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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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Architectural features of the PhoQ/PhoP network that govern bistability and priming.(A and B) Decoupling of phoQ (T281R) expression from PhoP-P control results in loss of bistability. Panel A is a schematic of the decoupled phoQ (T281R) strain. phoP is driven by its native promoters: the constitutive P2 and the PhoP-P responsive P1 promoters, whereas expression of phoQ (T281R) is governed by the PhoP-P independent, IPTG-inducible Ptrc promoter. Uninduced and fully induced cultures of the decoupled strain were washed twice in minimal medium without IPTG, diluted 105-fold into Minimal A medium containing 100 µM Mg2+ with indicated IPTG concentrations, grown to mid-exponential phase and imaged under a microscope (Methods). YFP/CFP distributions at various induction levels are shown in panel B. For each sample, the median value of YFP/CFP is represented as a horizontal line. Samples derived from uninduced and fully induced cultures are plotted in maroon and blue respectively. Inset shows the median YFP/CFP value as a function of IPTG concentration for samples derived from the induced (blue) and uninduced (maroon) cultures. (C and D) Deletion of the constitutive P2 promoter of the phoPQ operon reduces priming. Panel C is a schematic of the ΔP2 strain in which the constitutive P2 promoter of the phoPQ operon had been deleted. Overnight cultures of phoQ (T281R) OFF, ΔP2 OFF, and ΔP2 ON strains in indicated medium at 37°C were diluted 1000-fold into Minimal A medium with 100 µM Mg2+, grown to mid-exponential phase and imaged under a microscope (Methods). The percentage of ON cells in the images is plotted in panel D. Error bars indicate half the range of two independent experiments. The range is less than 0.5% in the instances where error bars are not visible.
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pgen-1003706-g006: Architectural features of the PhoQ/PhoP network that govern bistability and priming.(A and B) Decoupling of phoQ (T281R) expression from PhoP-P control results in loss of bistability. Panel A is a schematic of the decoupled phoQ (T281R) strain. phoP is driven by its native promoters: the constitutive P2 and the PhoP-P responsive P1 promoters, whereas expression of phoQ (T281R) is governed by the PhoP-P independent, IPTG-inducible Ptrc promoter. Uninduced and fully induced cultures of the decoupled strain were washed twice in minimal medium without IPTG, diluted 105-fold into Minimal A medium containing 100 µM Mg2+ with indicated IPTG concentrations, grown to mid-exponential phase and imaged under a microscope (Methods). YFP/CFP distributions at various induction levels are shown in panel B. For each sample, the median value of YFP/CFP is represented as a horizontal line. Samples derived from uninduced and fully induced cultures are plotted in maroon and blue respectively. Inset shows the median YFP/CFP value as a function of IPTG concentration for samples derived from the induced (blue) and uninduced (maroon) cultures. (C and D) Deletion of the constitutive P2 promoter of the phoPQ operon reduces priming. Panel C is a schematic of the ΔP2 strain in which the constitutive P2 promoter of the phoPQ operon had been deleted. Overnight cultures of phoQ (T281R) OFF, ΔP2 OFF, and ΔP2 ON strains in indicated medium at 37°C were diluted 1000-fold into Minimal A medium with 100 µM Mg2+, grown to mid-exponential phase and imaged under a microscope (Methods). The percentage of ON cells in the images is plotted in panel D. Error bars indicate half the range of two independent experiments. The range is less than 0.5% in the instances where error bars are not visible.

Mentions: To test whether elimination of PhoP-P dependent transcription of phoQ (T281R) abrogated bistability, we constructed a strain in which phoQ was deleted from its native locus and phoQ (T281R) was inserted at a phage attachment site under the control of the IPTG-inducible (and PhoP-P insensitive) Ptrc promoter (Figure 6A). We measured YFP/CFP in individual cells after 10 hours of growth (∼15 generations) at various IPTG concentrations starting from uninduced and fully induced populations of cells (Figure 6B). We did not observe any evidence of hysteresis in this strain: cultures started with both fully induced and uninduced cells converge to similar distributions for all IPTG concentrations tested (Figure 6B). Furthermore, distributions spanning intermediate values of YFP/CFP could be observed even after 15 generations of culture (i.e. populations did not converge to distributions with low and high modes). Taken together, these observations suggest that there is no bistability in the decoupled strain. The wide distributions seen at 12.5 µM and 25 µM IPTG can be attributed to a combination of noise in IPTG induction [23], [24] and the sensitivity of the system to induction level in this range (Figure 6B inset) and are also seen in our stochastic simulations (Figure S5D). The slightly lower median of samples derived from the uninduced culture compared with the induced culture could be a result of a stochastic effect that slows down induction kinetics in autoregulated systems [16]. We also determined that a strain similar to the one depicted in Figure 6A, but with phoQ (T281R) under the control of its native phoPQ promoter instead of Ptrc exhibited bistability (data not shown).


