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 i...

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Veröffentlicht in:The FEBS journal 2022-02, Vol.289 (4), p.1135-1148
Hauptverfasser: Varming, Anders Kokkenborg, Rasmussen, Kim Krighaar, Zong, Zhiyou, Thulstrup, Peter Waaben, Kilstrup, Mogens, Lo Leggio, Leila
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container_title The FEBS journal
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creator Varming, Anders Kokkenborg
Rasmussen, Kim Krighaar
Zong, Zhiyou
Thulstrup, Peter Waaben
Kilstrup, Mogens
Lo Leggio, Leila
description 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.
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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. 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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.</abstract><cop>England</cop><pub>Blackwell Publishing Ltd</pub><pmid>34665941</pmid><doi>10.1111/febs.16238</doi><tpages>14</tpages><orcidid>https://orcid.org/0000-0002-5135-0882</orcidid><oa>free_for_read</oa></addata></record>
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subjects Affinity
bacteriophage
Bacteriophages - genetics
Bacteriophages - metabolism
Binding
Binding Sites
Circular dichroism
Deoxyribonucleic acid
Dichroism
DNA
DNA - chemistry
DNA - metabolism
Domains
Electrophoresis
Electrophoretic mobility
flexible linker
Fluorimetry
Gene silencing
genetic switch
Isoelectric focusing
Life cycles
lysogeny
Models, Molecular
Molecular dynamics
Oligomerization
Phages
Regulatory proteins
repressor
Repressor Proteins - chemistry
Repressor Proteins - genetics
Repressor Proteins - metabolism
Scattering, Small Angle
Spectroscopy
X-Rays
title Flexible linker modulates the binding affinity of the TP901‐1 CI phage repressor to DNA
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