Figure 1. Evolution of a complex genetic switch from a simple integration-dependent genetic switch. (A) Circuitry diagram of an integration-dependent genetic switch composed of three genes: int, rep and cro. Int (blue circles) and Rep (purple circles) are C-terminally tagged (red boxes on 3′ of the genes, red asterisk on proteins) for degradation by host proteases (red circles). The P_Rep promoter drives leftward transcription of two lysogenic genes rep and int, while P_R drives rightward transcription of lytic genes to the right of the switch, the first of which is cro. The attP sequence, used for site-specific homologous recombination into the bacterial chromosome, is located within the open reading frame of rep. Integration stabilizes Rep by removing the C-terminal degradation tag. Rep has a negative effect on P_R expression by binding to the operator O_R. Cro (green circles) has a negative effect on lysogeny by an unknown mechanism. Int alone is capable of both integrative and excisive recombination and it is the expression of Int that controls directionality of recombination. (B) Addition of a new promoter (P_I) between rep and int leads to higher expression of int. This leads to prophage instability. Stability is restored by addition of a recombination directionality factor (RDF) followed by subsequent acquisition of an additional DNA binding domain in Int (dark blue box at the 5′ of the int gene, dark blue circle on Int) and arm-type binding sites in attP (xattPx). Control of directionality of recombination is now dependent on expression of Int and the RDF (pink circle). (C) A duplication event leads to a second copy of the attP-int cassette in the phage genome, leading to further evolution of the newly added cassette and loss of the int and attP functions in the original (grayed). With the addition of an RDF, direction of recombination is less responsive to Int concentrations and the C-terminal degradation tag on Int is lost (grayed box on 3′ of gene). As dependency on integration for immunity is lost, new means to control rep expression arise. Rep gains the ability to bind to RNA Polymerase and activate its own expression at P_Rep. Following this, the C-terminal degradation tag on Rep is lost (grayed box on 3′ of gene). This may lead to very high levels of Rep, which is downregulated by mutations that make repressor dependent on autoactivation. This leads to low lysogenization frequencies that are restored to normal levels by addition of a repressor establishment promoter (P_RE) that is activated by a host transcriptional activator (CII, turquoise circle), which is later acquired by the phage; CII can then acquire the ability to control int expression through P_I. Additions of negative autoregulation of Rep and cooperativity of Rep binding further fine-tune the switch. The final product is a genetic switch with circuitry resembling that of λ and no longer involving integration to determine the outcome of infection.