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The architecture of a prototypical bacterial signaling circuit enables a single point mutation to confer novel network properties.

Ram S, Goulian M - PLoS Genet. (2013)

Bottom Line: We describe a remarkable example of this versatility in the well-studied PhoQ/PhoP bacterial signaling network, which has an architecture found in many two-component systems.We found that a single point mutation that abolishes the phosphatase activity of the sensor kinase PhoQ results in a striking change in phenotype.Our results demonstrate the remarkable versatility of the prototypical two-component signaling architecture and highlight the tradeoffs in the particular case of the PhoQ/PhoP system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Even a single mutation can cause a marked change in a protein's properties. When the mutant protein functions within a network, complex phenotypes may emerge that are not intrinsic properties of the protein itself. Network architectures that enable such dramatic changes in function from a few mutations remain relatively uncharacterized. We describe a remarkable example of this versatility in the well-studied PhoQ/PhoP bacterial signaling network, which has an architecture found in many two-component systems. We found that a single point mutation that abolishes the phosphatase activity of the sensor kinase PhoQ results in a striking change in phenotype. The mutant responds to stimulus in a bistable manner, as opposed to the wild-type, which has a graded response. Mutant cells in on and off states have different morphologies, and their state is inherited over many generations. Interestingly, external conditions that repress signaling in the wild-type drive the mutant to the on state. Mathematical modeling and experiments suggest that the bistability depends on positive autoregulation of the two key proteins in the circuit, PhoP and PhoQ. The qualitatively different characteristics of the mutant come at a substantial fitness cost. Relative to the off state, the on state has a lower fitness in stationary phase cultures in rich medium (LB). However, due to the high inheritance of the on state, a population of on cells can be epigenetically trapped in a low-fitness state. Our results demonstrate the remarkable versatility of the prototypical two-component signaling architecture and highlight the tradeoffs in the particular case of the PhoQ/PhoP system.

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Phenotypic bimodality and hysteresis in the phoQ (T281R) mutant.(A) Schematic of the PhoQ/PhoP circuit. The phoPQ operon in transcribed and translated to produce PhoP and PhoQ proteins (cyan arrows). Post-tranlational interactions between PhoQ and PhoP generate PhoP-P, which upregulates the expression of the phoPQ operon and the yfp reporter (solid black arrows). The PhoQ phosphatase activity, which is absent in PhoQ (T281R), is depicted with a gray arrow. (B) Bimodal behavior of YFP colony fluorescence. Panel shows CFP and YFP channel images of a minimal medium plate on which an LB overnight culture of the phoQ (T281R) strain was spread. (C and D) Phenotypic hysteresis. Overnight cultures of phoQ (WT), phoQ (T281R) OFF, and phoQ (T281R) ON strains in LB were diluted 1000-fold into Minimal A medium with 100 µM Mg2, grown to mid-exponential phase and imaged by fluorescence microscopy (Methods). The PhoP-P state of the cell was determined by measuring YFP expression driven by the PhoP-P responsive mgrB promoter and normalized to CFP expression driven by a constitutive promoter. Cell widths were quantified by fitting phase image masks to ellipses and computing the minor axis length. Representative phase images of the three strains are shown in panel C and reveal morphological differences between OFF and ON cells. Fluorescence and cell width values for each cell are plotted in panel D with indicated colors distinguishing the three strains. Note that phoQ (T281R) OFF cells (maroon) have much lower fluorescence and are wider than phoQ (T281R) ON cells (blue).
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pgen-1003706-g001: Phenotypic bimodality and hysteresis in the phoQ (T281R) mutant.(A) Schematic of the PhoQ/PhoP circuit. The phoPQ operon in transcribed and translated to produce PhoP and PhoQ proteins (cyan arrows). Post-tranlational interactions between PhoQ and PhoP generate PhoP-P, which upregulates the expression of the phoPQ operon and the yfp reporter (solid black arrows). The PhoQ phosphatase activity, which is absent in PhoQ (T281R), is depicted with a gray arrow. (B) Bimodal behavior of YFP colony fluorescence. Panel shows CFP and YFP channel images of a minimal medium plate on which an LB overnight culture of the phoQ (T281R) strain was spread. (C and D) Phenotypic hysteresis. Overnight cultures of phoQ (WT), phoQ (T281R) OFF, and phoQ (T281R) ON strains in LB were diluted 1000-fold into Minimal A medium with 100 µM Mg2, grown to mid-exponential phase and imaged by fluorescence microscopy (Methods). The PhoP-P state of the cell was determined by measuring YFP expression driven by the PhoP-P responsive mgrB promoter and normalized to CFP expression driven by a constitutive promoter. Cell widths were quantified by fitting phase image masks to ellipses and computing the minor axis length. Representative phase images of the three strains are shown in panel C and reveal morphological differences between OFF and ON cells. Fluorescence and cell width values for each cell are plotted in panel D with indicated colors distinguishing the three strains. Note that phoQ (T281R) OFF cells (maroon) have much lower fluorescence and are wider than phoQ (T281R) ON cells (blue).

