Polynucleotide phosphorylase of Escherichia coli is required for the establishment of bacteriophage P4 immunity

Abstract

Bacteriophage P4's superinfection immunity mechanism is unique among those of other known bacteriophages in several respects: (i) the P4 immunity factor is not a protein but a short, stable RNA (CI RNA); (ii) in the prophage the expression of the replication operon is prevented by premature transcription termination rather than by repression of transcription initiation; (iii) transcription termination is controlled via RNA-RNA interactions between the CI RNA and two complementary target sequences on the nascent transcript; and (iv) the CI RNA is produced by processing of the same transcript it controls. It was thought that several host-encoded factors may participate in the molecular events required for P4 immunity expression, i.e., RNA processing, RNA-RNA interactions, and transcription termination. To identify such factors we searched for Escherichia coli mutations that affect P4 lysogenization. One such mutation, bfl-1, severely reduced P4's lysogenization frequency and delayed both the disappearance of the long transcripts that cover the entire replication operon and the appearance of the CI RNA. By physical mapping and genetic analysis we show that bfl-1 is allelic to pnp, which codes for polynucleotide phosphorylase, a 3-to-5 exonucleolytic enzyme. A previously isolated pnp null mutant (pnp-7) exhibited a phenotype similar to that of bfl-1. These results indicate that the polynucleotide phosphorylase of E. coli is involved with the maturation pathway of bacteriophage P4's RNA immunity factor.P4 is a satellite phage of Escherichia coli that depends on the genes of a helper phage, such as P2, for capsid and tail morphogenesis and cell lysis. Autonomous P4 replication requires the expression of the P4 ␣ gene, encoded in the distal part of the P4 left operon (Fig. 1A), and leads either to the lytic cycle (when a helper phage genome is present in the host cell) or to the multicopy-plasmid state (in the absence of the helper). The P4 genome may also integrate in the host cell chromosome and establish superinfection immunity that prevents expression of the left operon (for a review, see references 15 and 26).The P4 superinfection immunity mechanism is unique among the known bacteriophages in several aspects. First, the promoter (P LE ) responsible for the early expression of the left operon is not repressed in the prophage; thus, in order to prevent expression of P4 replication genes, premature transcription termination of the left operon is established at a Rho-dependent terminator about 480 nucleotides (nt) downstream of P LE (Fig. 1B) (5,10,16). Second, the P4 immunity factor is a small RNA, 69 nt long (CI RNA), which is produced by processing of the leader region of the left operon itself; RNase P is required for the correct maturation of the CI RNA 5Ј end (10,14,16). Third, transcription termination depends on interactions between the CI RNA and two complementary regions on the leader transcript of the left operon (10,16,36).To understand in detail the P4 immunity mechanism, several events should be clarifie...

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