Characterization of an unusual repressor encoded by mycobacteriophage BPs

Temperate phages are capable of both lytic and lysogenic growth. In lytic growth, phages utilize the host machinery to produce new virions and kill their host; during lysogeny, the phage genome integrates into the host chromosome and produces a repressor that prevents expression of lytic genes, thus maintaining the lysogenic state. A few phage genetic switches governing the lytic/lysogenic decision have been well characterized, most of which contain analogous regulatory proteins with similar functions. A new type of genetic switch, which uses phage integration directly in synthesizing the phage repressor, is described. This dependence on integration arises because of the unusual feature that the attP core, the site where strand exchange occurs during integration, is located within the repressor gene; in this study we are using the mycobacteriophage BPs as a model system. The BPs repressor gene, 33, encodes a 136-residue protein (gp33136) that we have shown is not active for immunity unless overexpressed. However, when the phage genome integrates into the bacterial chromosome, a stop codon is introduced at the attL junction site, creating a truncated, but active form of the repressor (gp33103), which is expressed within the lysogen. Electrophoretic mobility shift assays (EMSA) have shown that each form of the repressor binds specifically to two operators present within the 33-34 intergenic regulatory region, as well as to four other operators located across the BPs genome. Here we report studies done to map the sequence requirements for repressor function in repressing lytic growth and show that a previously reported 12 bp operator sequence represents one half of a full binding site. We also show that the BPs repressor does not seem to form inter-DNA bridges for transcriptional regulation, but evidence suggests that bending DNA plays a role. Characterization of the binding sites and behavior of the BPs repressor will deepen our understanding of this new class of integrations-dependent switch. The overarching goal, of which this project represents only a portion, is to dissect BPs so that we have a full understanding of its biology, what its genes do, how they are expressed, how expression is regulated and manipulated to confer immunity, and how it recognizes its mycobacterial hosts