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Evolutionary expansion of a regulatory network by counter-silencing.

Will WR, Bale DH, Reid PJ, Libby SJ, Fang FC - Nat Commun (2014)

Bottom Line: Conserved genes are regulated by classical PhoP-mediated activation and are invariant in promoter architecture, whereas horizontally acquired genes exhibit variable promoter architecture and are regulated by PhoP-mediated counter-silencing.Biochemical analyses show that a horizontally acquired promoter adopts different structures in the silenced and counter-silenced states, implicating the remodelling of the H-NS nucleoprotein filament and the subsequent restoration of open-complex formation as the central mechanism of counter-silencing.Our results indicate that counter-silencing is favoured in the regulatory integration of newly acquired genes because it is able to accommodate multiple promoter architectures.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington 98195, USA.

ABSTRACT
Horizontal gene transfer plays a major role in bacterial evolution. Successful acquisition of new genes requires their incorporation into existing regulatory networks. This study compares the regulation of conserved genes in the PhoPQ regulon of Salmonella enterica serovar Typhimurium with that of PhoPQ-regulated horizontally acquired genes, which are silenced by the histone-like protein H-NS. We demonstrate that PhoP upregulates conserved and horizontally acquired genes by distinct mechanisms. Conserved genes are regulated by classical PhoP-mediated activation and are invariant in promoter architecture, whereas horizontally acquired genes exhibit variable promoter architecture and are regulated by PhoP-mediated counter-silencing. Biochemical analyses show that a horizontally acquired promoter adopts different structures in the silenced and counter-silenced states, implicating the remodelling of the H-NS nucleoprotein filament and the subsequent restoration of open-complex formation as the central mechanism of counter-silencing. Our results indicate that counter-silencing is favoured in the regulatory integration of newly acquired genes because it is able to accommodate multiple promoter architectures.

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Reconstitution of the pagC counter-silencing circuit in vitro(a) H-NS strongly represses pagC in vitro. Transcriptional outputs are normalized to the 0 nM control reaction. (b) Reconstitution of pagC counter-silencing in vitro. A pagC-containing template was incubated in the presence of 600 nM H-NS, 100 nM SlyA, and 500 nM PhoP-P, as indicated. Transcriptional output is normalized to the H-NS reaction. Both SlyA and PhoP-P are required for counter-silencing. (c) SlyA and PhoP-P do not act as co-activators in the absence H-NS. A pagC-containing template was incubated in the presence of 100 nM SlyA, 500 nM PhoP-P (Fig. 2), or both as indicated. Transcriptional output is normalized the control reaction. Data represent the mean ± SEM; n = 3. (d) KMnO4 footprinting analysis indicates that RNAP is unable to form an open complex in the presence of H-NS. The addition of SlyA and PhoP-P restores open complex formation at the pagC promoter. Results are presented as a DDFA plot representing difference in peak height in relative fluorescent units (RFU) between the control (no RNAP) and the experimental samples. Peaks indicate regions of single stranded DNA caused by open complex formation. The relative distance to the transcriptional start site (TSS) is indicated on the horizontal axis. Data represent the mean ± SD; n=3. See also Supplementary Fig. 3 and Supplementary Fig. 4.
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Figure 3: Reconstitution of the pagC counter-silencing circuit in vitro(a) H-NS strongly represses pagC in vitro. Transcriptional outputs are normalized to the 0 nM control reaction. (b) Reconstitution of pagC counter-silencing in vitro. A pagC-containing template was incubated in the presence of 600 nM H-NS, 100 nM SlyA, and 500 nM PhoP-P, as indicated. Transcriptional output is normalized to the H-NS reaction. Both SlyA and PhoP-P are required for counter-silencing. (c) SlyA and PhoP-P do not act as co-activators in the absence H-NS. A pagC-containing template was incubated in the presence of 100 nM SlyA, 500 nM PhoP-P (Fig. 2), or both as indicated. Transcriptional output is normalized the control reaction. Data represent the mean ± SEM; n = 3. (d) KMnO4 footprinting analysis indicates that RNAP is unable to form an open complex in the presence of H-NS. The addition of SlyA and PhoP-P restores open complex formation at the pagC promoter. Results are presented as a DDFA plot representing difference in peak height in relative fluorescent units (RFU) between the control (no RNAP) and the experimental samples. Peaks indicate regions of single stranded DNA caused by open complex formation. The relative distance to the transcriptional start site (TSS) is indicated on the horizontal axis. Data represent the mean ± SD; n=3. See also Supplementary Fig. 3 and Supplementary Fig. 4.

Mentions: To test the hypothesis that PhoP-P acts as a counter-silencer rather than as an activator at non-group A genes, we reconstituted a counter-silencing circuit in vitro using the model group C pagC gene. To confirm the role of H-NS as a specific repressor of pagC, IVT analyses were performed on pagC in the presence of increasing H-NS concentrations. The transcriptional output of pagC decreased by more than ten-fold in the presence of 600 nM H-NS, whereas the group A phoP gene was only modestly repressed (Fig. 3a). Neither PhoP-P nor SlyA alone was capable of relieving H-NS silencing, whereas the two proteins in combination increased pagC transcription (Fig. 3b and Supplementary Fig. 4b). This increase was not due to the formation of a SlyA-PhoP-P activator complex, as no increase in mRNA was detectable in the absence of H NS (Fig. 3c), indicating that PhoP acts as a counter-silencer of pagC via a mechanism distinct from the classical transcriptional activation observed at the phoP promoter.


