Positive autoregulation of cI is a dispensable feature of the phage lambda gene regulatory circuitry

Abstract

Complex gene regulatory circuits contain many features that are likely to contribute to their operation. It is unclear, however, whether all these features are necessary for proper circuit behavior or whether certain ones are refinements that make the circuit work better but are dispensable for qualitatively normal behavior. We have addressed this question using the phage lambda regulatory circuit, which can persist in two stable states, the lytic state and the lysogenic state. In the lysogenic state, the CI repressor positively regulates its own expression by stimulating transcription from the P(RM) promoter. We tested whether this feature is an essential part of the regulatory circuitry. Several phages with a cI mutation preventing positive autoregulation and an up mutation in the P(RM) promoter showed near-normal behavior. We conclude that positive autoregulation is not necessary for proper operation of the lambda circuitry and speculate that it serves a partially redundant function of stabilizing a bistable circuit, a form of redundancy we term "circuit-level redundancy." We discuss our findings in the context of a two-stage model for evolution and elaboration of regulatory circuits from simpler to more complex forms.

FIG. 1.

Diagram of O_R region, and activities of CI. (A) Diagram of O_R. Locations of P_R and P_RM promoters are shown. Cro and CI bind to O_R1, O_R2, and O_R3, although the relative affinities differ; Cro binds most tightly to O_R3, while CI binds most tightly to O_R1. The proximal portions of the cI and cro genes are shown. The map is to scale. (B) Activities of CI. The N- and C-terminal domains of CI are indicated by the letters N and C, respectively. CI dimerizes with a dissociation constant of ∼10 nM. Dimers bind to three operators at O_R and to three other sites in the O_L region (depicted only at the bottom). Dimers bind cooperatively to adjacent operators, generally to O_R1 and O_R2 as shown. CI bound to O_R2 stimulates its own expression from P_RM. When CI is bound to two adjacent operators at O_R and at O_L (not shown for the unlooped form), the two tetramers can interact to form a loop as depicted. In a second step, two more dimers bind to the empty sites (depicted as O_R3 and O_L3); binding to O_R3 represses P_RM. Details of the contacts among CI dimers in the complex are not known; one likely possibility is depicted. Diagrams are not to scale.

FIG. 2.

Isolation of pools with mutated P_RM. (A) Sequence of the P_RM promoter, inverted from the usual order of the λ map. Locations of O_R3 and the end of O_R2 are shown in bold; the −35 and −10 regions of P_RM are underlined; the N′s represent positions that were randomized. Above the sequence is that of a consensus promoter; positions in which P_RM differs are underlined. (B) Genetic crosses. λ Δ(P_RM) was crossed with a pool of cells containing P_RM variants made as previously described (see Materials and Methods and Results). Maps of the O_R region in plasmids and phages are to scale. The dashed line between the two parents shows the double crossover yielding the desired recombinant, shown at the bottom. Ns and Bg, NsiI and BglII, respectively. (C) Selection and enrichment for cross progeny forming stable lysogens with a set point near the wild type (see text for details) m.o.i., multiplicity of infection.

FIG. 3.

UV induction of selected mutants with cI D38N. UV induction curves were determined for single lysogens as described in Materials and Methods. Results of typical experiments are shown; data were obtained in a single experiment. Here and in Fig. 4 to 6, mutants are referred to by giving the P_RM allele followed by the cI allele; for example, NP2 D38N is shorthand for P_RM-NP2 cI D38N. WT, wild type.

FIG. 4.

Activities of selected promoters and responses to various levels of CI D38N or wild-type (WT) CI. Single lysogens of phages bearing P_RM::lacZ protein fusions were prepared. For each, three host strains were used. The first, JL6994, contained no CI; the second and third contained pJWL709 or pA3B2, which are regulated by the Lac repressor and make low levels of CI D38N or wild-type CI, respectively. A small amount of CI is made in the absence of IPTG. Cells were grown in the presence of the indicated levels of IPTG, and β-galactosidase levels were measured as described in Materials and Methods. Results of a typical experiment are shown. (A) Activity in the presence of an O_L variant located distal to the lacZ reporter with CI D38N. (B) Activity in the presence of O_L with wild-type CI. (C) Activity in the absence of O_L with cI D38N.

FIG. 5.

UV induction and promoter activities of RP mutants. (A) UV induction of single lysogens. All phages carried cI D38R and the indicated promoter allele, except for the wild type (WT). (B) UV induction of single lysogens. Except for the wild type, all phages carried cI D38N and the indicated promoter allele. (C) Promoter activity of D38R suppressors in the presence of CI D38R. Single lysogens of phages bearing P_RM::lacZ protein fusions were prepared and assayed as described in legend to Fig. 4, except that the second strain carried pJWL710, which expresses CI D38R. In the wild-type control, wild-type CI was provided from plasmid pA3B2. All phages carried a variant of O_L located distal to the lacZ reporter. For each curve, the P_RM allele in the reporter fusion is given, followed by the allele of cI carried on the plasmid. (D and E) Promoter activities of D38R suppressors in the presence of the wild type (D) or CI D38N (E). The experiment was as in panel C, except that cells with the CI-bearing plasmid carried pJWL709 or pA3B2, which expresses CI D38N or wild-type CI, respectively. Curves are labeled as for panel C.

FIG. 6.

Promoter activities of P_RM variants with weak responses to CI, and UV induction of prophages bearing these promoters. Experiments were carried out as described in the legends to Fig. 3 and 4, except that CI was wild type (WT) in all cases. (A and B) Promoter activity in the absence (A) or presence (B) of a distal O_L site. The P_RM-MD114 fusion carried a silent mutation in Leu7 of cI and hence cannot be compared directly to the data in panel D, since it is possible that this change affects the apparent promoter strength (for a similar mutation, see reference 32). Data are presented to illustrate the slight response to CI for this promoter. (C and D) UV induction curves for derivatives that are induced at low (C) or higher (D) set points. All data were obtained in a single experiment and are separated for clarity. (E) Evolutionary pathways among P_RM variants. Alleles differing by a single base change are connected by single lines.