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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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Long-term culturing demonstrates the metastability of the ON state in LB.Seven independent lineages of phoQ (T281R) OFF and phoQ (T281R) ON were established and maintained as depicted in detail in Figure S4. OFF and ON colonies were inoculated in LB and grown for 24 hours to generate a Day 1 culture. For each lineage, the Day 1 culture was diluted 106-fold to generate a corresponding Day 2 culture, and this procedure was repeated for a total of 5 days. Day 1, 3, and 5 cultures were also diluted and plated in duplicate on minimal medium plates. The mean percentage of YFP-bright colonies on these plates was plotted in maroon (phoQ (T281R) OFF) or blue (phoQ (T281R) ON) with different symbols representing independent lineages. Representative merged images of Day 3 plates are shown on the right. These were constructed by merging the background-subtracted CFP image with its corresponding background-subtracted YFP image as the red and green channels respectively.
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pgen-1003706-g005: Long-term culturing demonstrates the metastability of the ON state in LB.Seven independent lineages of phoQ (T281R) OFF and phoQ (T281R) ON were established and maintained as depicted in detail in Figure S4. OFF and ON colonies were inoculated in LB and grown for 24 hours to generate a Day 1 culture. For each lineage, the Day 1 culture was diluted 106-fold to generate a corresponding Day 2 culture, and this procedure was repeated for a total of 5 days. Day 1, 3, and 5 cultures were also diluted and plated in duplicate on minimal medium plates. The mean percentage of YFP-bright colonies on these plates was plotted in maroon (phoQ (T281R) OFF) or blue (phoQ (T281R) ON) with different symbols representing independent lineages. Representative merged images of Day 3 plates are shown on the right. These were constructed by merging the background-subtracted CFP image with its corresponding background-subtracted YFP image as the red and green channels respectively.

Mentions: As expected, lineages inoculated with phoQ (T281R) OFF yielded mostly OFF colonies on the assay plates (Figure 5). Lineages inoculated with ON colonies, however, showed a different pattern. These remained close to 100% ON for several days (Figure 5, see Day 3 time point), but on Day 5, an appreciable fraction of YFP-dim colonies could be seen in 6 out of 7 lineages. Furthermore, the fraction of YFP-dim colonies obtained in the different lineages was not the same. This divergence highlights both the stochastic nature and the low probability of ON→OFF switching. Since all the ON lineages are likely to converge to a mostly OFF state eventually, the ON lineage in LB is metastable – a long-lived, but not truly stable state due to the competitive advantage of the OFF state. We note that the ON→OFF transitions seen in the ON lineages are unlikely to be the result of mutational events since the YFP-dim colonies obtained in these lineages could be primed ON by overnight growth in minimal medium with 10 mM Mg2+ (1 dim colony was tested per lineage, 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)

Long-term culturing demonstrates the metastability of the ON state in LB.Seven independent lineages of phoQ (T281R) OFF and phoQ (T281R) ON were established and maintained as depicted in detail in Figure S4. OFF and ON colonies were inoculated in LB and grown for 24 hours to generate a Day 1 culture. For each lineage, the Day 1 culture was diluted 106-fold to generate a corresponding Day 2 culture, and this procedure was repeated for a total of 5 days. Day 1, 3, and 5 cultures were also diluted and plated in duplicate on minimal medium plates. The mean percentage of YFP-bright colonies on these plates was plotted in maroon (phoQ (T281R) OFF) or blue (phoQ (T281R) ON) with different symbols representing independent lineages. Representative merged images of Day 3 plates are shown on the right. These were constructed by merging the background-subtracted CFP image with its corresponding background-subtracted YFP image as the red and green channels respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003706-g005: Long-term culturing demonstrates the metastability of the ON state in LB.Seven independent lineages of phoQ (T281R) OFF and phoQ (T281R) ON were established and maintained as depicted in detail in Figure S4. OFF and ON colonies were inoculated in LB and grown for 24 hours to generate a Day 1 culture. For each lineage, the Day 1 culture was diluted 106-fold to generate a corresponding Day 2 culture, and this procedure was repeated for a total of 5 days. Day 1, 3, and 5 cultures were also diluted and plated in duplicate on minimal medium plates. The mean percentage of YFP-bright colonies on these plates was plotted in maroon (phoQ (T281R) OFF) or blue (phoQ (T281R) ON) with different symbols representing independent lineages. Representative merged images of Day 3 plates are shown on the right. These were constructed by merging the background-subtracted CFP image with its corresponding background-subtracted YFP image as the red and green channels respectively.
Mentions: As expected, lineages inoculated with phoQ (T281R) OFF yielded mostly OFF colonies on the assay plates (Figure 5). Lineages inoculated with ON colonies, however, showed a different pattern. These remained close to 100% ON for several days (Figure 5, see Day 3 time point), but on Day 5, an appreciable fraction of YFP-dim colonies could be seen in 6 out of 7 lineages. Furthermore, the fraction of YFP-dim colonies obtained in the different lineages was not the same. This divergence highlights both the stochastic nature and the low probability of ON→OFF switching. Since all the ON lineages are likely to converge to a mostly OFF state eventually, the ON lineage in LB is metastable – a long-lived, but not truly stable state due to the competitive advantage of the OFF state. We note that the ON→OFF transitions seen in the ON lineages are unlikely to be the result of mutational events since the YFP-dim colonies obtained in these lineages could be primed ON by overnight growth in minimal medium with 10 mM Mg2+ (1 dim colony was tested per lineage, 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