Derepression of prophage P2 by satellite phage P4: cloning of the P4 epsilon gene and identification of its product

Abstract

Escherichia coli phage P4 lacks all of the genetic information necessary for capsid, tail, and lysis functions. P4 is therefore dependent on a helper phage, such as P2, for lytic propagation. During P4 superinfection of a P2 lysogen, the P2 prophage is derepressed by the action of the P4-encoded gene. We have cloned the gene and identified the 10-kDa E protein. The gene product is the only P4 protein required to derepress prophage P2, which leads to in situ P2 DNA replication. A two-plasmid derepression assay system has been developed to examine the derepression activity of E. The reporter plasmid contains the two face-to-face promoters, Pe and Pc, involved in the lysis-lysogeny transcriptional switch of phage P2 and the immunity repressor C. The Pe promoter is coupled to a cat reporter gene. In the construct, the C repressor is transcribed from the Pc promoter and represses the Pe promoter, which mimics the in situ-repressed P2 prophage. The E protein is supplied in trans from a compatible plasmid in which the gene is under the control of the T7 promoter. We show here that in the two-plasmid assay system, induction of the E protein derepresses the Pe promoter. The ash9 mutation, which is located upstream of the gene, enhances the E-mediated derepression of the Pe promoter. The purified E protein shows no specific DNA binding activity, and the implications of this are discussed.

Characterization of the Developmental Switch Region of Bacteriophage P2 Hy dis

“…Upon a P4 infection of a P2 lysogenic cell, the P2 prophage becomes derepressed by the P4 E antirepressor (Liu et al, 1997). The surface of the P2 C repressor that interacts with the P4 E antirepressor has been localized by a phage epitope display technique (Liu et al, 1998) and mutational analysis (Renberg- Eriksson et al, 2000).…”

“…This region is well conserved in the P2 Hy dis C repressor; only 2 of 10 amino acid residues are different and they are conserved with respect to hydrophobicity. To analyze derepression of the P2 Hy dis phage, the activity of the Pe promoter in pEE915 (C-Pe-Pc-cat) was measured after isopropyl ␤-D-thiogalactoside induction of the E antirepressor from pEE804 in BL21(DE3) cells, as described previously (Liu et al, 1997), and compared to the level obtained with the equivalent P2 reporter construct. To quantitate the levels of CAT expression, cell extracts were prepared from samples taken before and 3.2 ϫ 10 8 3.6 ϫ 10 5 11 ϫ 10 Ϫ4 pEE720 (P2 Cox)…”

“…The late gene functions can be provided by a coinfecting P2 or P2-related phage or by a P2 prophage. In the latter case, the P4 E protein has been shown to turn the transcriptional switch of P2 from lysogenic to lytic mode (Liu et al, 1997), and it has been shown that E binds to the P2 C immunity repressor and thereby acts as an antirepressor (Liu et al, 1998). The developmental switch of P2 contains two mutually exclusive promoters, the early promoter Pe and the repressor promoter Pc, and the two repressors C and Cox (Lundqvist and Haggård-Ljungquist et al, 1987).…”

Interacting interfaces of the P4 antirepressor E and the P2 immunity repressor C

“…Growth was continued and samples were collected after 60, 120 and 180 min. Cell extracts were prepared as described previously (Liu et al, 1997). The total protein concentrations of the samples were determined (Bradford, 1976) and activity was assayed (Gorman et al, 1982).…”

Translation of Two Nested Genes in Bacteriophage P4 Controls Immunity-Specific Transcription Termination

“…These data suggest that the Ash Ϫ phenotype is correlated with overexpression of one or more genes of the P4 left operon. A possible candidate is the P4 ε gene product, which is required for derepression of P2 prophage (18,31). It might be hypothesized that greater production of the ε protein may be required to derepress a P3 prophage.…”