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The role of coupled positive feedback in the expression of the SPI1 type three secretion system in Salmonella.

Saini S, Ellermeier JR, Slauch JM, Rao CV - PLoS Pathog. (2010)

Bottom Line: While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown.This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal.Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

ABSTRACT
Salmonella enterica serovar Typhimurium is a common food-borne pathogen that induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). The genes encoding the SPI1 T3SS are tightly regulated by a network of interacting transcriptional regulators involving three coupled positive feedback loops. While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown. To determine the function of this circuit, we measured gene expression dynamics at both population and single-cell resolution in a number of SPI1 regulatory mutants. Using these data, we constructed a mathematical model of the SPI1 gene circuit. Analysis of the model predicted that the circuit serves two functions. The first is to place a threshold on SPI1 activation, ensuring that the genes encoding the T3SS are expressed only in response to the appropriate combination of environmental and cellular cues. The second is to amplify SPI1 gene expression. To experimentally test these predictions, we rewired the SPI1 genetic circuit by changing its regulatory architecture. This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal. Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics.

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HilC and RtsA amplify SPI1 gene expression.(A) Comparison of time-course dynamics for PhilD (pSS074, black) and PhilA (pSS077, red) promoter activities in wild type (solid lines) and a ΔhilC ΔrtsA mutant (CR350, dashed lines) as determined using luciferase transcriptional reporters. (B and C) Comparison of PhilA (pSS055, B) and PhilD (pSS072, C) promoter activities in wild type (black) and a ΔhilC ΔrtsA mutant (CR350, grey) as determined using GFP transcriptional reporters and flow cytometry. Note that the loss of HilC and RtsA causes both a delay and decrease in PhilA promoter activity whereas it causes only a decrease in activity in the case of the PhilD promoter. Experiments were performed as described in Figure 1.
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ppat-1001025-g003: HilC and RtsA amplify SPI1 gene expression.(A) Comparison of time-course dynamics for PhilD (pSS074, black) and PhilA (pSS077, red) promoter activities in wild type (solid lines) and a ΔhilC ΔrtsA mutant (CR350, dashed lines) as determined using luciferase transcriptional reporters. (B and C) Comparison of PhilA (pSS055, B) and PhilD (pSS072, C) promoter activities in wild type (black) and a ΔhilC ΔrtsA mutant (CR350, grey) as determined using GFP transcriptional reporters and flow cytometry. Note that the loss of HilC and RtsA causes both a delay and decrease in PhilA promoter activity whereas it causes only a decrease in activity in the case of the PhilD promoter. Experiments were performed as described in Figure 1.

Mentions: Unlike HilD, the HilC and RtsA proteins are not absolutely required for HilA expression. Yet, these two proteins can independently induce transcription from the PhilA promoter when constitutively expressed from an ectopic promoter [16]. To understand the role of these two proteins in regulating SPI1, we compared gene expression in wild type and a ΔhilC ΔrtsA mutant using the luciferase reporters (Figure 3A). Deleting these two regulators decreases the activity of the PhilD and PhilA promoters. Moreover, in the ΔhilC ΔrtsA mutant, there is also a delay in the induction of the PhilA promoter. This delay becomes more apparent when we normalize the luminescence measurements with respect to their maximal values (Figure S3A). When we measured gene expression at single-cell resolution using flow cytometry, we again observed a switch-like response in the ΔhilC ΔrtsA mutant (Figure 3B). The main difference relative to wild type was that the transition from the “off” to “on” state occurred more slowly in the absence of HilC and RtsA. Also, the activity of the PhilA promoter in the “on” state was lower in the ΔhilC ΔrtsA mutant than in wild type. With the PhilD promoter, we did not observe any change in the timing of promoter activation in the ΔhilC ΔrtsA mutant relative to wild type (Figure 3C and S3A). Rather, we observed only a decrease in the level of PhilD promoter activity associated with the “on” state. Similar results for both promoters are observed in the single deletion mutants, though the overall effect is small, indicating that HilC and RtsA additively contribute to SPI1 gene expression (Figure S3B–E). Based on these results, we conclude that HilC and RtsA serve two functions in the SPI1 circuit. First, HilC and RtsA amplify HilA and HilD expression, in the sense that HilA and HilD expression is reduced the absence of HilC and RtsA. Second, HilC and RtsA accelerate the transition of HilA expression from the “off” to the “on” state.