Mentions: A previous study reported that the phoQ (T281R) mutation results in a broad distribution of PhoP-regulated transcription in a population of E. coli cells [13]. To explore the origin of this heterogeneity, we engineered a phoQ (T281R) strain with the mutation at the native phoPphoQ locus. The strain also contained a PhoP-P responsive promoter controlling yfp transcription and a constitutive promoter controlling cfp, allowing the use of YFP fluorescence to infer PhoP-P levels (Figure 1A and Methods). Two fluorescent colony phenotypes could be discerned on agar plates: YFP-dim, which we designated phoQ (T281R) OFF, and YFP-bright, which we designated phoQ (T281R) ON (Figure 1B). Considering that each colony is composed of hundreds of millions of cells that originated from a single cell, the appearance of two distinct colony phenotypes suggests that individual cells have two phenotypic states and that these states are heritable.


The architecture of a prototypical bacterial signaling circuit enables a single point mutation to confer novel network properties.

Ram S, Goulian M - PLoS Genet. (2013)

Phenotypic bimodality and hysteresis in the phoQ (T281R) mutant.(A) Schematic of the PhoQ/PhoP circuit. The phoPQ operon in transcribed and translated to produce PhoP and PhoQ proteins (cyan arrows). Post-tranlational interactions between PhoQ and PhoP generate PhoP-P, which upregulates the expression of the phoPQ operon and the yfp reporter (solid black arrows). The PhoQ phosphatase activity, which is absent in PhoQ (T281R), is depicted with a gray arrow. (B) Bimodal behavior of YFP colony fluorescence. Panel shows CFP and YFP channel images of a minimal medium plate on which an LB overnight culture of the phoQ (T281R) strain was spread. (C and D) Phenotypic hysteresis. Overnight cultures of phoQ (WT), phoQ (T281R) OFF, and phoQ (T281R) ON strains in LB were diluted 1000-fold into Minimal A medium with 100 µM Mg2, grown to mid-exponential phase and imaged by fluorescence microscopy (Methods). The PhoP-P state of the cell was determined by measuring YFP expression driven by the PhoP-P responsive mgrB promoter and normalized to CFP expression driven by a constitutive promoter. Cell widths were quantified by fitting phase image masks to ellipses and computing the minor axis length. Representative phase images of the three strains are shown in panel C and reveal morphological differences between OFF and ON cells. Fluorescence and cell width values for each cell are plotted in panel D with indicated colors distinguishing the three strains. Note that phoQ (T281R) OFF cells (maroon) have much lower fluorescence and are wider than phoQ (T281R) ON cells (blue).
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pgen-1003706-g001: Phenotypic bimodality and hysteresis in the phoQ (T281R) mutant.(A) Schematic of the PhoQ/PhoP circuit. The phoPQ operon in transcribed and translated to produce PhoP and PhoQ proteins (cyan arrows). Post-tranlational interactions between PhoQ and PhoP generate PhoP-P, which upregulates the expression of the phoPQ operon and the yfp reporter (solid black arrows). The PhoQ phosphatase activity, which is absent in PhoQ (T281R), is depicted with a gray arrow. (B) Bimodal behavior of YFP colony fluorescence. Panel shows CFP and YFP channel images of a minimal medium plate on which an LB overnight culture of the phoQ (T281R) strain was spread. (C and D) Phenotypic hysteresis. Overnight cultures of phoQ (WT), phoQ (T281R) OFF, and phoQ (T281R) ON strains in LB were diluted 1000-fold into Minimal A medium with 100 µM Mg2, grown to mid-exponential phase and imaged by fluorescence microscopy (Methods). The PhoP-P state of the cell was determined by measuring YFP expression driven by the PhoP-P responsive mgrB promoter and normalized to CFP expression driven by a constitutive promoter. Cell widths were quantified by fitting phase image masks to ellipses and computing the minor axis length. Representative phase images of the three strains are shown in panel C and reveal morphological differences between OFF and ON cells. Fluorescence and cell width values for each cell are plotted in panel D with indicated colors distinguishing the three strains. Note that phoQ (T281R) OFF cells (maroon) have much lower fluorescence and are wider than phoQ (T281R) ON cells (blue).
