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Phenotypic heterogeneity in mycobacterial stringent response.

Ghosh S, Sureka K, Ghosh B, Bose I, Basu J, Kundu M - BMC Syst Biol (2011)

Bottom Line: Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time.This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise.The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Bose Institute, Kolkata, India.

ABSTRACT

Background: A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity.

Results: In the present study, we characterise quantitatively the single cell promoter activity of the three key genes, namely, mprA, sigE and rel, in the stringent response pathway with gfp as the reporter. The origin of bimodality in the GFP distribution lies in two stable expression states, i.e., bistability. We develop a theoretical model to study the dynamics of the stringent response pathway. The model incorporates a recently proposed mechanism of bistability based on positive feedback and cell growth retardation due to protein synthesis. Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time. This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise.

Conclusions: The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.

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Related in: MedlinePlus

Specific growth rate μ versus GFP fluorescence intensity xGFP fitted with an expression similar to that given in Eq. (1). The values of  and θGFP are  and θGFP = 0.317. The data points correspond to the growth period of 16-23 hours.
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Figure 3: Specific growth rate μ versus GFP fluorescence intensity xGFP fitted with an expression similar to that given in Eq. (1). The values of and θGFP are and θGFP = 0.317. The data points correspond to the growth period of 16-23 hours.

Mentions: Thus, the decay rate of proteins has the form -γeff x = -(γ + μ)x where μ is given by Eq. (1). There are alternative explanations for the origin of the nonlinear decay term, e.g., the synthesis of a protein may retard cell growth if it is toxic to the cell [33]. In the case of mycobacteria, there is some experimental evidence of cell growth retardation brought about by protein synthesis. The response regulator MprA has an essential role in the stringent response pathway leading to persistence of mycobacteria under nutrient deprivation. Inactivation of the regulator in an mprA insertion mutant resulted in reduced persistence in a murine model but the growth of the mutant was proved to be significantly higher than that observed in the cases of the wild-type species [38,39]. Our experimental data (Figure 3) provide further support to the hypothesis that MprA synthesis leads to reduced specific growth rate. The data points represent GFP fluorescence intensity with gfp fused to the mprA promoter. The GFP acts as a reporter of the mprA promoter activity culminating in MprA (also MprB) synthesis. The data points shown in Figure 3 are those that correspond to the growth period of 16-23 hours in Figure 2.


Phenotypic heterogeneity in mycobacterial stringent response.

Ghosh S, Sureka K, Ghosh B, Bose I, Basu J, Kundu M - BMC Syst Biol (2011)

Specific growth rate μ versus GFP fluorescence intensity xGFP fitted with an expression similar to that given in Eq. (1). The values of  and θGFP are  and θGFP = 0.317. The data points correspond to the growth period of 16-23 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045321&req=5

Figure 3: Specific growth rate μ versus GFP fluorescence intensity xGFP fitted with an expression similar to that given in Eq. (1). The values of and θGFP are and θGFP = 0.317. The data points correspond to the growth period of 16-23 hours.
Mentions: Thus, the decay rate of proteins has the form -γeff x = -(γ + μ)x where μ is given by Eq. (1). There are alternative explanations for the origin of the nonlinear decay term, e.g., the synthesis of a protein may retard cell growth if it is toxic to the cell [33]. In the case of mycobacteria, there is some experimental evidence of cell growth retardation brought about by protein synthesis. The response regulator MprA has an essential role in the stringent response pathway leading to persistence of mycobacteria under nutrient deprivation. Inactivation of the regulator in an mprA insertion mutant resulted in reduced persistence in a murine model but the growth of the mutant was proved to be significantly higher than that observed in the cases of the wild-type species [38,39]. Our experimental data (Figure 3) provide further support to the hypothesis that MprA synthesis leads to reduced specific growth rate. The data points represent GFP fluorescence intensity with gfp fused to the mprA promoter. The GFP acts as a reporter of the mprA promoter activity culminating in MprA (also MprB) synthesis. The data points shown in Figure 3 are those that correspond to the growth period of 16-23 hours in Figure 2.

Bottom Line: Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time.This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise.The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Bose Institute, Kolkata, India.

ABSTRACT

Background: A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity.

Results: In the present study, we characterise quantitatively the single cell promoter activity of the three key genes, namely, mprA, sigE and rel, in the stringent response pathway with gfp as the reporter. The origin of bimodality in the GFP distribution lies in two stable expression states, i.e., bistability. We develop a theoretical model to study the dynamics of the stringent response pathway. The model incorporates a recently proposed mechanism of bistability based on positive feedback and cell growth retardation due to protein synthesis. Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time. This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise.

Conclusions: The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.

Show MeSH
Related in: MedlinePlus