Repression of the lysogenic PR promoter in bacteriophage TP901-1 through binding of a CI-MOR complex to a composite OM-OR operator

Abstract

A functional genetic switch from the lactococcal bacteriophage TP901-1, deciding which of two divergently transcribing promoters becomes most active and allows this bi-stable decision to be inherited in future generations requires a DNA region of less than 1 kb. The fragment encodes two repressors, CI and MOR, transcribed from the PR and PL promoters respectively. CI can repress the transcription of the mor gene at three operator sites (OR, OL, and OD), leading to the immune state. Repression of the cI gene, leading to the lytic (anti-immune) state, requires interaction between CI and MOR by an unknown mechanism, but involving a CI:MOR complex. A consensus for putative MOR binding sites (OM sites), and a common topology of three OM sites adjacent to the OR motif was here identified in diverse phage switches that encode CI and MOR homologs, in a search for DNA sequences similar to the TP901-1 switch. The OR site and all putative OM sites are important for establishment of the anti-immune repression of PR, and a putative DNA binding motif in MOR is needed for establishment of the anti-immune state. Direct evidence for binding between CI and MOR is here shown by pull-down experiments, chemical crosslinking, and size exclusion chromatography. The results are consistent with two possible models for establishment of the anti-immune repression of cI expression at the PR promoter.

Revealing the mechanism of repressor inactivation during switching of a temperate bacteriophage

“…Recently, additional evidence in favor of this hypothesis has been presented, first of all by demonstration of a direct interaction between CI and MOR (7). Secondly, a series of mutagenesis experiments verified the role of an evolutionary conserved composite OM1-OM2-OR-OM3 operator site as a plausible binding site for the CI:MOR complex (Fig.…”

“…1A). In fact, mutations at all sites (OM1, OM2, OR and OM3, carried out one by one), decrease repression of PR in the anti-immune state (7). The OM sites are non-palindromic, however, OM2 and OM3 are present on opposite strands to form a palindromic OM2-ORL-ORR-OM3 site.…”

“…Based on these experiments, an updated model of the lytic state was proposed where CI:MOR complexes of different stoichiometries bind to the OM1-OM2-OR-OM3 operator ( Fig. 1D) (7). Importantly, statistical thermodynamical modeling of the TP901-1 genetic switch, supported by measurements of the activity of the lysogenic PR promoter as function of the concentration of CI in the absence and presence of MOR, suggested that binding of a CI:MOR complex to DNA is not the sole action of MOR and that MOR in addition inhibits CI binding to DNA (8).…”

“…The CI-MOR complex has been proposed to bind to the evolutionary conserved OM1-OM2-OR-OM3 operator site to repress transcription from the lysogenic promoter, PR (7). To verify this hypothesis and obtain insight into the importance of the OM sites, we used 15 N-labeled MOR, CI-NTD and pre-formed CI-NTD:MOR complex and added DNA sequences corresponding to OL (used as a control), OR, OM1-OM2 and OM2-OR-OM3.…”

“…Phylogenetic analysis of the TP901-1 switch region at the DNA level has previously offered structural insights into the CI protein showing that primarily the N-terminal domain of CI that is involved in DNA and MOR binding has been conserved in switches carrying mor genes and OM binding sites (7). To gain more insight into the evolution of the CI:MOR interactions, the primary sequences of CI and MOR were used as templates in the web server Fig.…”