Evolutionary expansion of a regulatory network by counter-silencing.

Will WR, Bale DH, Reid PJ, Libby SJ, Fang FC - Nat Commun (2014)

Reconstitution of the pagC counter-silencing circuit in vitro(a) H-NS strongly represses pagC in vitro. Transcriptional outputs are normalized to the 0 nM control reaction. (b) Reconstitution of pagC counter-silencing in vitro. A pagC-containing template was incubated in the presence of 600 nM H-NS, 100 nM SlyA, and 500 nM PhoP-P, as indicated. Transcriptional output is normalized to the H-NS reaction. Both SlyA and PhoP-P are required for counter-silencing. (c) SlyA and PhoP-P do not act as co-activators in the absence H-NS. A pagC-containing template was incubated in the presence of 100 nM SlyA, 500 nM PhoP-P (Fig. 2), or both as indicated. Transcriptional output is normalized the control reaction. Data represent the mean ± SEM; n = 3. (d) KMnO4 footprinting analysis indicates that RNAP is unable to form an open complex in the presence of H-NS. The addition of SlyA and PhoP-P restores open complex formation at the pagC promoter. Results are presented as a DDFA plot representing difference in peak height in relative fluorescent units (RFU) between the control (no RNAP) and the experimental samples. Peaks indicate regions of single stranded DNA caused by open complex formation. The relative distance to the transcriptional start site (TSS) is indicated on the horizontal axis. Data represent the mean ± SD; n=3. See also Supplementary Fig. 3 and Supplementary Fig. 4.
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Figure 3: Reconstitution of the pagC counter-silencing circuit in vitro(a) H-NS strongly represses pagC in vitro. Transcriptional outputs are normalized to the 0 nM control reaction. (b) Reconstitution of pagC counter-silencing in vitro. A pagC-containing template was incubated in the presence of 600 nM H-NS, 100 nM SlyA, and 500 nM PhoP-P, as indicated. Transcriptional output is normalized to the H-NS reaction. Both SlyA and PhoP-P are required for counter-silencing. (c) SlyA and PhoP-P do not act as co-activators in the absence H-NS. A pagC-containing template was incubated in the presence of 100 nM SlyA, 500 nM PhoP-P (Fig. 2), or both as indicated. Transcriptional output is normalized the control reaction. Data represent the mean ± SEM; n = 3. (d) KMnO4 footprinting analysis indicates that RNAP is unable to form an open complex in the presence of H-NS. The addition of SlyA and PhoP-P restores open complex formation at the pagC promoter. Results are presented as a DDFA plot representing difference in peak height in relative fluorescent units (RFU) between the control (no RNAP) and the experimental samples. Peaks indicate regions of single stranded DNA caused by open complex formation. The relative distance to the transcriptional start site (TSS) is indicated on the horizontal axis. Data represent the mean ± SD; n=3. See also Supplementary Fig. 3 and Supplementary Fig. 4.
Mentions: To test the hypothesis that PhoP-P acts as a counter-silencer rather than as an activator at non-group A genes, we reconstituted a counter-silencing circuit in vitro using the model group C pagC gene. To confirm the role of H-NS as a specific repressor of pagC, IVT analyses were performed on pagC in the presence of increasing H-NS concentrations. The transcriptional output of pagC decreased by more than ten-fold in the presence of 600 nM H-NS, whereas the group A phoP gene was only modestly repressed (Fig. 3a). Neither PhoP-P nor SlyA alone was capable of relieving H-NS silencing, whereas the two proteins in combination increased pagC transcription (Fig. 3b and Supplementary Fig. 4b). This increase was not due to the formation of a SlyA-PhoP-P activator complex, as no increase in mRNA was detectable in the absence of H NS (Fig. 3c), indicating that PhoP acts as a counter-silencer of pagC via a mechanism distinct from the classical transcriptional activation observed at the phoP promoter.

Bottom Line: Conserved genes are regulated by classical PhoP-mediated activation and are invariant in promoter architecture, whereas horizontally acquired genes exhibit variable promoter architecture and are regulated by PhoP-mediated counter-silencing.Biochemical analyses show that a horizontally acquired promoter adopts different structures in the silenced and counter-silenced states, implicating the remodelling of the H-NS nucleoprotein filament and the subsequent restoration of open-complex formation as the central mechanism of counter-silencing.Our results indicate that counter-silencing is favoured in the regulatory integration of newly acquired genes because it is able to accommodate multiple promoter architectures.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington 98195, USA.

ABSTRACT
Horizontal gene transfer plays a major role in bacterial evolution. Successful acquisition of new genes requires their incorporation into existing regulatory networks. This study compares the regulation of conserved genes in the PhoPQ regulon of Salmonella enterica serovar Typhimurium with that of PhoPQ-regulated horizontally acquired genes, which are silenced by the histone-like protein H-NS. We demonstrate that PhoP upregulates conserved and horizontally acquired genes by distinct mechanisms. Conserved genes are regulated by classical PhoP-mediated activation and are invariant in promoter architecture, whereas horizontally acquired genes exhibit variable promoter architecture and are regulated by PhoP-mediated counter-silencing. Biochemical analyses show that a horizontally acquired promoter adopts different structures in the silenced and counter-silenced states, implicating the remodelling of the H-NS nucleoprotein filament and the subsequent restoration of open-complex formation as the central mechanism of counter-silencing. Our results indicate that counter-silencing is favoured in the regulatory integration of newly acquired genes because it is able to accommodate multiple promoter architectures.

Show MeSH
Related in: MedlinePlus