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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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DNase I DDFA of the pagC promoter regionIn vitro DNase I footprinting studies were performed on the pagC promoter region with H-NS, SlyA, and PhoP-P, at concentrations of 600 nM, 100 nM, and 500 nM respectively, as indicated. DNA-protein complexes were incubated at room temperature before digestion with DNase I. 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 the H-NS + SlyA + PhoP-P reactions, representing the difference between the H-NS control and the experimental sample (b). The relative distance in base pairs to the TSS is indicated on the horizontal axis. Peaks indicate regions of hypersensitivity, typical of bent or distorted DNA, whereas valleys indicate protected regions, typical of protein binding sites. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See Supplementary Fig. 5 for representative raw chromatograms.
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Figure 4: DNase I DDFA of the pagC promoter regionIn vitro DNase I footprinting studies were performed on the pagC promoter region with H-NS, SlyA, and PhoP-P, at concentrations of 600 nM, 100 nM, and 500 nM respectively, as indicated. DNA-protein complexes were incubated at room temperature before digestion with DNase I. 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 the H-NS + SlyA + PhoP-P reactions, representing the difference between the H-NS control and the experimental sample (b). The relative distance in base pairs to the TSS is indicated on the horizontal axis. Peaks indicate regions of hypersensitivity, typical of bent or distorted DNA, whereas valleys indicate protected regions, typical of protein binding sites. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See Supplementary Fig. 5 for representative raw chromatograms.

Mentions: DNase I footprinting shows that H-NS binds extensively to the promoter region under silencing conditions (-117 to +16) (Fig. 4 and Supplementary Fig. 8). PhoP binds to a discrete site located between positions −83 and −60, resulting in two hypersensitive sites at −70 and −68. Increases in sensitivity to DNase I-mediated cleavage are typically the result of a distortion or bend in the DNA duplex, resulting in the extrusion and increased digestion of individual bases, suggesting that PhoP induces the formation of a bend at this site. SlyA binds extensively across the pagC promoter region, (protected sites at −117, −104, −83, −35, and −11), as well as downstream of the H-NS bound region at +59. SlyA also induces a modest change in sensitivity near position −83, which overlaps the promoter-distal half of the PhoP binding site. Increases in sensitivity are observed at −38 and +35, indicating that SlyA binding also bends or distorts the DNA duplex. In the presence of H-NS, neither SlyA nor PhoP-P was observed to bind upstream of the promoter in the H-NS-bound region, although SlyA binding was observed downstream of the promoter at +59. However, under counter-silencing conditions SlyA and PhoP-P act cooperatively to form the PhoP-P-induced bend at −70 and −68, with additional sensitive sites at −49 and −38. Strikingly, most H-NS was not displaced despite pronounced changes in the structure of the nucleoprotein complex.


Evolutionary expansion of a regulatory network by counter-silencing.

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

DNase I DDFA of the pagC promoter regionIn vitro DNase I footprinting studies were performed on the pagC promoter region with H-NS, SlyA, and PhoP-P, at concentrations of 600 nM, 100 nM, and 500 nM respectively, as indicated. DNA-protein complexes were incubated at room temperature before digestion with DNase I. 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 the H-NS + SlyA + PhoP-P reactions, representing the difference between the H-NS control and the experimental sample (b). The relative distance in base pairs to the TSS is indicated on the horizontal axis. Peaks indicate regions of hypersensitivity, typical of bent or distorted DNA, whereas valleys indicate protected regions, typical of protein binding sites. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See Supplementary Fig. 5 for representative raw chromatograms.
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Related In: Results  -  Collection

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Figure 4: DNase I DDFA of the pagC promoter regionIn vitro DNase I footprinting studies were performed on the pagC promoter region with H-NS, SlyA, and PhoP-P, at concentrations of 600 nM, 100 nM, and 500 nM respectively, as indicated. DNA-protein complexes were incubated at room temperature before digestion with DNase I. 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 the H-NS + SlyA + PhoP-P reactions, representing the difference between the H-NS control and the experimental sample (b). The relative distance in base pairs to the TSS is indicated on the horizontal axis. Peaks indicate regions of hypersensitivity, typical of bent or distorted DNA, whereas valleys indicate protected regions, typical of protein binding sites. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See Supplementary Fig. 5 for representative raw chromatograms.
Mentions: DNase I footprinting shows that H-NS binds extensively to the promoter region under silencing conditions (-117 to +16) (Fig. 4 and Supplementary Fig. 8). PhoP binds to a discrete site located between positions −83 and −60, resulting in two hypersensitive sites at −70 and −68. Increases in sensitivity to DNase I-mediated cleavage are typically the result of a distortion or bend in the DNA duplex, resulting in the extrusion and increased digestion of individual bases, suggesting that PhoP induces the formation of a bend at this site. SlyA binds extensively across the pagC promoter region, (protected sites at −117, −104, −83, −35, and −11), as well as downstream of the H-NS bound region at +59. SlyA also induces a modest change in sensitivity near position −83, which overlaps the promoter-distal half of the PhoP binding site. Increases in sensitivity are observed at −38 and +35, indicating that SlyA binding also bends or distorts the DNA duplex. In the presence of H-NS, neither SlyA nor PhoP-P was observed to bind upstream of the promoter in the H-NS-bound region, although SlyA binding was observed downstream of the promoter at +59. However, under counter-silencing conditions SlyA and PhoP-P act cooperatively to form the PhoP-P-induced bend at −70 and −68, with additional sensitive sites at −49 and −38. Strikingly, most H-NS was not displaced despite pronounced changes in the structure of the nucleoprotein complex.

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