The lysis-lysogeny decision of bacteriophage 933W: a 933W repressor-mediated long-distance loop has no role in regulating 933W P(RM) activity

Abstract

Our data show that unlike bacteriophage λ, repressor bound at O(L) of bacteriophage 933W has no role in regulation of 933W repressor occupancy of 933W O(R)3 or the transcriptional activity of 933W P(RM). This finding suggests that a cooperative long-range loop between repressor tetramers bound at O(R) and O(L) does not form in bacteriophage 933W. Nonetheless, 933W forms lysogens, and 933W prophage display a threshold response to UV induction similar to related lambdoid phages. Hence, the long-range loop thought to be important for constructing a threshold response in lambdoid bacteriophages is dispensable. The lack of a loop requires bacteriophage 933W to use a novel strategy in regulating its lysis-lysogeny decisions. As part of this strategy, the difference between the repressor concentrations needed to bind O(R)2 and activate 933W P(RM) transcription or bind O(R)3 and repress transcription from P(RM) is <2-fold. Consequently, P(RM) is never fully activated, reaching only ∼25% of the maximum possible level of repressor-dependent activation before repressor-mediated repression occurs. The 933W repressor also apparently does not bind cooperatively to the individual sites in O(R) and O(L). This scenario explains how, in the absence of DNA looping, bacteriophage 933W displays a threshold effect in response to DNA damage and suggests how 933W lysogens behave as "hair triggers" with spontaneous induction occurring to a greater extent in this phage than in other lambdoid phages.

Fig. 1.

Structure of the 933W immunity region. The positions of O_R1, O_R2, and O_R3, of O_L1 and O_L2, and of the repressor (cI) and cro genes (Cro) are indicated by boxes; the transcription start sites of P_L, P_RM, and P_R are indicated by bent arrows.

Fig. 2.

Demonstration of the λimm^933W threshold response to UV induction, similar to that of λimm⁴³⁴. Lysogens containing either 933W (λimm^933W) or 434 (λimm⁴³⁴) immunity regions were subjected to either no UV light or increasing doses of UV light. Lysogens were then diluted and incubated with aeration at 37°C for 2 h to allow phage induction. Phage titers were determined by plating on MG1655. Cell numbers were determined by plating aliquots of cells on LB plates prior to the 2-h incubation. Phage numbers are expressed as the fraction of the maximal amount of phage-forming units (PFU) per cell-forming units (CFU). Error bars represent standard deviations calculated from the averages of 5 replicate experiments.

Fig. 3.

Deletion of 933W O_L does not affect repressor occupancy at O_R3. (A) Representative DMS footprinting gel. DMS methylation of 933W genomic DNA template was used to detect repressor protection of O_R sites. DMS was either not added (lanes 1 and 2) to lysogenic cells (lanes 3 to 6) or isolated genomic DNA from lysogens was added (lanes 7 to 10) and allowed to react at 37°C for 5 min. Isolated genomic DNA in all lanes was subjected to primer extension using a radiolabeled DNA primer complementary to regions just outside of the 933W O_R and Taq polymerase (see Materials and Methods). Locations of guanine bases in O_R3, O_R2, and O_R1 sensitive to methylation by DMS are indicated. For the in vivo DMS treatment (lanes 3 to 6), DMS was added to overnight cultures of λimm^933W (wt) or λimm^933WΔOL (ΔO_L) grown at 37°C. The level of 933W repressor was determined by either endogenous lysogen levels (lanes 3 and 5) or endogenous levels plus additional repressor expressed from p933WR (lanes 4 and 6). No DMS was used in lanes 1 and 2. For the in vitro DMS treatment (lanes 7 to 10), DMS was added to purified genomic DNA isolated from λimm^933W (lanes 7 and 8) or λimm^933WΔOL (lanes 9 and 10) lysogens. Lanes 7 and 9 had no 933W repressor present upon DMS addition. In lanes 8 and 10, saturating amounts of purified 933W repressor were added to genomic DNA prior to the DMS methylation reaction. (B) Quantification of in vitro DMS methylation intensities of guanines in O_R3 (see panel A, lanes 7 to 10). (C) Quantification of in vivo DMS methylation intensities of guanines in O_R3 (see panel A, lanes 3 to 6). In panels B and C, intensities were normalized to the reactivity at position 1′. Error bars in panels B and C represent standard deviations derived from 4 replicate experiments.

