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A Novel Feedback Loop That Controls Bimodal Expression of Genetic Competence.

Gamba P, Jonker MJ, Hamoen LW - PLoS Genet. (2015)

Bottom Line: The protein influences the expression of many genes, however, Kre is only found in bacteria that contain a ComK homologue and, importantly, kre expression itself is downregulated by ComK.The evolutionary significance of this new feedback loop for the reduction of transcriptional noise in comK expression is discussed.Our findings show the importance of mRNA stability in bimodal regulation, a factor that requires more attention when studying and modelling this non-deterministic developmental mechanism.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom.

ABSTRACT
Gene expression can be highly heterogeneous in isogenic cell populations. An extreme type of heterogeneity is the so-called bistable or bimodal expression, whereby a cell can differentiate into two alternative expression states. Stochastic fluctuations of protein levels, also referred to as noise, provide the necessary source of heterogeneity that must be amplified by specific genetic circuits in order to obtain a bimodal response. A classical model of bimodal differentiation is the activation of genetic competence in Bacillus subtilis. The competence transcription factor ComK activates transcription of its own gene, and an intricate regulatory network controls the switch to competence and ensures its reversibility. However, it is noise in ComK expression that determines which cells activate the ComK autostimulatory loop and become competent for genetic transformation. Despite its important role in bimodal gene expression, noise remains difficult to investigate due to its inherent stochastic nature. We adapted an artificial autostimulatory loop that bypasses all known ComK regulators to screen for possible factors that affect noise. This led to the identification of a novel protein Kre (YkyB) that controls the bimodal regulation of ComK. Interestingly, Kre appears to modulate the induction of ComK by affecting the stability of comK mRNA. The protein influences the expression of many genes, however, Kre is only found in bacteria that contain a ComK homologue and, importantly, kre expression itself is downregulated by ComK. The evolutionary significance of this new feedback loop for the reduction of transcriptional noise in comK expression is discussed. Our findings show the importance of mRNA stability in bimodal regulation, a factor that requires more attention when studying and modelling this non-deterministic developmental mechanism.

No MeSH data available.


Related in: MedlinePlus

Transposon insertion in ykyB increases the activation of an artificial ComK feedback loop.Strains PG401 (amyE::PcomG-lacZ-gfp, PcomG-comK, ΔmecA) and PG401-Tn4 (amyE::PcomG-lacZ-gfp, PcomG-comK, ΔmecA, ykyB:Tn) were grown on nutrient agar plates (A) or competence medium plates (B) supplemented with X-gal. Cells from plates were imaged by fluorescent light microscopy. Insets show related phase contrast images and arbitrary GFP colour intensity scales. Pictures and microscopy images were taken after overnight incubation at 37°C.
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pgen.1005047.g002: Transposon insertion in ykyB increases the activation of an artificial ComK feedback loop.Strains PG401 (amyE::PcomG-lacZ-gfp, PcomG-comK, ΔmecA) and PG401-Tn4 (amyE::PcomG-lacZ-gfp, PcomG-comK, ΔmecA, ykyB:Tn) were grown on nutrient agar plates (A) or competence medium plates (B) supplemented with X-gal. Cells from plates were imaged by fluorescent light microscopy. Insets show related phase contrast images and arbitrary GFP colour intensity scales. Pictures and microscopy images were taken after overnight incubation at 37°C.

Mentions: Previously, we have shown that an artificial autostimulatory ComK feedback loop shows a bimodal expression pattern that closely resembles the wild-type pattern of ComK expression (Fig 1) [7]. Theoretical modelling has shown that a simple positive feedback loop can produce a bimodal response if there is sufficient noise in the expression of the activator and a threshold level for activation [11]. Binding of ComK to DNA is highly cooperative and presumably this non-linear reaction determines the hypersensitive response of the positive feedback loop to small fluctuations of ComK levels [34]. This leaves expression noise as an important determinant of the fraction of ComK expressing cells and therefore of the bimodal distribution. To identify possible factors that influence the bimodal outcome of this artificial feedback loop, we constructed a lacZ-gfp operon that is driven by the comG promoter. This reporter enables the screening of mutants on plate as well as by fluorescence light microscopy, which makes it possible to distinguish differences in cellular ComK levels from differences in the frequency of ComK expressing cells. The PcomG-lacZ-gfp reporter was integrated at the ectopic amyE locus and combined with the artificial ComK feedback loop resulting in strain PG401 (PcomG-comK, ΔmecA, PcomG-lacZ-gfp). On nutrient agar plates containing X-gal, PG401 colonies developed a faint blue colour after 2 days of incubation. However, PG401 colonies develop a clear blue colour after overnight incubation on competence medium plates (Fig 2), indicating that medium composition still influences the artificial ComK feedback loop. This is surprising since this loop was constructed in such way that none of the known competence regulators are able to influence its activity (Fig 1). Nevertheless, the effect on nutrient agar plates could be used to our advantage since it facilitates the selection of mutants with different lacZ, thus ComK, activities.


