Flexible linker modulates the binding affinity of the TP901‐1 CI phage repressor to DNA

Temperate bacteriophages can switch between two life cycles following infection of a host bacterium: the lytic or lysogenic life cycle. The choice between these is controlled by a bistable genetic switch. We investigated the genetic switch of the lactococcal temperate bacteriophage, TP901‐1, which is controlled by two regulatory proteins, the Clear 1 (CI) repressor and modulator of repression (MOR) antirepressor. CI consists of a DNA‐binding N‐terminal domain and a C‐terminal domain responsible for oligomerization, connected by a flexible interdomain linker. Full‐length CI is hexameric, whereas the truncated version CI with 58 C‐terminal residues truncated (CIΔ58), missing the second C‐terminal subdomain, is dimeric, but binds with the same affinity as full‐length CI to the OL operator site, responsible for lytic genes transcription repression. Three variants of CIΔ58 with shorter, longer, and PP substituted linkers were produced and confirmed by circular dichroism spectroscopy and nanodifferential scanning fluorimetry to be well folded. With small‐angle X‐ray scattering, we delineated the conformational space sampled by the variants and wild‐type in solution and found that shortening and lengthening the linker decrease and increase this, respectively, as also substantiated by molecular dynamics and as intended. Isoelectric focusing electrophoresis confirmed that all variants are able to bind to the MOR antirepressor. However, using electrophoretic mobility shift assays, we showed that shortening and lengthening the linker lead to a 94 and 17 times decrease in affinity to OL, respectively. Thus, an appropriate linker length appears to be crucial for appropriate DNA‐binding and subsequent TP901‐1 genetic switch function. The CI repressor of the lactococcal temperate bacteriophage TP901‐1 controls the genetic switch by binding DNA operator sites. We characterized a truncated variant of CI, termed CIΔ58, and variants with mutations in the linker region using a variety of biochemical, biophysical, and computational techniques and found that shortening and lengthening of the linker lead to a 94 and 17 times decrease in affinity for DNA, respectively, highlighting its functional importance.