Fig. 4.

Deletion of 933W O_L does not affect in vivo levels of P_RM transcripts. Quantitative real-time PCR was used to determine P_RM cDNA levels produced from reverse transcription of total RNA extracted from λimm^933W and λimm^933WΔOL lysogens producing wild-type levels of 933W repressor (containing pET17b and pGP1-2) or excess 933W repressor (+++-containing p933WR and pGP1-2). Amounts of P_RM transcripts were normalized to the amount of UmuC transcripts, determined in parallel reverse transcription reactions (see Materials and Methods). Differences between the amounts of transcript without or with excess repressor were significant (P < 0.0001). The amount of transcripts from O_L⁺ and ΔO_L lysogens were not significantly different (P = 0.34), regardless of the absence or presence of the repressor-producing plasmid. Data are derived from eight replicate experiments.

Fig. 5.

Sequences and intrinsic affinities of 933W for separate naturally occurring 933W operators. (A) Binding of 933W repressor to individual naturally occurring binding sites. Various concentrations of 933W repressor were mixed with radiolabeled binding site-containing DNA, and protein-DNA complex formation was detected as described in Materials and Methods. Points represent averages of ≥8 replicate experiments. Lines represent best nonlinear least-squares fits to the data based on a hyperbolic equation. (B) The sequences and dissociation constants (K_D [lswb]± standard deviation]) of 933W repressor binding to O_R1, O_R2, O_R3, O_L1, and O_L2. Dissociation constants were derived from data shown in panel A and determined as described in Materials and Methods. Standard deviations were calculated from the averages of ≥8 replicate experiments.

Fig. 6.

DNase I footprinting of complexes between the 933W repressor and 933W O_R. Shown is a representative phosphorimage of a representative gel. DNA templates containing 933W O_R radioactively labeled were partially digested with DNase I in the presence of increasing amounts of the 933W repressor. Lane 1 shows the DNase I cleavage pattern of the DNA in the absence of added repressor. In lanes 2 to 13, repressor concentrations were increased in 1.5-fold steps starting at 0.17 nM protein. The arrows identify positions of protected bands used in measuring site occupancies of O_R1, O_R2, and O_R3.

Fig. 7.

O_R2 and O_R3 are required for regulation of P_RM transcription by repressor. (A) Representative transcription gels. 933W DNA templates containing wild-type (wt) O_R or O_R regions bearing mutations in either O_R2 (O_R2⁻) or O_R3 (O_R3⁻) were transcribed in vitro in the absence of repressor (lane 1) and at repressor concentrations increased in 2-fold steps (lanes 2 to 7), starting with 4.5 nM protein. Positions of transcripts initiated from P_R and P_RM are indicated. The 933W repressor was incubated with DNA template at 25°C for 10 min, followed by addition of E. coli RNA polymerase. The reaction mixture was transferred to 37°C for 10 min before the transcription reaction was initiated by the addition of nucleotides and heparin. (B) Graphical representation of the amount of P_RM transcript synthesized as a function of 933W repressor concentration from the template bearing wild-type O_R or templates bearing a mutation in O_R3 or O_R2. The transcript amounts are quantified as a percentage of maximal P_R transcription as a function of 933W repressor concentration. Error bars represent standard deviations calculated from the averages of at least three replicate experiments. (C) Numerical simulation of transcription data using O_R occupancy data calculated from dissociation constants given in Fig. 5 (see also Materials and Methods). The lines represent simulated data. Points are the measured P_RM activity, as described for panel B.

Fig. 8.

Alignments of the CTDs of the 933W repressor with three other lambdoid phage repressors known to cooperatively bind DNA. Multiple sequence alignments were performed using ClustalW (24). Shaded boxes were used to demonstrate the output based on residue matches (black) and functional similarities (gray). This program was written by Kay Hofmann and Michael D. Baron. Numbers indicate the residue within the protein that defines the beginning of the CTD. Black dots indicate residues in the λ repressor that contribute to cooperative interactions between repressor dimers.