Gianpiero Sironi

Bacteriophage P4 immunity controlled by small RNAs via transcription termination

Satellite bacteriophage P4 immunity is encoded within a short DNA region 357 bp long containing the promoter PLE and 275 bp downstream. PLE is active both in the early post‐infection phase, when genes necessary for P4 lytic cycle are transcribed from this promoter, and in the lysogenic condition, when expression of the above genes is prevented by prophage immunity.

Genetic analysis of the immunity region of phage‐plasmid P4

In the prophage P4, expression of the early genes is prevented by premature termination of transcription from the constitutive promoter Ple. In order to identify the region coding for the immunity determinant, we cloned several fragments of P4 DNA and tested their ability to confer immunity to P4 superinfection. A 357 bp long fragment (P4 8418‐8774) is sufficient to confer immunity to an infecting P4 phage and to complement the immunity‐defective P4 cl405 mutant, both in the presence and in the absence of the helper phage P2.

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.

Regulation of the plasmid state of the genetic element P4

SummaryAfter infection of sensitive cells in the absence of a helper phage, the satellite bacteriophage P4 enters a temporary phase of uncommitted replication followed by commitment to either the repressed-integrated condition or the derepressed-high copy number mode of replication. The transient phase and the stable plasmid condition differ from each other in the pattern of protein synthesis, in the rate of P4 DNA replication and in the expression of some gene functions. The regulatory condition characteristic of the P4 plasmid state affects a superinfecting genome, preventing the establishment of the P4 immune condition.

Plasmid mode of propagation of the genetic element P4

The satellite bacteriophage P4, in the presence of a helper phage, can enter either the lytic or the lysogenic cycle. In the absence of the helper, P4 can integrate in the bacterial chromosome. In addition, the partially immunity-insensitive mutant P4 vir1 can be maintained as a plasmid. We have found that in the absence of the helper, P4 wt also can be maintained as a plasmid, and that both P4 wt and P4 vir1 have two options for their intracellular propagation: a repressed-integrated or a derepressed-high copy number plasmid mode of maintenance. In the repressed mode, the P4 wt genome is only found integrated into the bacterial chromosome, while the P4 vir1 is found also as a low copy number plasmid coexisting with the integrated P4 vir1 genome. The clones carrying P4 in the derepressed-high copy number plasmid state are obtained at low frequency (0.3%) upon infection with P4 wt, while the vir1 mutation increases this frequency about 300-fold. Such clones can be distinguished easily because of their typical colony morphology (rosettes), due to the presence of filamentous cells. Filamentation of the bacterial host suggests that the presence of derepressed P4 genomes in high copy number interferes with the normal cell division mechanism. The derepressed clones are rather stable, but may revert spontaneously to the repressed state. Spontaneous transition from the repressed to the derepressed state was not observed; however, it can be induced by P2 or P4 vir1 superinfection of P4 wt and P4 vir1 lysogens or by growing the P4 vir1 lysogens up to the late exponential phase. The ability of P4 to choose either of two stable states and the potential to shift between these two modes of propagation indicate that the synthesis of the immunity repressor is regulated.

X-ray sensitivity of Escherichia coli lysogenic for bacteriophage P2.

SummaryStrains of Escherichia coli C or K lysogenic for the non-inducible phage P2 show a lower survival following X-ray irradiation as compared to nonlysogenic strains. This difference in X-ray sensitivity is not accompanied by a significant difference in X-ray induced mutability. The capacity of X-irradiated P2 lysogens to multiply any of a number of unirradiated infecting phages is severely impaired. These effects of X-ray treatment can be most simply explained as a consequence of the fact that protein and RNA syntheses are strongly inhibited in P2 lysogens after X-irradiation. All the above events specifically occurring in X-rayed P2 lysogens are dependent on the P2 gene old.

Evidence of cell fragility caused by gene kil following λ induction

Abstract Escherichia coli cells carrying λ cI857 prophage lyse 40 min after λ thermoinduction; the lysis depends on the λ genes Q , R , and S . If chloramphenicol (CAP) is added within 20 min after λ cI857 induction, an early, unproductive lysis occurs. This lysis is independent of the genes int, rex, O , P , Q , and all late genes. Instead, early lysis depends upon the kil gene. The early lysis is under the positive control of λ gene N and the negative control of gene cro . One or more events specifically connected with λ induction appear to be necessary for the occurrence of early lysis, since early lysis cannot be observed after λ infection. Induced λ kil + lysogens are more sensitive to osmotic shock than induced λ kil - lysogens. CAP-induced early lysis can be prevented in a hypertonic medium. These results suggest that induction of λ causes an osmotic fragility due to a damage of the cell envelope which requires repair; in the absence of protein synthesis the cell envelope is not repaired and cell lysis ensues.