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. 2007 Oct 1;21(19):2461-72.
doi: 10.1101/gad.1584907.

Cro's role in the CI Cro bistable switch is critical for {lambda}'s transition from lysogeny to lytic development

Rachel A Schubert  1 , Ian B Dodd, J Barry Egan, Keith E Shearwin
Affiliations

Cro's role in the CI Cro bistable switch is critical for {lambda}'s transition from lysogeny to lytic development

Rachel A Schubert et al. Genes Dev. .

Abstract

CI represses cro; Cro represses cI. This double negative feedback loop is the core of the classical CI-Cro epigenetic switch of bacteriophage lambda. Despite the classical status of this switch, the role in lambda development of Cro repression of the P(RM) promoter for CI has remained unclear. To address this, we created binding site mutations that strongly impaired Cro repression of P(RM) with only minimal effects on CI regulation of P(RM). These mutations had little impact on lambda development after infection but strongly inhibited the transition from lysogeny to the lytic pathway. We demonstrate that following inactivation of CI by ultraviolet treatment of lysogens, repression of P(RM) by Cro is needed to prevent synthesis of new CI that would otherwise significantly impede lytic development. Thus a bistable CI-Cro circuit reinforces the commitment to a developmental transition.

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Figures

Figure 1.
Figure 1.
Cro repression of PRM. (A) Regulation of lytic and lysogenic promoters by Cro. (B) Repression of PRM by single-copy cro in cis. The chromosomally integrated PRM.lacZ transcriptional reporter carries the OR.cro region inserted so that lacZ is transcribed from PRM but is translated from the wild-type lacZ ribosome-binding site (RBS). Wild-type Cro, or a mutant protein carrying a deletion within its HTH DNA-binding motif (croΔ21), is expressed in cis from PR. (C) Repression of CI expression by single-copy cro in cis. The PRM.cI translational reporter is as in B, except that codon 20 of cI is fused to codon 10 of lacZ, such that lacZ is transcribed from PRM and translated from the cI RBS. Errors are 95% confidence intervals.
Figure 2.
Figure 2.
OR mutations relieving Cro repression of PRM. (A) The PRM-OR-PR region and the mutations used in this study. The c21, r1, r314A, c12, and r204 mutations change the ΔG of Cro binding to OR1 by +1.7, +0.4, +1.7, +1.3, and +1.4 kcal/mol, respectively (Takeda et al. 1989), and the ΔG of CI binding to OR1 by −0.2, +2.9, +1.0, −0.8, and +0.3 kcal/mol, respectively (Sarai and Takeda 1989). (B) Activity of the PRM. lacZ reporter (croΔ21 as in Fig. 1B) carrying KO mutations in OR3 and OR2, in response to Cro supplied from an IPTG-inducible expression plasmid (or an empty vector, dashed curve). LacZ activities have been normalized to aid comparison of repression with the OR2-KO mutation, which decreases PRM intrinsic activity by approximately twofold. (C) Effect of OR mutations on repression of PRM by Cro expressed from a single-copy cro gene in cis. The PRM.lacZ reporters are as in Figure 1B, except that Δcro here indicates truncation of cro at the +43 position of PR. Fold repression is calculated without subtraction of background. (B,C) Errors are 95% confidence intervals.
Figure 3.
Figure 3.
Effects of the mutations on other aspects of OR regulation. Response of PRM.lacZ reporters (A) and PR.lacZ reporters (B,C) to CI (A,C), or Cro (B) supplied by IPTG-inducible plasmids. The λ fragment extends from the +62 of PRM to the +43 of PR. The inset in C shows reporter expression with CI supplied from a λatt80 prophage. (D) Translational fusion reporters showing the effect of the OR mutations on cI translation from PRE mRNA. λ PRE is substituted by the constitutive 186 pB promoter. (A–D) Error bars and ranges are 95% confidence limits.
Figure 4.
Figure 4.
Behavior of the OR mutant phages. (A) Phage production after infection. C600 cells were infected at a phage:bacterium ratio <0.01 with λWT or OR mutant phages and assayed for IC (lysing cells + FP) at various times. Values are normalized to preburst averages. (B) CI Western blotting of extracts from C600 nonlysogenic cells (non) or single C600 lysogens of λWT or OR mutant phages. (Note that there is a cross-reacting host band running just below CI). Values are CI levels relative to wild type (with 95% confidence limits) obtained by quantitation of the blots (n = 8 or 9). (C) Phage production after UV irradiation (10 J/m2) of C600 lysogens. (D) Temperature induction of λ.cIts and λ.cIts.OR3-x3 prophages. C600 lysogens were transferred from 30°C to 39°C and samples were assayed for FPs. (E) UV induction (as in C) of λ.OR3-r1 and λ.OR3-KO prophages and their PRE− derivatives. For the OR3-r1 PRE+ and OR3-r1 PRE− phages, respectively, 56% and 50% of lysogens were induced, producing 26 and 32 phage per irradiated lysogen. For the OR3-KO PRE+ and OR3-KO PRE− phages, respectively, 18% and 30% of lysogens were induced, producing on average 0.6 and 0.9 phage per irradiated lysogen.
Figure 5.
Figure 5.
Effect of the OR3-x3 mutation on UV induction of the CI–Cro switch. (A) Structure of the chromosomally integrated cI.PRM.PR.cro.lacZ reporter constructs. LacZ activity from PR (B), and Western blotting of CI (C) following 10 J/m2 UV treatment of the cro+ reporters. (D) Relative LacZ activities in the OR3+ and OR3-x3 reporters after different UV doses. The strains carrying the croHTH− mutation showed that the OR3-x3 mutation made no difference to PR LacZ activity in the absence of Cro (open symbols, 10 J/m2 UV treatment).
Figure 6.
Figure 6.
Repression of PRM by single-copy cro in cis in the presence of CI. The cro+ curve was obtained using the PRM.lacZ transcriptional reporter in Figure 1B. The reporter for the Δcro curve is the same except that the cro gene is truncated at the +43 position of PR. CI was supplied with an IPTG-inducible expression plasmid system, with 150 μM IPTG producing a PRM response equivalent to that at lysogenic CI levels (Dodd et al. 2001). Errors are 95% confidence intervals. The dashed line shows the response of a PR.lacZ (Δcro) reporter to CI (LacZ values divided by 5) (Dodd et al. 2004).
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References

    1. Adhya S., Gottesman M., Gottesman M. Promoter occlusion: Transcription through a promoter may inhibit its activity. Cell. 1982;29:939–944. - PubMed
    1. Atsumi S., Little J.W., Little J.W. Role of the lytic repressor in prophage induction of phage λ as analyzed by a module-replacement approach. Proc. Natl. Acad. Sci. 2006;103:4558–4563. - PMC - PubMed
    1. Baek K., Svenningsen S., Eisen H., Sneppen K., Brown S., Svenningsen S., Eisen H., Sneppen K., Brown S., Eisen H., Sneppen K., Brown S., Sneppen K., Brown S., Brown S. Single-cell analysis of λ immunity regulation. J. Mol. Biol. 2003;334:363–372. - PubMed
    1. Bailone A., Levine A., Devoret R., Levine A., Devoret R., Devoret R. Inactivation of prophage λ repressor in vivo. J. Mol. Biol. 1979;131:553–572. - PubMed
    1. Court D.L., Oppenheim A.B., Adhya S.L., Oppenheim A.B., Adhya S.L., Adhya S.L. A new look at bacteriophage λ genetic networks. J. Bacteriol. 2007;189:298–304. - PMC - PubMed

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