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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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UV laser DDFA of the pagC promoter regionPlasmid DNA containing the pagC region was incubated in the presence of 600 nM H-NS, 100 nM SlyA, and 500 nM PhoP-P as indicated and exposed to a single pulse of 266 nm radiation. Crosslinking was quantified by fluorescent primer extension. The relative distance to the TSS is indicated in base pairs on the horizontal axis. Results are presented as DDFA plots, representing the difference in fluorescent peak height (RFU) between the protein-free control and the experimental sample (a). DDFA plots are also shown for the H-NS + SlyA, H-NS + PhoP-P, and H-NS + SlyA + PhoP-P reactions representing the difference between the H-NS control and the experimental sample (b). Peaks represent a protein-induced change in DNA structure, typically due to increased intramolecular contacts or DNA-protein crosslinks. Valleys also represent a change in DNA structure, due to decreased intramolecular contacts. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See Supplementary Fig. 6 for representative raw chromatograms.
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Figure 5: UV laser DDFA of the pagC promoter regionPlasmid DNA containing the pagC region was incubated in the presence of 600 nM H-NS, 100 nM SlyA, and 500 nM PhoP-P as indicated and exposed to a single pulse of 266 nm radiation. Crosslinking was quantified by fluorescent primer extension. The relative distance to the TSS is indicated in base pairs on the horizontal axis. Results are presented as DDFA plots, representing the difference in fluorescent peak height (RFU) between the protein-free control and the experimental sample (a). DDFA plots are also shown for the H-NS + SlyA, H-NS + PhoP-P, and H-NS + SlyA + PhoP-P reactions representing the difference between the H-NS control and the experimental sample (b). Peaks represent a protein-induced change in DNA structure, typically due to increased intramolecular contacts or DNA-protein crosslinks. Valleys also represent a change in DNA structure, due to decreased intramolecular contacts. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See Supplementary Fig. 6 for representative raw chromatograms.

Mentions: UV laser footprinting, wherein a high intensity UV (266 nm) laser pulse probes DNA structural changes in response to protein binding34, suggests that H-NS-dependent structural changes are limited to a discrete region upstream of the transcription start site (TSS) (−111 to −43), henceforth referred to as the upstream regulatory region (URR) (Fig. 5 and Supplementary Fig. 9). Decreased cross-linking in the URR suggests fewer intramolecular contacts between adjacent bases, consistent with the “stiffening” mode of H-NS binding14. As recent studies indicate that stiffening is required for silencing35, the structure of the URR may be important for the regulation of pagC transcription. Although both proteins altered the URR structure in the absence of H-NS, neither PhoP nor SlyA alone were capable of altering the URR structure in the presence of H-NS. However, SlyA was observed by DNase I footprinting to bind DNA downstream of the TSS (+38 to +59) and outside of the H-NS-bound region independently of PhoP-P. Under counter-silencing conditions, PhoP appeared to act cooperatively with SlyA to alter the URR structure at −84 and −77, resembling SlyA-bound DNA at those sites in the absence of H-NS. These results corroborate and extend the findings of the DNase I footprinting analysis, demonstrating that counter-silencing of pagC results when SlyA alters the structure of H-NS-bound DNA in the URR, thereby allowing PhoP-P to bend the DNA and facilitate open complex formation.


Evolutionary expansion of a regulatory network by counter-silencing.

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

UV laser DDFA of the pagC promoter regionPlasmid DNA containing the pagC region was incubated in the presence of 600 nM H-NS, 100 nM SlyA, and 500 nM PhoP-P as indicated and exposed to a single pulse of 266 nm radiation. Crosslinking was quantified by fluorescent primer extension. The relative distance to the TSS is indicated in base pairs on the horizontal axis. Results are presented as DDFA plots, representing the difference in fluorescent peak height (RFU) between the protein-free control and the experimental sample (a). DDFA plots are also shown for the H-NS + SlyA, H-NS + PhoP-P, and H-NS + SlyA + PhoP-P reactions representing the difference between the H-NS control and the experimental sample (b). Peaks represent a protein-induced change in DNA structure, typically due to increased intramolecular contacts or DNA-protein crosslinks. Valleys also represent a change in DNA structure, due to decreased intramolecular contacts. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See Supplementary Fig. 6 for representative raw chromatograms.
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Figure 5: UV laser DDFA of the pagC promoter regionPlasmid DNA containing the pagC region was incubated in the presence of 600 nM H-NS, 100 nM SlyA, and 500 nM PhoP-P as indicated and exposed to a single pulse of 266 nm radiation. Crosslinking was quantified by fluorescent primer extension. The relative distance to the TSS is indicated in base pairs on the horizontal axis. Results are presented as DDFA plots, representing the difference in fluorescent peak height (RFU) between the protein-free control and the experimental sample (a). DDFA plots are also shown for the H-NS + SlyA, H-NS + PhoP-P, and H-NS + SlyA + PhoP-P reactions representing the difference between the H-NS control and the experimental sample (b). Peaks represent a protein-induced change in DNA structure, typically due to increased intramolecular contacts or DNA-protein crosslinks. Valleys also represent a change in DNA structure, due to decreased intramolecular contacts. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See Supplementary Fig. 6 for representative raw chromatograms.
Mentions: UV laser footprinting, wherein a high intensity UV (266 nm) laser pulse probes DNA structural changes in response to protein binding34, suggests that H-NS-dependent structural changes are limited to a discrete region upstream of the transcription start site (TSS) (−111 to −43), henceforth referred to as the upstream regulatory region (URR) (Fig. 5 and Supplementary Fig. 9). Decreased cross-linking in the URR suggests fewer intramolecular contacts between adjacent bases, consistent with the “stiffening” mode of H-NS binding14. As recent studies indicate that stiffening is required for silencing35, the structure of the URR may be important for the regulation of pagC transcription. Although both proteins altered the URR structure in the absence of H-NS, neither PhoP nor SlyA alone were capable of altering the URR structure in the presence of H-NS. However, SlyA was observed by DNase I footprinting to bind DNA downstream of the TSS (+38 to +59) and outside of the H-NS-bound region independently of PhoP-P. Under counter-silencing conditions, PhoP appeared to act cooperatively with SlyA to alter the URR structure at −84 and −77, resembling SlyA-bound DNA at those sites in the absence of H-NS. These results corroborate and extend the findings of the DNase I footprinting analysis, demonstrating that counter-silencing of pagC results when SlyA alters the structure of H-NS-bound DNA in the URR, thereby allowing PhoP-P to bend the DNA and facilitate open complex formation.

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