that are essential for P2 itself. P4 does not depend on the two known P2 genes, A and B, with early functions essential for P2. P4 also does not require the function of any of the nonessential P2 genes tested. In most of the experiments P4 had to transactivate its helper, usually a P2 prophage, to obtain expression of the late gene functions of the helper. Genetic evidence indicates that replication of helper (i.e., P2) DNA is not required for this transactivation to occur. However, transactivation by P4 may extend to P2 genes A and B. This is suggested by the observation that defects in P2 genes A or B cause a delay in the lysis of P4 infected cells lysogenic for P2. It is known that, in the absence of P4, the expression of late P2 gene functions depends on the expression of genes A and B, needed for P2 DNA replication. Thus, it is concluded that the mechanism P4 employs to transactivate P2 genes is different from the mechanism by which P2 itself activates its genes with late functions. This conclusion is further supported by the finding that P4, but not P2, can transactivate genes of a related, heteroimmune prophage.

Replication of bacteriophage P4 DNA in a nonlysogenic host.

Abstract Bacteriophage P4 can replicate its DNA in the absence of a helper genome upon which it depends for the completion of its lytic cycle of reproduction. A major fraction of such intracellular DNA consists of double-stranded, covalently closed circular molecules. P4 DNA replication does not depend on the REP function of the host.

Some morphological properties of P4 bacteriophage and P4 DNA

Abstract P4 phage morphology and P4 DNA lengths have been studied by electron microscopy. Although P4 phage appears to contain the relatively complex structures of P2 phage (head, tail with contractile sheath and tail fibers), it is unusual in that its DNA content and head diameter are very much less than for P2 phage. The tails of P4 and P2 appear to be indistinguishable. These findings are consistent with the fact that P4 requires a helper genome to undergo a complete lytic growth cycle.

Mutual derepression in the P2–P4 bacteriophage system☆

Abstract P4 lysogens are derepressed by infection with P2 as is evident from the induction of P4 phage production by P2 infection of P4 lysogens. Mutations interfering with P2 DNA synthesis greatly increase the P4 yield, but DNA replication by the infecting phage does not preclude P4 induction. Of four P2 mutations known to block spontaneous phage production by P2 lysogens, three ( cox-2, cox-3, cox-4 ) also prevent derepression of P4 lysogens by P2. These studies also revealed that, under certain conditions, P4 can complement P2 mutants deficient in gene B function which is required for P2 DNA replication. P2 lysogens are derepressed by infection with P4. This derepression leads to a P2 gene A-dependent P2 prophage replication and, in P2 lysogens mixedly infected with P2 and P4, also to the replication of the DNA of the coinfecting P2. If the derepressing P4 is deficient in gene a function, then derepression of the P2 lysogen will lead to the production of 10 or more P2 phages per cell, provided that P2 prophage excision is enhanced by the P2 nip mutation or is bypassed by infecting the P2 lysogen with P2 as well as with P4. Gene α proficient P4 interferes with the P2 phage production by P2 lysogens; possible causes for this interference are considered, including P4 DNA replication which is known to require gene a function.

Lysogenization by satellite phage P4

Abstract Satellite phage P4 attaches to the E. coli K-12 chromosome at a preferred site near 96 min. The attachment site on the P4 genome is 31.4–36.5% from the left cohesive end, which has been redefined as the end with the same base sequence as the P2 helper phage left end. The P4 int gene is in the region from 27.5 to 31.4%. Immunity-sensitive clear plaque mutants define two P4 genes. One is likely to code for repressor and maps very near the P4 gene which is needed to derepress a helper prophage. The other is probably not involved in lysogenization and maps near the left end of the P4 genome, among nonessential genes.

Inheritance of Prophage P2 in Superinfection Experiments.

Abstract Genetically marked P2 phage was used to superinfect cells of Escherichia coli (or Shigella dysenteriae ) which were lysogenic for P2 and hence immune to the superinfecting phage. The genetic incorporation of the superinfecting P2 was studied by examining the progeny of the superinfected cells. The results obtained are in agreement with the previously established rule that genetic incorporation occurs most frequently at the preferred location (I) either by prophage addition or by prophage substitution. Besides complete substitution, partial (= single marker) substitution was also observed, but at a lower frequency than complete substitution. The frequency of genetic incorporation was found to increase linearly with the multiplicity of superinfection for small multiplicities. The probability of genetic incorporation of a superinfecting phage is decreased by the presence of a prophage in location I; this hindrance does not depend on the immune specificity of such prophage. It is assumed that in immune cells multiplication of prophage precursors (= preprophages) is prevented. The probability of genetic incorporation of a preprophage in the absence of steric hindrance (5%) compares well with the probabilities found in other systems for the genetic incorporation of bacterial markers in transduction experiments. Weak virulent mutants of P2 and P2 Hy dis can become prophages, but only in cells already lysogenic for a temperate phage of the same immunity class.

Multiplication of bacteriophage P4 in the absence of replication of the DNA of its helper

Abstract Multiplication of the helper-dependent phage P4 occurs without detectable helper (= P2) DNA synthesis in P4 infected mitomycin C-pretreated cells lysogenic for P2 and also in mitomycin C-pretreated nonlysogenic REP- cells mixedly infected with P4 and P2. These findings are consistent with the hypothesis that P4 selectively activates expression of the helper genes with late functions.

A transactivation mutant of satellite phage P4

Abstract A mutant of satellite phage P4, δ6, was isolated as a thermosensitive mutant on the basis of its plaque-forming ability on Escherichia coli C(P2+). Complementation tests show that this mutant defines a new P4 gene, called δ. Unlike P4 wild type, P4 δ6 depends on the gene A function of its P2 helper at all temperatures, whether the helper is supplied as a prophage or as a coinfecting phage. The P2 gene A product is required both for P2 DNA replication and for P2 late gene expression (Lindahl, G. (1970) Virology 42, 522–533). P2 DNA replication is critical for growth of P4 δ6, since host mutations which block P2 DNA replication prevent production of P4 δ6 progeny, whereas they allow P4+ to multiply. The δ6 mutation makes P4 unable to activate P2 late mRNA synthesis from nonreplicating P2 phage. Thus, P4 δ6 is defective in transactivation.

The gene fimU affects expression of Salmonella typhimurium type 1 fimbriae and is related to the Escherichia coli tRNA gene argU

The gene fimU, located on a recombinant plasmid carrying the Salmonella typhimurium type 1 fimbrial gene cluster is closely related to the Escherichia coli tRNA gene argU. The fimU gene complements an E. coli argU mutant that is a P2 lysogen, thereby allowing the phage P4 to grow in this strain but preventing the growth of phage lambda. In addition, fimU was shown to be involved in fimbrial expression since transformants of the E. coli argU mutant could produce fimbriae only in the presence of fimU but not in its absence, whereas in an E. coli argU+ strain fimbriation did not require the fimU gene.