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

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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.

Original languageEnglish
JournalFEBS Journal
Volume289
Issue number4
Pages (from-to)1135-1148
Number of pages14
ISSN1742-464X
DOIs
Publication statusPublished - 2022

Bibliographical note

Funding Information:
We thank Alina Vitaliyivna Kulakova for help with analyzing the SAXS data and Yusuf Theibich for help with running the IEF electrophoresis. We also thank Karin Hammer for helpful discussions throughout the TP901‐1 switch project. AKV was funded by a PhD scholarship from the Lundbeck Foundation (grant R249‐2017‐977). ZZ was funded by a grant from Novo Nordisk Foundation (NNF17OC0027698) to LLL. Additional funding for materials was provided by a grant from the Danish Council for Independent Research (4002‐00107) to LLL). Molecular dynamics simulations were performed at the Danish National Supercomputer for Life Sciences Computerome 2.0, installed at the National Life Science Center at Technical University of Denmark. We acknowledge the European Synchrotron Radiation Facility for provision of beam time on BM29, and we would like to thank the beamline staff for assistance with collecting SAXS data. Travel to synchrotrons was supported by the DANSCATT program, funded by the Danish Ministry for Higher Education and Science and the European Community's Seventh Framework Programme (FP7/2007–2013) under BioStruct‐X (grant agreement no. 283570).

Publisher Copyright:
© 2021 Federation of European Biochemical Societies

    Research areas

  • bacteriophage, flexible linker, genetic switch, lysogeny, repressor

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