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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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SPI1 gene expression is hierarchical and exhibits a switch-like transition from the “off” to the “on” state.(A) Diagram of the SPI1 gene circuit. HilA is the master SPI1 regulator as it activates the expression of the genes encoding the T3SS. HilA, in turn, is regulated by HilC, HilD, and RtsA. These three regulators can independently activate HilA expression. They can also activate their own expression and that of each other's. HilE represses the activity of HilD by binding to it and preventing it from activating its target promoters. (B) Time-course dynamics of PhilD (pSS074), PhilC (pSS075), PrtsA (pSS076), and PhilA (pSS077) promoter activities in wild-type cells as determined using luciferase transcriptional reporters. To induce SPI1 gene expression, cells were first grown overnight in LB/no salt and then sub-cultured into fresh LB/1% NaCl conditions to an OD of 0.05 and grown statically. Luminescence values were normalized with the OD600 absorbance to account for cell density. Average promoter activities from three independent experiments on separate days are reported. For each experiment, six samples were tested. Error-bars indicate standard deviation. (C) Dynamics of PhilA (pSS055) promoter activity in wild-type cells as determined using green fluorescent protein (GFP) transcriptional fusions and flow cytometry. The SPI1 gene expression was induced as described above. Samples were collected at the indicated times and arrested in their respective state by adding chloramphenicol. Approximately 30,000 cell measurements were used to construct each histogram. As a control, we expressed GFP from a constitutive promoter and observed continuous, rheostatic-like expression dynamics and a homogenous response in the population (Figure S1E). Strain genotypes and plasmid descriptions are provided in Tables 1 and 2.
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ppat-1001025-g001: SPI1 gene expression is hierarchical and exhibits a switch-like transition from the “off” to the “on” state.(A) Diagram of the SPI1 gene circuit. HilA is the master SPI1 regulator as it activates the expression of the genes encoding the T3SS. HilA, in turn, is regulated by HilC, HilD, and RtsA. These three regulators can independently activate HilA expression. They can also activate their own expression and that of each other's. HilE represses the activity of HilD by binding to it and preventing it from activating its target promoters. (B) Time-course dynamics of PhilD (pSS074), PhilC (pSS075), PrtsA (pSS076), and PhilA (pSS077) promoter activities in wild-type cells as determined using luciferase transcriptional reporters. To induce SPI1 gene expression, cells were first grown overnight in LB/no salt and then sub-cultured into fresh LB/1% NaCl conditions to an OD of 0.05 and grown statically. Luminescence values were normalized with the OD600 absorbance to account for cell density. Average promoter activities from three independent experiments on separate days are reported. For each experiment, six samples were tested. Error-bars indicate standard deviation. (C) Dynamics of PhilA (pSS055) promoter activity in wild-type cells as determined using green fluorescent protein (GFP) transcriptional fusions and flow cytometry. The SPI1 gene expression was induced as described above. Samples were collected at the indicated times and arrested in their respective state by adding chloramphenicol. Approximately 30,000 cell measurements were used to construct each histogram. As a control, we expressed GFP from a constitutive promoter and observed continuous, rheostatic-like expression dynamics and a homogenous response in the population (Figure S1E). Strain genotypes and plasmid descriptions are provided in Tables 1 and 2.

Mentions: The master regulator for the SPI1 gene circuit is HilA, a transcription factor that contains a DNA-binding motif belonging to the OmpR/ToxR family [4] and a large C-terminal domain of unknown function [25]. HilA activates the expression of the genes encoding the structural components of the SPI1 T3SS [4], [26], [27], [28]. HilA also activates the expression of an AraC-like transcription factor, InvF, involved in regulating the expression of the SPI1 secreted effector proteins and their cognate chaperones [29], [30]. HilA expression, in turn, is regulated by three AraC-like transcription factors - HilC, HilD, and RtsA – with homologous DNA binding domains [22], [31], [32]. Both hilC and hilD are encoded within SPI1 whereas rtsA is encoded elsewhere on the chromosome. These three transcription factors can independently activate HilA expression. They can also activate each others' and their own expression [16]. Specifically, HilC, HilD, and RtsA are all capable of individually activating the PhilA PhilC, PhilD, and PrtsA promoters. These auto-regulatory interactions result in three coupled positive feedback loops comprising HilC, HilD, and RtsA, the output of each capable of activating HilA expression (Figure 1A). Of the three, HilD is dominant, as there is no HilA expression in its absence [33]. This reflects the fact that many activating signals, both environmental and intracellular, affect SPI1 gene expression by modifying the activity of HilD protein [16], [18], [19], [23], [34], [35]. In addition to positive regulation, SPI1 gene expression is also subject to negative regulation. HilE, a protein of unknown structure encoded outside SPI1, binds HilD [34] and prevents it from activating its target promoters.


