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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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Passage through stationary phase and High Mg2+ cause OFF cells to prime to ON state.Priming is defined as the deterministic conversion of OFF cells to the ON state. (A and B) Passage through stationary phase primes OFF cells in minimal medium. Starting from OFF cells, two lineages (STA and EXP) were established in minimal medium with various magnesium concentrations as shown schematically in panel A and in detail in Figure S2. Cells from these lineages were imaged under a microscope as described in Methods and the percentage of ON cells in the mid-exponential culture obtained at the end was plotted (panel B). (C and D) Overnight culture at slow growth rates and high Mg2+ results in priming. Overnight cultures inoculated with OFF colonies were set up in indicated conditions, diluted 1000-fold into Minimal A medium with 100 µM Mg2+, grown to mid-exponential phase at 37°C (even for 30°C overnight cultures) and imaged under a microscope (Methods). The percentage of ON cells present in the images of mid-exponential cultures is shown. Panel C shows the effect of increasing [Mg2+] in LB, while panel D documents the effect of changing growth rate in low [Mg2+] media. In panels B, C and 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-g002: Passage through stationary phase and High Mg2+ cause OFF cells to prime to ON state.Priming is defined as the deterministic conversion of OFF cells to the ON state. (A and B) Passage through stationary phase primes OFF cells in minimal medium. Starting from OFF cells, two lineages (STA and EXP) were established in minimal medium with various magnesium concentrations as shown schematically in panel A and in detail in Figure S2. Cells from these lineages were imaged under a microscope as described in Methods and the percentage of ON cells in the mid-exponential culture obtained at the end was plotted (panel B). (C and D) Overnight culture at slow growth rates and high Mg2+ results in priming. Overnight cultures inoculated with OFF colonies were set up in indicated conditions, diluted 1000-fold into Minimal A medium with 100 µM Mg2+, grown to mid-exponential phase at 37°C (even for 30°C overnight cultures) and imaged under a microscope (Methods). The percentage of ON cells present in the images of mid-exponential cultures is shown. Panel C shows the effect of increasing [Mg2+] in LB, while panel D documents the effect of changing growth rate in low [Mg2+] media. In panels B, C and 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 experimentally validate the insights from the simple model, we reasoned that changing [Mg2+] could be used to modulate the PhoP→PhoP-P flux (the kinase rate equivalent). Accordingly, we grew cultures of mostly OFF cells in minimal media with different [Mg2+] and maintained them exclusively in the exponential phase by serial dilution (growth rates were similar over the [Mg2+] range tested). Surprisingly, we found that high [Mg2+] (10 mM) drove all OFF cells to ON, whereas low [Mg2+] (100 µM or 1 mM) preserved the OFF state (EXP lineages, Figure 2A–B). This, in effect, represents a reversal of sensitivity to Mg2+ in the phoQ (T281R) strain as the wild-type strain is repressed (produces lower PhoP-P) by high Mg2+. If a portion of the same batch of OFF cells that was maintained in the exponential phase in the EXP lineages was instead passaged through stationary phase (effectively, a slow growth phase) and subsequently diluted and grown to mid-exponential phase, then even the low [Mg2+] lineages turned mostly ON (STA lineages, Figure 2A–B). Note that the different outcomes of STA and EXP lineages at low [Mg2+] represent yet another manifestation of hysteresis, since both lineages have identical starting points (same culture of OFF cells) and similar end points (mid-exponential cultures with the same [Mg2+]), but different history between start and end.


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)

Passage through stationary phase and High Mg2+ cause OFF cells to prime to ON state.Priming is defined as the deterministic conversion of OFF cells to the ON state. (A and B) Passage through stationary phase primes OFF cells in minimal medium. Starting from OFF cells, two lineages (STA and EXP) were established in minimal medium with various magnesium concentrations as shown schematically in panel A and in detail in Figure S2. Cells from these lineages were imaged under a microscope as described in Methods and the percentage of ON cells in the mid-exponential culture obtained at the end was plotted (panel B). (C and D) Overnight culture at slow growth rates and high Mg2+ results in priming. Overnight cultures inoculated with OFF colonies were set up in indicated conditions, diluted 1000-fold into Minimal A medium with 100 µM Mg2+, grown to mid-exponential phase at 37°C (even for 30°C overnight cultures) and imaged under a microscope (Methods). The percentage of ON cells present in the images of mid-exponential cultures is shown. Panel C shows the effect of increasing [Mg2+] in LB, while panel D documents the effect of changing growth rate in low [Mg2+] media. In panels B, C and 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750022&req=5

pgen-1003706-g002: Passage through stationary phase and High Mg2+ cause OFF cells to prime to ON state.Priming is defined as the deterministic conversion of OFF cells to the ON state. (A and B) Passage through stationary phase primes OFF cells in minimal medium. Starting from OFF cells, two lineages (STA and EXP) were established in minimal medium with various magnesium concentrations as shown schematically in panel A and in detail in Figure S2. Cells from these lineages were imaged under a microscope as described in Methods and the percentage of ON cells in the mid-exponential culture obtained at the end was plotted (panel B). (C and D) Overnight culture at slow growth rates and high Mg2+ results in priming. Overnight cultures inoculated with OFF colonies were set up in indicated conditions, diluted 1000-fold into Minimal A medium with 100 µM Mg2+, grown to mid-exponential phase at 37°C (even for 30°C overnight cultures) and imaged under a microscope (Methods). The percentage of ON cells present in the images of mid-exponential cultures is shown. Panel C shows the effect of increasing [Mg2+] in LB, while panel D documents the effect of changing growth rate in low [Mg2+] media. In panels B, C and 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 experimentally validate the insights from the simple model, we reasoned that changing [Mg2+] could be used to modulate the PhoP→PhoP-P flux (the kinase rate equivalent). Accordingly, we grew cultures of mostly OFF cells in minimal media with different [Mg2+] and maintained them exclusively in the exponential phase by serial dilution (growth rates were similar over the [Mg2+] range tested). Surprisingly, we found that high [Mg2+] (10 mM) drove all OFF cells to ON, whereas low [Mg2+] (100 µM or 1 mM) preserved the OFF state (EXP lineages, Figure 2A–B). This, in effect, represents a reversal of sensitivity to Mg2+ in the phoQ (T281R) strain as the wild-type strain is repressed (produces lower PhoP-P) by high Mg2+. If a portion of the same batch of OFF cells that was maintained in the exponential phase in the EXP lineages was instead passaged through stationary phase (effectively, a slow growth phase) and subsequently diluted and grown to mid-exponential phase, then even the low [Mg2+] lineages turned mostly ON (STA lineages, Figure 2A–B). Note that the different outcomes of STA and EXP lineages at low [Mg2+] represent yet another manifestation of hysteresis, since both lineages have identical starting points (same culture of OFF cells) and similar end points (mid-exponential cultures with the same [Mg2+]), but different history between start and end.

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