A simulation model of Escherichia coli osmoregulatory switch using E-CELL system

Abstract

Background: Bacterial signal transduction mechanism referred to as a "two component regulatory systems" contributes to the overall adaptability of the bacteria by regulating the gene expression. Osmoregulation is one of the well-studied two component regulatory systems comprising of the sensor, EnvZ and the cognate response regulator, OmpR, which together control the expression of OmpC and OmpF porins in response to the osmolyte concentration.

Results: A quantitative model of the osmoregulatory switch operative in Escherichia coli was constructed by integrating the enzyme rate equations using E-CELL system. Using the substance reactor logic of the E-CELL system, a total of 28 reactions were defined from the injection of osmolyte till the regulated expression of porins by employing the experimental kinetic constants as reported in literature. In the case of low osmolarity, steady state production of OmpF and repression of OmpC was significant. In this model we show that the steady state - production of OmpF is dramatically reduced in the high osmolarity medium. The rate of OmpC production increased after sucrose addition, which is comparable with literature results. The relative porin production seems to be unaltered with changes in cell volume changes, ATP, EnvZ and OmpR at low and high osmolarity conditions. But the reach of saturation was rapid at high and low osmolarity with altered levels of the above components.

Conclusions: The E-CELL system allows us to perform virtual experiments on the bacterial osmoregulation model. This model does not take into account interaction with other networks in the cell. It suggests that the regulation of OmpF and OmpC is a direct consequence of the level of OmpRP in the cell and is dependent on the way in which OmpRP interacts with ompF and ompC regulatory regions. The preliminary simulation experiment indicates that both reaching steady state expression and saturation is delayed in the case of OmpC compared to OmpF. Experimental analysis will help improve the model. The model captures the basic features of the generally accepted view of EnvZ-OmpR signaling and is a reasonable starting point for building sophisticated models and explaining quantitative features of the system.

Figure 1

Two Component Regulatory Systems. The first component, sensor kinase autophosphorylates and transfers phosphate to the response regulator. These are also called the HAP systems indicating the involvement of Histidine of sensor kinase and Aspartate of Response regulators playing a key role in signal transduction.

Figure 2

Molecular Model of Osmoregulatory switch operative at High osmolarity in Escherichia coli. With the injection stimulus of sucrose as osmolyte the sensor EnvZ is triggered to take up either kinase or phosphatase activity. In high osmolarity conditions the higher osmolyte medium (NB (Nutrient Broth)+20% sucrose) makes the kinase activity of EnvZ (EnvZk) predominate the phosphatase activity resulting in the formation of EnvZkpOmpR complex. Finally after the phosphotransfer of phosphate group to OmpR, the promoter sites of OmpF are occupied in cooperative manner with F1, F1F2 and F1F2F3, high affinity binding sites. The cellular concentration of OmpRP makes it available for the OmpC promoter C1, C1C2, C1C2C3. Additionally OmpRP binds to F4 after binding F1F2F3 promoter directly repressing OmpF expression, thereby facilitating OmpC expression.

Figure 3

Molecular Model of Osmoregulatory switch operative at Low Osmolarity in Escherichia coli. The low osmolarity conditions favour the phosphatase activity of EnvZ. The Phosphatase domain (EnvZp) upon autophosphorylation leads to formation of EnvZppOmpR complex and later favours the dephosphorylation of the cellular OmpRP thus making OmpRP available only for cooperative binding to the high affinity promoter of OmpF namely F1, F1F2, and F1F2F3 favouring OmpF expression.

Figure 4

Porin production at Low and High osmolarity conditions Indicated in the X-axis is the time in seconds and number of molecules of the component in the osmoregulatory switch in Y-axis. (a) The start of simulation with OmpF (indicated in blue) synthesis gradually triggered. By mid term of simulation steady state production and final saturation of OmpF molecules could be seen. Shown in b is the OmpC (indicated in pink) production and reaching saturation levels at high osmolarity conditions.

Figure 5

Regulator-promoter complex formation simulation The intermediate products of simulation namely the regulator-promoter complexes for regulation of (a) OmpF: f1omprp – blue; f1f2omprp – pink; f1f2f3omprp – yellow and (b) OmpC: c1omprp-yellow; c1c2omprp-pink; c1c2c3omprp-blue respectively.

Figure 6

Schematic representation of steps in creation of simulation model using E-CELL system