Sequence Tolerance of the Phage λ P _RM Promoter: Implications for Evolution of Gene Regulatory Circuitry

Abstract

Much of the gene regulatory circuitry of phage centers on a complex region called the O R region. This ϳ100-bp region is densely packed with regulatory sites, including two promoters and three repressor-binding sites. The dense packing of this region is likely to impose severe constraints on its ability to change during evolution, raising the question of how the specific arrangement of sites and their exact sequences could evolve to their present form. Here we ask whether the sequence of a cis-acting site can be widely varied while retaining its function; if it can, evolution could proceed by a larger number of paths. To help address this question, we developed a cloning vector that allowed us to clone fragments spanning the O R region. By using this vector, we carried out intensive mutagenesis of the P RM promoter, which drives expression of CI repressor and is activated by CI itself. We made a pool of fragments in which 8 of the 12 positions in the ؊35 and ؊10 regions were randomized and cloned this pool into the vector, making a pool of P RM variant phage. About 10% of the P RM variants were able to lysogenize, suggesting that the regulatory circuitry is compatible with a wide range of P RM sequences. Analysis of several of these phages indicated a range of behaviors in prophage induction. Several isolates had induction properties similar to those of the wild type, and their promoters resembled the wild type in their responses to CI. We term this property of different sequences allowing roughly equivalent function "sequence tolerance " and discuss its role in the evolution of gene regulatory circuitry.Complex gene regulatory circuits can have a large number of interlocking components. This degree of interconnectivity raises two issues. First, how did these circuits evolve? Second, how can we understand the behavior of these existing circuits and predict their behavior in the face of small changes in parameters such as promoter strength? For these and other reasons, we have been analyzing the behavior of the regulatory circuitry of phage in the intact system. This system is probably the best-understood complex circuit (38, 39). Most, if not all, of the regulatory interactions have been identified, and most of these are well characterized at the mechanistic level. Previous analysis of this circuit generally has been carried out in uncoupled systems (such as the use of reporter genes and fusions with the lac promoter), an approach necessary to disentangle the causality of this system. With a circuit diagram in hand, it is now possible to return to the intact circuit and ask how particular changes affect the overall operation of the system.Many of the critical interactions in the circuit center on a complex regulatory region termed the O R region (Fig. 1C). This ϳ100-bp region is densely packed with cis-acting sites, including two promoters and three sites to which both the CI and Cro repressors can bind (38). In addition, the promoters and repressor-binding sites overlap extensively. CI and Cro regulate the expr...

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

“…Phage strain JL351 was used as wild type (27). It has a set point for prophage induction roughly half that of wild-type due to a silent XhoI site early in cI.…”

“…This site is present in the phage analyzed here. JL387 was JL351 v1 v3 and was used as the cloning vector for the O R region as described (18,27). A cII Ϫ derivative of AWCF was isolated by crossing AWCF with pJWL848 (see Supporting Text, which is published as supporting information on the PNAS web site), isolating clear plaques, and verifying the presence of the cII mutation by sequencing.…”

“…Bacterial strain JL6142 ⌬(lacIPOZYA) (18) was used as wild type. JL7223 was an hfl Ϫ derivative of JL6142 made by transduction with P1 clr100 Cm by using JL6039 hflA150 (linked to Tn10) as donor (27), selecting for tetracycline resistance and screening for the Hfl phenotype as described in ref. 27.…”

“…3A). Previous evidence (27,31) indicates that the set point is determined in part by the strength of P RM ; presumably, this affects the level of CI and the ability of P RM to provide more CI as CI levels fall and negative autoregulation is relieved. In AWCF, however, P RM is the same as that of wild type, and its expression is not markedly affected by dimeric LacR.…”

“…One might expect that any variant forming turbid plaques could lysogenize, but conditions differ in a plaque from those in an isolated cell, because lysogens in a plaque are often reinfected by other phages (see ref. 27). Among the six lysogenizing variants, the four W-variants could form single lysogens, as judged by a PCR test (see Materials and Methods).…”