Structural basis of the bacteriophage <scp>TP</scp>901‐1 <scp>CI</scp> repressor dimerization and interaction with <scp>DNA</scp>

Rasmussen, Kim Krighaar; Varming, Anders K.; Schmidt, Signe; Frandsen, Kristian E. H.; Thulstrup, Peter W.; Jensen, Malene Ringkjøbing; Leggio, Leila Lo

doi:10.1002/1873-3468.13060

Cited by 5 publications

(18 citation statements)

References 44 publications

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“…In terms of domain organization, the CI proteins from TP901-1, l and 186 are similar containing an N-terminal DNA binding domain but displaying a different quaternary structure with full-length CI TP901-1 being hexameric, CI l octameric and CI 186 heptameric (25,26). Furthermore, the C-terminal domains are of predominantly β-fold in l and 186, but helical in TP901-1 (2,3). In the l phage, the repressor CI and the anti-repressor Cro compete for binding to multiple operator sites in the divergent promoter region leading to CI-mediated repression of the lytic promoter or Cro repression of the lysogenic promoter.…”

Section: Discussionmentioning

confidence: 99%

“…1B) (1,2). Full-length CI has been shown to be hexameric (a trimer of dimers) at high protein concentrations, while a shorter construct encompassing residues 1-122 of CI (CI-D58) has been shown to be dimeric mediated by dimerization of CI-CTD1 through a pair of helical hooks (3). MOR is a single domain protein of 72 amino acids of previously unknown structure (Fig.…”

Section: Significance Statementmentioning

confidence: 99%

“…On the basis of this and experimental small angle X-ray scattering (SAXS) data, we have obtained a model of CI-D58 in complex with the full operator site, where the two N-terminal domains bind to the two adjacent, major grooves on the DNA and a flexible linker connects these domains to the dimeric helical hook structure located at the C-terminus of CI-D58 ( Fig. 5E) (3). Superimposing the structure of the MOR:CI-NTD complex onto one of the CI-NTD of the CI-D58:OL complex leads to a likely steric clash between MOR and the other CI-NTD ( Fig.…”

Section: Fig S2a-d)mentioning

confidence: 99%

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

Rasmussen

Palencia

Varming

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion and rotating frame R1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.

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Section: Discussionmentioning

confidence: 99%

Section: Significance Statementmentioning

confidence: 99%

Section: Fig S2a-d)mentioning

confidence: 99%

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

Rasmussen

Palencia

Varming

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…1B) [9,10]. CI has been shown to be hexameric in solution (trimer of dimers) at higher concentrations, while a shorter truncated construct containing CI-NTD, the flexible linker, and CI-CTD 1 (CIΔ58, 1-122) have been shown to be dimeric in solution, mediated by the helical hooks of the CI-CTD 1 dimerization domain [11,12].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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 DNAbinding 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 O L 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 O L , respectively. Thus, an appropriate linker length appears to be crucial for appropriate DNAbinding and subsequent TP901-1 genetic switch function.

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“…This overall operator topology appears to be shared among many lactococcal and streptococcal bacteriophage switches, despite the fact that the operator sequences do not follow the same consensus 7 . The CI monomer consists of an N-terminal DNA binding region 8 , a helical hook region which mediates dimerization 9 and a C-terminal helical region of unknown structure necessary for multimerization 3 . Upon binding of CI dimers to each of these operators in the lysogenic state of the TP901-1 switch, the P L promoter is almost fully repressed ensuring low concentrations of MOR and lytic proteins, while the P R promoter is only partially auto-repressed Figure 1.…”

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confidence: 99%

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

Pedersen

Neergaard

Cassias

et al. 2020

Sci Rep

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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.

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Structural basis of the bacteriophage TP901‐1 CI repressor dimerization and interaction with DNA

Cited by 5 publications

References 44 publications

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

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

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

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

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