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Structured and Dynamic Disordered Domains Regulate the Activity of a Multifunctional Anti-σ Factor.

Herrou J, Willett JW, Crosson S - MBio (2015)

Bottom Line: We further demonstrate that NepR strongly stimulates the rate of PhyR phosphorylation in vitro and that this effect requires the structured and disordered domains of NepR.We conclude that structured and intrinsically disordered domains of NepR coordinately control multiple functions in the GSR signaling pathway, including core protein partner switch interactions and pathway activation by phosphorylation.Our results provide evidence for a new layer of GSR regulatory control in which NepR directly modulates PhyR phosphorylation and, hence, activation of the GSR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.

No MeSH data available.


Related in: MedlinePlus

Functional analysis of full-length and truncated NepR alleles as regulators of GSR transcription in C. crescentus. (A) Measured β-galactosidase activity from the σT-dependent PsigU-lacZ reporter plasmid. β-Galactosidase activities were measured in WT and ΔnepR ΔsigT backgrounds containing a plasmid expressing sigT from a xylose-inducible promoter (Pxyl-sigT), in the presence (+) or absence (−) of sigT inducer (0.2% xylose) and the presence (+) or absence (−) of osmotic upshock stress (150 mM sucrose). Empty vector (EV) controls (Pxyl and Pvan) are also included. (B) σT-dependent transcription measured in a ΔnepR ΔsigT strain expressing sigT from Pxyl-sigT. Transcription was assayed as a function of full-length (nepRFL) and terminally truncated (nepRSV) nepR alleles expressed from a vanillate-inducible promoter (Pvan-nepRFL or Pvan-nepRSV). Boundaries of expressed nepR alleles are as follows: nepRFL, M1 to E68; nepRSV, Q31 to E62. β-Galactosidase activities were assayed in the presence (+) and absence (−) of nepR induction (0.5 mM vanillate) and in the presence (+) and absence (−) of osmotic upshock (150 mM sucrose). Transcription was compared to an empty vector (EV) control strain. Stability and function of HA-tagged nepR alleles were further evaluated by dot blotting and β-galactosidase transcriptional assays described in Fig. S4 in the supplemental material. All assays were performed in triplicate; error bars represent standard deviations.
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fig3: Functional analysis of full-length and truncated NepR alleles as regulators of GSR transcription in C. crescentus. (A) Measured β-galactosidase activity from the σT-dependent PsigU-lacZ reporter plasmid. β-Galactosidase activities were measured in WT and ΔnepR ΔsigT backgrounds containing a plasmid expressing sigT from a xylose-inducible promoter (Pxyl-sigT), in the presence (+) or absence (−) of sigT inducer (0.2% xylose) and the presence (+) or absence (−) of osmotic upshock stress (150 mM sucrose). Empty vector (EV) controls (Pxyl and Pvan) are also included. (B) σT-dependent transcription measured in a ΔnepR ΔsigT strain expressing sigT from Pxyl-sigT. Transcription was assayed as a function of full-length (nepRFL) and terminally truncated (nepRSV) nepR alleles expressed from a vanillate-inducible promoter (Pvan-nepRFL or Pvan-nepRSV). Boundaries of expressed nepR alleles are as follows: nepRFL, M1 to E68; nepRSV, Q31 to E62. β-Galactosidase activities were assayed in the presence (+) and absence (−) of nepR induction (0.5 mM vanillate) and in the presence (+) and absence (−) of osmotic upshock (150 mM sucrose). Transcription was compared to an empty vector (EV) control strain. Stability and function of HA-tagged nepR alleles were further evaluated by dot blotting and β-galactosidase transcriptional assays described in Fig. S4 in the supplemental material. All assays were performed in triplicate; error bars represent standard deviations.

Mentions: We next sought to assess the functional implications of our two-hybrid interaction data (Fig. 2) using GSR-dependent transcription in C. crescentus cells as a functional readout. We first attempted to generate a strain of C. crescentus in which the chromosomal copy of nepR was deleted. We were unable to delete nepR alone but were able to simultaneously delete nepR and sigT. Similar results have been described in Sphingomonas species (14) and in Sinorhizobium meliloti, where nepR could not be deleted unless its cognate ecfG was first deleted (23). We successfully transformed the C. crescentus ΔnepR ΔsigT double deletion mutant with a xylose-inducible allele of sigT (Pxyl-sigT) and an empty plasmid containing the vanillate-inducible promoter (Pvan), which enabled us to assess σT-dependent transcription as a function of induced σT expression (Fig. 3A).