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)

SPI1 gene expression is hierarchical and exhibits a switch-like transition from the “off” to the “on” state.(A) Diagram of the SPI1 gene circuit. HilA is the master SPI1 regulator as it activates the expression of the genes encoding the T3SS. HilA, in turn, is regulated by HilC, HilD, and RtsA. These three regulators can independently activate HilA expression. They can also activate their own expression and that of each other's. HilE represses the activity of HilD by binding to it and preventing it from activating its target promoters. (B) Time-course dynamics of PhilD (pSS074), PhilC (pSS075), PrtsA (pSS076), and PhilA (pSS077) promoter activities in wild-type cells as determined using luciferase transcriptional reporters. To induce SPI1 gene expression, cells were first grown overnight in LB/no salt and then sub-cultured into fresh LB/1% NaCl conditions to an OD of 0.05 and grown statically. Luminescence values were normalized with the OD600 absorbance to account for cell density. Average promoter activities from three independent experiments on separate days are reported. For each experiment, six samples were tested. Error-bars indicate standard deviation. (C) Dynamics of PhilA (pSS055) promoter activity in wild-type cells as determined using green fluorescent protein (GFP) transcriptional fusions and flow cytometry. The SPI1 gene expression was induced as described above. Samples were collected at the indicated times and arrested in their respective state by adding chloramphenicol. Approximately 30,000 cell measurements were used to construct each histogram. As a control, we expressed GFP from a constitutive promoter and observed continuous, rheostatic-like expression dynamics and a homogenous response in the population (Figure S1E). Strain genotypes and plasmid descriptions are provided in Tables 1 and 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2912647&req=5

ppat-1001025-g001: SPI1 gene expression is hierarchical and exhibits a switch-like transition from the “off” to the “on” state.(A) Diagram of the SPI1 gene circuit. HilA is the master SPI1 regulator as it activates the expression of the genes encoding the T3SS. HilA, in turn, is regulated by HilC, HilD, and RtsA. These three regulators can independently activate HilA expression. They can also activate their own expression and that of each other's. HilE represses the activity of HilD by binding to it and preventing it from activating its target promoters. (B) Time-course dynamics of PhilD (pSS074), PhilC (pSS075), PrtsA (pSS076), and PhilA (pSS077) promoter activities in wild-type cells as determined using luciferase transcriptional reporters. To induce SPI1 gene expression, cells were first grown overnight in LB/no salt and then sub-cultured into fresh LB/1% NaCl conditions to an OD of 0.05 and grown statically. Luminescence values were normalized with the OD600 absorbance to account for cell density. Average promoter activities from three independent experiments on separate days are reported. For each experiment, six samples were tested. Error-bars indicate standard deviation. (C) Dynamics of PhilA (pSS055) promoter activity in wild-type cells as determined using green fluorescent protein (GFP) transcriptional fusions and flow cytometry. The SPI1 gene expression was induced as described above. Samples were collected at the indicated times and arrested in their respective state by adding chloramphenicol. Approximately 30,000 cell measurements were used to construct each histogram. As a control, we expressed GFP from a constitutive promoter and observed continuous, rheostatic-like expression dynamics and a homogenous response in the population (Figure S1E). Strain genotypes and plasmid descriptions are provided in Tables 1 and 2.
Mentions: The master regulator for the SPI1 gene circuit is HilA, a transcription factor that contains a DNA-binding motif belonging to the OmpR/ToxR family [4] and a large C-terminal domain of unknown function [25]. HilA activates the expression of the genes encoding the structural components of the SPI1 T3SS [4], [26], [27], [28]. HilA also activates the expression of an AraC-like transcription factor, InvF, involved in regulating the expression of the SPI1 secreted effector proteins and their cognate chaperones [29], [30]. HilA expression, in turn, is regulated by three AraC-like transcription factors - HilC, HilD, and RtsA – with homologous DNA binding domains [22], [31], [32]. Both hilC and hilD are encoded within SPI1 whereas rtsA is encoded elsewhere on the chromosome. These three transcription factors can independently activate HilA expression. They can also activate each others' and their own expression [16]. Specifically, HilC, HilD, and RtsA are all capable of individually activating the PhilA PhilC, PhilD, and PrtsA promoters. These auto-regulatory interactions result in three coupled positive feedback loops comprising HilC, HilD, and RtsA, the output of each capable of activating HilA expression (Figure 1A). Of the three, HilD is dominant, as there is no HilA expression in its absence [33]. This reflects the fact that many activating signals, both environmental and intracellular, affect SPI1 gene expression by modifying the activity of HilD protein [16], [18], [19], [23], [34], [35]. In addition to positive regulation, SPI1 gene expression is also subject to negative regulation. HilE, a protein of unknown structure encoded outside SPI1, binds HilD [34] and prevents it from activating its target promoters.

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