The role of coupled positive feedback in the expression of the SPI1 type three secretion system in Salmonella.

Saini S, Ellermeier JR, Slauch JM, Rao CV - PLoS Pathog. (2010)

HilC and RtsA amplify SPI1 gene expression.(A) Comparison of time-course dynamics for PhilD (pSS074, black) and PhilA (pSS077, red) promoter activities in wild type (solid lines) and a ΔhilC ΔrtsA mutant (CR350, dashed lines) as determined using luciferase transcriptional reporters. (B and C) Comparison of PhilA (pSS055, B) and PhilD (pSS072, C) promoter activities in wild type (black) and a ΔhilC ΔrtsA mutant (CR350, grey) as determined using GFP transcriptional reporters and flow cytometry. Note that the loss of HilC and RtsA causes both a delay and decrease in PhilA promoter activity whereas it causes only a decrease in activity in the case of the PhilD promoter. Experiments were performed as described in Figure 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2912647&req=5

ppat-1001025-g003: HilC and RtsA amplify SPI1 gene expression.(A) Comparison of time-course dynamics for PhilD (pSS074, black) and PhilA (pSS077, red) promoter activities in wild type (solid lines) and a ΔhilC ΔrtsA mutant (CR350, dashed lines) as determined using luciferase transcriptional reporters. (B and C) Comparison of PhilA (pSS055, B) and PhilD (pSS072, C) promoter activities in wild type (black) and a ΔhilC ΔrtsA mutant (CR350, grey) as determined using GFP transcriptional reporters and flow cytometry. Note that the loss of HilC and RtsA causes both a delay and decrease in PhilA promoter activity whereas it causes only a decrease in activity in the case of the PhilD promoter. Experiments were performed as described in Figure 1.
Mentions: Unlike HilD, the HilC and RtsA proteins are not absolutely required for HilA expression. Yet, these two proteins can independently induce transcription from the PhilA promoter when constitutively expressed from an ectopic promoter [16]. To understand the role of these two proteins in regulating SPI1, we compared gene expression in wild type and a ΔhilC ΔrtsA mutant using the luciferase reporters (Figure 3A). Deleting these two regulators decreases the activity of the PhilD and PhilA promoters. Moreover, in the ΔhilC ΔrtsA mutant, there is also a delay in the induction of the PhilA promoter. This delay becomes more apparent when we normalize the luminescence measurements with respect to their maximal values (Figure S3A). When we measured gene expression at single-cell resolution using flow cytometry, we again observed a switch-like response in the ΔhilC ΔrtsA mutant (Figure 3B). The main difference relative to wild type was that the transition from the “off” to “on” state occurred more slowly in the absence of HilC and RtsA. Also, the activity of the PhilA promoter in the “on” state was lower in the ΔhilC ΔrtsA mutant than in wild type. With the PhilD promoter, we did not observe any change in the timing of promoter activation in the ΔhilC ΔrtsA mutant relative to wild type (Figure 3C and S3A). Rather, we observed only a decrease in the level of PhilD promoter activity associated with the “on” state. Similar results for both promoters are observed in the single deletion mutants, though the overall effect is small, indicating that HilC and RtsA additively contribute to SPI1 gene expression (Figure S3B–E). Based on these results, we conclude that HilC and RtsA serve two functions in the SPI1 circuit. First, HilC and RtsA amplify HilA and HilD expression, in the sense that HilA and HilD expression is reduced the absence of HilC and RtsA. Second, HilC and RtsA accelerate the transition of HilA expression from the “off” to the “on” state.

Bottom Line: While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown.This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal.Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

ABSTRACT
Salmonella enterica serovar Typhimurium is a common food-borne pathogen that induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). The genes encoding the SPI1 T3SS are tightly regulated by a network of interacting transcriptional regulators involving three coupled positive feedback loops. While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown. To determine the function of this circuit, we measured gene expression dynamics at both population and single-cell resolution in a number of SPI1 regulatory mutants. Using these data, we constructed a mathematical model of the SPI1 gene circuit. Analysis of the model predicted that the circuit serves two functions. The first is to place a threshold on SPI1 activation, ensuring that the genes encoding the T3SS are expressed only in response to the appropriate combination of environmental and cellular cues. The second is to amplify SPI1 gene expression. To experimentally test these predictions, we rewired the SPI1 genetic circuit by changing its regulatory architecture. This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal. Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics.

Show MeSH
Related in: MedlinePlus