Mentions: A previous study reported that the phoQ (T281R) mutation results in a broad distribution of PhoP-regulated transcription in a population of E. coli cells [13]. To explore the origin of this heterogeneity, we engineered a phoQ (T281R) strain with the mutation at the native phoPphoQ locus. The strain also contained a PhoP-P responsive promoter controlling yfp transcription and a constitutive promoter controlling cfp, allowing the use of YFP fluorescence to infer PhoP-P levels (Figure 1A and Methods). Two fluorescent colony phenotypes could be discerned on agar plates: YFP-dim, which we designated phoQ (T281R) OFF, and YFP-bright, which we designated phoQ (T281R) ON (Figure 1B). Considering that each colony is composed of hundreds of millions of cells that originated from a single cell, the appearance of two distinct colony phenotypes suggests that individual cells have two phenotypic states and that these states are heritable.

Bottom Line: We describe a remarkable example of this versatility in the well-studied PhoQ/PhoP bacterial signaling network, which has an architecture found in many two-component systems.We found that a single point mutation that abolishes the phosphatase activity of the sensor kinase PhoQ results in a striking change in phenotype.Our results demonstrate the remarkable versatility of the prototypical two-component signaling architecture and highlight the tradeoffs in the particular case of the PhoQ/PhoP system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Even a single mutation can cause a marked change in a protein's properties. When the mutant protein functions within a network, complex phenotypes may emerge that are not intrinsic properties of the protein itself. Network architectures that enable such dramatic changes in function from a few mutations remain relatively uncharacterized. We describe a remarkable example of this versatility in the well-studied PhoQ/PhoP bacterial signaling network, which has an architecture found in many two-component systems. We found that a single point mutation that abolishes the phosphatase activity of the sensor kinase PhoQ results in a striking change in phenotype. The mutant responds to stimulus in a bistable manner, as opposed to the wild-type, which has a graded response. Mutant cells in on and off states have different morphologies, and their state is inherited over many generations. Interestingly, external conditions that repress signaling in the wild-type drive the mutant to the on state. Mathematical modeling and experiments suggest that the bistability depends on positive autoregulation of the two key proteins in the circuit, PhoP and PhoQ. The qualitatively different characteristics of the mutant come at a substantial fitness cost. Relative to the off state, the on state has a lower fitness in stationary phase cultures in rich medium (LB). However, due to the high inheritance of the on state, a population of on cells can be epigenetically trapped in a low-fitness state. Our results demonstrate the remarkable versatility of the prototypical two-component signaling architecture and highlight the tradeoffs in the particular case of the PhoQ/PhoP system.

Show MeSH
Related in: MedlinePlus