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Sequence Tolerance of the Phage (cid:2) P RM Promoter: Implications for Evolution of Gene Regulatory Circuitry†

Much of the gene regulatory circuitry of phage (cid:1) centers on a complex region called the O R region. This (cid:3) 100-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 (cid:1) 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 (cid:2) 35 and (cid:2) 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 (cid:1) 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 from 5 to 12 J/m 2 , a difference we attribute to changes in the spectral properties of the lamp relative to those of the meter (International Light model IL1400A) used to measure its output. Second, phage titers were determined with lawns made from exponentially growing cells (JL2497 was grown to 2 (cid:11) 10 8 /ml in LBMM, centrifuged, and resuspended in 1/10 volume of TMG [27]), rather than the starved overnight lawns used in the previous studies. This change gave higher and more reproducible titers.