Role of the lytic repressor in prophage induction of phage lambda as analyzed by a module-replacement approach

Abstract

Using a module exchange approach, we have tested a long-standing model for the role of Cro repressor in lambda prophage induction. This epigenetic switch from lysogeny to the lytic state occurs on activation of the host SOS system, which leads to specific cleavage of CI repressor. It has been proposed that Cro repressor, which operates during lytic growth and which we shall term the lytic repressor, is crucial to prophage induction. In this view, Cro binds to the O(R)3 operator, thereby repressing the cI gene and making the switch irreversible. Here we tested this model by replacing lambda Cro with a dimeric form of Lac repressor and adding several lac operators. This approach allowed us to regulate the function of the lytic repressor at will and to prevent it from repressing cI, because lac repressor could not repress P(RM) in our constructs. Repression of cI by the lytic repressor was not required for prophage induction to occur. However, our evidence suggests that this binding can make induction more efficient, particularly at intermediate levels of DNA damage that otherwise cause induction of only a fraction of the population. These results indicate that this strategy of module exchange will have broad applications for analysis of gene regulatory circuits.

Fig. 1.

Design of λlacI. (A) Diagram of the phage λ regulatory circuit. (+) and (−) indicate activation and repression of promoter, respectively (LacR can repress P_RM when a lacO variant is in the position of O_R3). CIII and CII (not depicted) are expressed from P_L and P_R, respectively. Expression of P_L is controlled by CI binding to two operators, O_L1 and O_L2, which lie to the right of P_L in positions analogous to O_R1 and O_R2, respectively. (B) Patterns of occupancy by CI and Cro at moderate protein levels. CI stabilizes the lysogenic state by repressing P_R and stimulating P_RM. At higher levels, CI binds O_R3 (cf. ref. 10), repressing P_RM. Cro bound to O_R3 represses P_RM. At higher levels, Cro binds to O_R2 and/or O_R1, repressing P_R. (C) Design of λlacI^dim. Cro was replaced with lacI^dim. Alleles of lacO were installed at P_L and P_R; at the position of O_R3, some variants had a lacO allele, and some had O_R3. The same set of lacO alleles was used at all three sites. Several alleles of the SD sequence for lacI were used. [Modified with permission from ref. (Copyright 1992, Cold Spring Harbor Lab. Press).]

Fig. 2.

Effect of IPTG on lysogenization frequency after single infection. (A) Lysogenization frequency without (open bar) or with IPTG (black bar, 10⁻⁵ M; gray bar, 10⁻⁴ M; stippled bar, 10⁻³ M). Cells were infected at an moi of ≈0.01; values are the percentage of infected cells giving rise to Kan^R colonies. (B) IPTG concentration-shift experiment (black bar, 10⁻⁷ M IPTG; hatched bar, 10⁻⁵ M IPTG). Frequencies observed at 10⁻⁷ and 10⁻⁵ M IPTG were approximately the same as seen without IPTG and with 10⁻³ M IPTG, respectively (see A).

Fig. 3.

UV dose responses for prophage induction. Exponentially growing cultures of lysogens were centrifuged, suspended in TMG, and irradiated (see Methods) at the indicated doses; aliquots were diluted 10-fold in LBGM, shaken for 2 h at 37°C, treated with CHCl₃, and titered. Values given are the average for three independent experiments. (A) Dose responses for wild type (λJL351) and the indicated variants. (B) Effect of IPTG on burst size. An AWCA lysogen was induced without IPTG (crosses) or with 10⁻⁵ M (open diamonds) or 10⁻⁴ M (open squares) IPTG. (C) Induction of AWCA lysogen in reciprocal IPTG-concentration-shift experiment. IPTG concentration shifted from 10⁻⁴ M to 10⁻⁵ M (open circles) and from 10⁻⁵ M to 10⁻⁴ M (open triangles) at indicated times after UV irradiation.

Fig. 4.

Response of P_RM from AWCF to presence of dimeric and wild-type LacR. (A) Design of the assay. The P_RM promoter from AWCF was fused to lacZ. The P_RM::lacZ protein fusion was carried on a prophage. Strains contained two plasmids. The first was either pBR322 or carried a fusion of the lacI^q promoter (an up-promoter mutation of the natural lacI promoter) driving lacI^dim or lacI⁺. The second plasmid was pGB2 or a lacI^q::cI fusion. (B) Cells were grown in the absence (open bar) or presence (black bar, 10⁻⁴ M; gray bar, 10⁻³ M) of IPTG, and β-galactosidase levels were measured as described in Materials and Methods. None, no plasmids were present. Values given are the average of three separate experiments.