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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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ON cells have a competitive disadvantage in stationary phase in LB.Overnight cultures of chloramphenicol-sensitive (CmS) and chloramphenicol-resistant (CmR) variants of phoQ (T281R) OFF and phoQ (T281R) ON were set up independently in LB. For each competition experiment, 1 ml of CmS and CmR overnight cultures were mixed and co-cultured for an additional 10 hours in stationary phase. Initial and final total and CmR populations were quantified by plating appropriate dilutions on LB and LB+chloramphenicol plates (see Figure S3 for a detailed protocol). Colony counts were used to compute a competitive ratio (CR), defined as CR = (C(10)L(0))/(C(0)L(10)), where C(T) and L(T) denote the counts on chloramphenicol and LB plates at time T respectively. Note that CR is different from the competitive index (CI) that is frequently used to quantify the outcome of competition experiments (CI is the quotient of final and initial ratios of the population of the two competing strains). In our experimental design, CR has an upper bound of ∼2, whereas CI is, in principle, an unbounded quantity. In competitions between CmS ON and CmR OFF cells, the final CmS population is below the detection limit and CI cannot be computed. Symbols indicate CR values obtained from 4 independent competition experiments. The solid, black line represents the median. Dashed, black line indicates a neutral competitive ratio of 1. Note that a CR of 2 (dashed, maroon line) corresponds to a near-elimination of the CmS population.
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pgen-1003706-g004: ON cells have a competitive disadvantage in stationary phase in LB.Overnight cultures of chloramphenicol-sensitive (CmS) and chloramphenicol-resistant (CmR) variants of phoQ (T281R) OFF and phoQ (T281R) ON were set up independently in LB. For each competition experiment, 1 ml of CmS and CmR overnight cultures were mixed and co-cultured for an additional 10 hours in stationary phase. Initial and final total and CmR populations were quantified by plating appropriate dilutions on LB and LB+chloramphenicol plates (see Figure S3 for a detailed protocol). Colony counts were used to compute a competitive ratio (CR), defined as CR = (C(10)L(0))/(C(0)L(10)), where C(T) and L(T) denote the counts on chloramphenicol and LB plates at time T respectively. Note that CR is different from the competitive index (CI) that is frequently used to quantify the outcome of competition experiments (CI is the quotient of final and initial ratios of the population of the two competing strains). In our experimental design, CR has an upper bound of ∼2, whereas CI is, in principle, an unbounded quantity. In competitions between CmS ON and CmR OFF cells, the final CmS population is below the detection limit and CI cannot be computed. Symbols indicate CR values obtained from 4 independent competition experiments. The solid, black line represents the median. Dashed, black line indicates a neutral competitive ratio of 1. Note that a CR of 2 (dashed, maroon line) corresponds to a near-elimination of the CmS population.

Mentions: The high PhoP-P level in the ON state has pleiotropic effects on the cell. As presented in Figure 1C–D, cell morphology is affected in the ON state. We also determined that the ON state has a lower fitness in stationary phase in LB. To explore fitness differences between ON and OFF cells, we performed competitions using chloramphenicol resistant (CmR) and sensitive (CmS) phoQ(T281R) strains prepared in the ON and OFF states (Figure 4, S3 and Methods). When ON and OFF cells were competed for 10 hours in stationary phase in LB, the ON fraction in the population showed a significant decrease (competitive ratio, Figure 4, columns 2 and 4). In contrast, competitions between CmS and CmR OFF cells or between CmS and CmR ON cells yielded a near-neutral competitive ratio (Figure 4, columns 1 and 3 respectively).


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)

ON cells have a competitive disadvantage in stationary phase in LB.Overnight cultures of chloramphenicol-sensitive (CmS) and chloramphenicol-resistant (CmR) variants of phoQ (T281R) OFF and phoQ (T281R) ON were set up independently in LB. For each competition experiment, 1 ml of CmS and CmR overnight cultures were mixed and co-cultured for an additional 10 hours in stationary phase. Initial and final total and CmR populations were quantified by plating appropriate dilutions on LB and LB+chloramphenicol plates (see Figure S3 for a detailed protocol). Colony counts were used to compute a competitive ratio (CR), defined as CR = (C(10)L(0))/(C(0)L(10)), where C(T) and L(T) denote the counts on chloramphenicol and LB plates at time T respectively. Note that CR is different from the competitive index (CI) that is frequently used to quantify the outcome of competition experiments (CI is the quotient of final and initial ratios of the population of the two competing strains). In our experimental design, CR has an upper bound of ∼2, whereas CI is, in principle, an unbounded quantity. In competitions between CmS ON and CmR OFF cells, the final CmS population is below the detection limit and CI cannot be computed. Symbols indicate CR values obtained from 4 independent competition experiments. The solid, black line represents the median. Dashed, black line indicates a neutral competitive ratio of 1. Note that a CR of 2 (dashed, maroon line) corresponds to a near-elimination of the CmS population.
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Related In: Results  -  Collection

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

pgen-1003706-g004: ON cells have a competitive disadvantage in stationary phase in LB.Overnight cultures of chloramphenicol-sensitive (CmS) and chloramphenicol-resistant (CmR) variants of phoQ (T281R) OFF and phoQ (T281R) ON were set up independently in LB. For each competition experiment, 1 ml of CmS and CmR overnight cultures were mixed and co-cultured for an additional 10 hours in stationary phase. Initial and final total and CmR populations were quantified by plating appropriate dilutions on LB and LB+chloramphenicol plates (see Figure S3 for a detailed protocol). Colony counts were used to compute a competitive ratio (CR), defined as CR = (C(10)L(0))/(C(0)L(10)), where C(T) and L(T) denote the counts on chloramphenicol and LB plates at time T respectively. Note that CR is different from the competitive index (CI) that is frequently used to quantify the outcome of competition experiments (CI is the quotient of final and initial ratios of the population of the two competing strains). In our experimental design, CR has an upper bound of ∼2, whereas CI is, in principle, an unbounded quantity. In competitions between CmS ON and CmR OFF cells, the final CmS population is below the detection limit and CI cannot be computed. Symbols indicate CR values obtained from 4 independent competition experiments. The solid, black line represents the median. Dashed, black line indicates a neutral competitive ratio of 1. Note that a CR of 2 (dashed, maroon line) corresponds to a near-elimination of the CmS population.
Mentions: The high PhoP-P level in the ON state has pleiotropic effects on the cell. As presented in Figure 1C–D, cell morphology is affected in the ON state. We also determined that the ON state has a lower fitness in stationary phase in LB. To explore fitness differences between ON and OFF cells, we performed competitions using chloramphenicol resistant (CmR) and sensitive (CmS) phoQ(T281R) strains prepared in the ON and OFF states (Figure 4, S3 and Methods). When ON and OFF cells were competed for 10 hours in stationary phase in LB, the ON fraction in the population showed a significant decrease (competitive ratio, Figure 4, columns 2 and 4). In contrast, competitions between CmS and CmR OFF cells or between CmS and CmR ON cells yielded a near-neutral competitive ratio (Figure 4, columns 1 and 3 respectively).

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