A Novel Feedback Loop That Controls Bimodal Expression of Genetic Competence.

Gamba P, Jonker MJ, Hamoen LW - PLoS Genet. (2015)

Transposon insertion in ykyB increases the activation of an artificial ComK feedback loop.Strains PG401 (amyE::PcomG-lacZ-gfp, PcomG-comK, ΔmecA) and PG401-Tn4 (amyE::PcomG-lacZ-gfp, PcomG-comK, ΔmecA, ykyB:Tn) were grown on nutrient agar plates (A) or competence medium plates (B) supplemented with X-gal. Cells from plates were imaged by fluorescent light microscopy. Insets show related phase contrast images and arbitrary GFP colour intensity scales. Pictures and microscopy images were taken after overnight incubation at 37°C.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482431&req=5

pgen.1005047.g002: Transposon insertion in ykyB increases the activation of an artificial ComK feedback loop.Strains PG401 (amyE::PcomG-lacZ-gfp, PcomG-comK, ΔmecA) and PG401-Tn4 (amyE::PcomG-lacZ-gfp, PcomG-comK, ΔmecA, ykyB:Tn) were grown on nutrient agar plates (A) or competence medium plates (B) supplemented with X-gal. Cells from plates were imaged by fluorescent light microscopy. Insets show related phase contrast images and arbitrary GFP colour intensity scales. Pictures and microscopy images were taken after overnight incubation at 37°C.
Mentions: Previously, we have shown that an artificial autostimulatory ComK feedback loop shows a bimodal expression pattern that closely resembles the wild-type pattern of ComK expression (Fig 1) [7]. Theoretical modelling has shown that a simple positive feedback loop can produce a bimodal response if there is sufficient noise in the expression of the activator and a threshold level for activation [11]. Binding of ComK to DNA is highly cooperative and presumably this non-linear reaction determines the hypersensitive response of the positive feedback loop to small fluctuations of ComK levels [34]. This leaves expression noise as an important determinant of the fraction of ComK expressing cells and therefore of the bimodal distribution. To identify possible factors that influence the bimodal outcome of this artificial feedback loop, we constructed a lacZ-gfp operon that is driven by the comG promoter. This reporter enables the screening of mutants on plate as well as by fluorescence light microscopy, which makes it possible to distinguish differences in cellular ComK levels from differences in the frequency of ComK expressing cells. The PcomG-lacZ-gfp reporter was integrated at the ectopic amyE locus and combined with the artificial ComK feedback loop resulting in strain PG401 (PcomG-comK, ΔmecA, PcomG-lacZ-gfp). On nutrient agar plates containing X-gal, PG401 colonies developed a faint blue colour after 2 days of incubation. However, PG401 colonies develop a clear blue colour after overnight incubation on competence medium plates (Fig 2), indicating that medium composition still influences the artificial ComK feedback loop. This is surprising since this loop was constructed in such way that none of the known competence regulators are able to influence its activity (Fig 1). Nevertheless, the effect on nutrient agar plates could be used to our advantage since it facilitates the selection of mutants with different lacZ, thus ComK, activities.

Bottom Line: The protein influences the expression of many genes, however, Kre is only found in bacteria that contain a ComK homologue and, importantly, kre expression itself is downregulated by ComK.The evolutionary significance of this new feedback loop for the reduction of transcriptional noise in comK expression is discussed.Our findings show the importance of mRNA stability in bimodal regulation, a factor that requires more attention when studying and modelling this non-deterministic developmental mechanism.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom.

ABSTRACT
Gene expression can be highly heterogeneous in isogenic cell populations. An extreme type of heterogeneity is the so-called bistable or bimodal expression, whereby a cell can differentiate into two alternative expression states. Stochastic fluctuations of protein levels, also referred to as noise, provide the necessary source of heterogeneity that must be amplified by specific genetic circuits in order to obtain a bimodal response. A classical model of bimodal differentiation is the activation of genetic competence in Bacillus subtilis. The competence transcription factor ComK activates transcription of its own gene, and an intricate regulatory network controls the switch to competence and ensures its reversibility. However, it is noise in ComK expression that determines which cells activate the ComK autostimulatory loop and become competent for genetic transformation. Despite its important role in bimodal gene expression, noise remains difficult to investigate due to its inherent stochastic nature. We adapted an artificial autostimulatory loop that bypasses all known ComK regulators to screen for possible factors that affect noise. This led to the identification of a novel protein Kre (YkyB) that controls the bimodal regulation of ComK. Interestingly, Kre appears to modulate the induction of ComK by affecting the stability of comK mRNA. The protein influences the expression of many genes, however, Kre is only found in bacteria that contain a ComK homologue and, importantly, kre expression itself is downregulated by ComK. The evolutionary significance of this new feedback loop for the reduction of transcriptional noise in comK expression is discussed. Our findings show the importance of mRNA stability in bimodal regulation, a factor that requires more attention when studying and modelling this non-deterministic developmental mechanism.

No MeSH data available.


Related in: MedlinePlus