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)

Architectural features of the PhoQ/PhoP network that govern bistability and priming.(A and B) Decoupling of phoQ (T281R) expression from PhoP-P control results in loss of bistability. Panel A is a schematic of the decoupled phoQ (T281R) strain. phoP is driven by its native promoters: the constitutive P2 and the PhoP-P responsive P1 promoters, whereas expression of phoQ (T281R) is governed by the PhoP-P independent, IPTG-inducible Ptrc promoter. Uninduced and fully induced cultures of the decoupled strain were washed twice in minimal medium without IPTG, diluted 105-fold into Minimal A medium containing 100 µM Mg2+ with indicated IPTG concentrations, grown to mid-exponential phase and imaged under a microscope (Methods). YFP/CFP distributions at various induction levels are shown in panel B. For each sample, the median value of YFP/CFP is represented as a horizontal line. Samples derived from uninduced and fully induced cultures are plotted in maroon and blue respectively. Inset shows the median YFP/CFP value as a function of IPTG concentration for samples derived from the induced (blue) and uninduced (maroon) cultures. (C and D) Deletion of the constitutive P2 promoter of the phoPQ operon reduces priming. Panel C is a schematic of the ΔP2 strain in which the constitutive P2 promoter of the phoPQ operon had been deleted. Overnight cultures of phoQ (T281R) OFF, ΔP2 OFF, and ΔP2 ON strains in indicated medium at 37°C were diluted 1000-fold into Minimal A medium with 100 µM Mg2+, grown to mid-exponential phase and imaged under a microscope (Methods). The percentage of ON cells in the images is plotted in panel D. Error bars indicate half the range of two independent experiments. The range is less than 0.5% in the instances where error bars are not visible.
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pgen-1003706-g006: Architectural features of the PhoQ/PhoP network that govern bistability and priming.(A and B) Decoupling of phoQ (T281R) expression from PhoP-P control results in loss of bistability. Panel A is a schematic of the decoupled phoQ (T281R) strain. phoP is driven by its native promoters: the constitutive P2 and the PhoP-P responsive P1 promoters, whereas expression of phoQ (T281R) is governed by the PhoP-P independent, IPTG-inducible Ptrc promoter. Uninduced and fully induced cultures of the decoupled strain were washed twice in minimal medium without IPTG, diluted 105-fold into Minimal A medium containing 100 µM Mg2+ with indicated IPTG concentrations, grown to mid-exponential phase and imaged under a microscope (Methods). YFP/CFP distributions at various induction levels are shown in panel B. For each sample, the median value of YFP/CFP is represented as a horizontal line. Samples derived from uninduced and fully induced cultures are plotted in maroon and blue respectively. Inset shows the median YFP/CFP value as a function of IPTG concentration for samples derived from the induced (blue) and uninduced (maroon) cultures. (C and D) Deletion of the constitutive P2 promoter of the phoPQ operon reduces priming. Panel C is a schematic of the ΔP2 strain in which the constitutive P2 promoter of the phoPQ operon had been deleted. Overnight cultures of phoQ (T281R) OFF, ΔP2 OFF, and ΔP2 ON strains in indicated medium at 37°C were diluted 1000-fold into Minimal A medium with 100 µM Mg2+, grown to mid-exponential phase and imaged under a microscope (Methods). The percentage of ON cells in the images is plotted in panel D. Error bars indicate half the range of two independent experiments. The range is less than 0.5% in the instances where error bars are not visible.
Mentions: To test whether elimination of PhoP-P dependent transcription of phoQ (T281R) abrogated bistability, we constructed a strain in which phoQ was deleted from its native locus and phoQ (T281R) was inserted at a phage attachment site under the control of the IPTG-inducible (and PhoP-P insensitive) Ptrc promoter (Figure 6A). We measured YFP/CFP in individual cells after 10 hours of growth (∼15 generations) at various IPTG concentrations starting from uninduced and fully induced populations of cells (Figure 6B). We did not observe any evidence of hysteresis in this strain: cultures started with both fully induced and uninduced cells converge to similar distributions for all IPTG concentrations tested (Figure 6B). Furthermore, distributions spanning intermediate values of YFP/CFP could be observed even after 15 generations of culture (i.e. populations did not converge to distributions with low and high modes). Taken together, these observations suggest that there is no bistability in the decoupled strain. The wide distributions seen at 12.5 µM and 25 µM IPTG can be attributed to a combination of noise in IPTG induction [23], [24] and the sensitivity of the system to induction level in this range (Figure 6B inset) and are also seen in our stochastic simulations (Figure S5D). The slightly lower median of samples derived from the uninduced culture compared with the induced culture could be a result of a stochastic effect that slows down induction kinetics in autoregulated systems [16]. We also determined that a strain similar to the one depicted in Figure 6A, but with phoQ (T281R) under the control of its native phoPQ promoter instead of Ptrc exhibited bistability (data not shown).

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