Structured and Dynamic Disordered Domains Regulate the Activity of a Multifunctional Anti-σ Factor.

Herrou J, Willett JW, Crosson S - MBio (2015)

Functional analysis of full-length and truncated NepR alleles as regulators of GSR transcription in C. crescentus. (A) Measured β-galactosidase activity from the σT-dependent PsigU-lacZ reporter plasmid. β-Galactosidase activities were measured in WT and ΔnepR ΔsigT backgrounds containing a plasmid expressing sigT from a xylose-inducible promoter (Pxyl-sigT), in the presence (+) or absence (−) of sigT inducer (0.2% xylose) and the presence (+) or absence (−) of osmotic upshock stress (150 mM sucrose). Empty vector (EV) controls (Pxyl and Pvan) are also included. (B) σT-dependent transcription measured in a ΔnepR ΔsigT strain expressing sigT from Pxyl-sigT. Transcription was assayed as a function of full-length (nepRFL) and terminally truncated (nepRSV) nepR alleles expressed from a vanillate-inducible promoter (Pvan-nepRFL or Pvan-nepRSV). Boundaries of expressed nepR alleles are as follows: nepRFL, M1 to E68; nepRSV, Q31 to E62. β-Galactosidase activities were assayed in the presence (+) and absence (−) of nepR induction (0.5 mM vanillate) and in the presence (+) and absence (−) of osmotic upshock (150 mM sucrose). Transcription was compared to an empty vector (EV) control strain. Stability and function of HA-tagged nepR alleles were further evaluated by dot blotting and β-galactosidase transcriptional assays described in Fig. S4 in the supplemental material. All assays were performed in triplicate; error bars represent standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4551977&req=5

fig3: Functional analysis of full-length and truncated NepR alleles as regulators of GSR transcription in C. crescentus. (A) Measured β-galactosidase activity from the σT-dependent PsigU-lacZ reporter plasmid. β-Galactosidase activities were measured in WT and ΔnepR ΔsigT backgrounds containing a plasmid expressing sigT from a xylose-inducible promoter (Pxyl-sigT), in the presence (+) or absence (−) of sigT inducer (0.2% xylose) and the presence (+) or absence (−) of osmotic upshock stress (150 mM sucrose). Empty vector (EV) controls (Pxyl and Pvan) are also included. (B) σT-dependent transcription measured in a ΔnepR ΔsigT strain expressing sigT from Pxyl-sigT. Transcription was assayed as a function of full-length (nepRFL) and terminally truncated (nepRSV) nepR alleles expressed from a vanillate-inducible promoter (Pvan-nepRFL or Pvan-nepRSV). Boundaries of expressed nepR alleles are as follows: nepRFL, M1 to E68; nepRSV, Q31 to E62. β-Galactosidase activities were assayed in the presence (+) and absence (−) of nepR induction (0.5 mM vanillate) and in the presence (+) and absence (−) of osmotic upshock (150 mM sucrose). Transcription was compared to an empty vector (EV) control strain. Stability and function of HA-tagged nepR alleles were further evaluated by dot blotting and β-galactosidase transcriptional assays described in Fig. S4 in the supplemental material. All assays were performed in triplicate; error bars represent standard deviations.
Mentions: We next sought to assess the functional implications of our two-hybrid interaction data (Fig. 2) using GSR-dependent transcription in C. crescentus cells as a functional readout. We first attempted to generate a strain of C. crescentus in which the chromosomal copy of nepR was deleted. We were unable to delete nepR alone but were able to simultaneously delete nepR and sigT. Similar results have been described in Sphingomonas species (14) and in Sinorhizobium meliloti, where nepR could not be deleted unless its cognate ecfG was first deleted (23). We successfully transformed the C. crescentus ΔnepR ΔsigT double deletion mutant with a xylose-inducible allele of sigT (Pxyl-sigT) and an empty plasmid containing the vanillate-inducible promoter (Pvan), which enabled us to assess σT-dependent transcription as a function of induced σT expression (Fig. 3A).

Bottom Line: We further demonstrate that NepR strongly stimulates the rate of PhyR phosphorylation in vitro and that this effect requires the structured and disordered domains of NepR.We conclude that structured and intrinsically disordered domains of NepR coordinately control multiple functions in the GSR signaling pathway, including core protein partner switch interactions and pathway activation by phosphorylation.Our results provide evidence for a new layer of GSR regulatory control in which NepR directly modulates PhyR phosphorylation and, hence, activation of the GSR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.

No MeSH data available.


Related in: MedlinePlus