Part:BBa_K2401003
PchR: Pyochelin Regulator
The Pyochelin-Complex Regulated Promotor (PchR) is the native pyochelin promotor of Pseudomonas aeruginosa (here: Pseudomonas aeruginosa PAO1) that is activated by its own product complexed with metal-ions, the native pyochelin-iron complex.
The Pyochelin-Complex Regulated Promotor is most likely a leaky promoter that induces the Pyochelin synthesis if the Pyochelin-iron3+-complex binds to the receptor. Most siderophore-receptor-complexes need to be leaky since the siderophore production is only necessary under certain circumstances and self-induced. In this context, the leakiness is not to be seen as a flaw but a feature, including a biosensor for iron3+-ions in the expression system that makes sure that no unnecessary metabolic load is exhibited. Therefore, the iron acquisition method is highly efficient and only expressed when the circumstances demand it.
Our goal was to characterize the promoter and measure if pyochelin-gallium3+ and Nor-pyochelin-gallium3+ complexes induce the native promoter to estimate the metabolic excess overload that our antibiotic-compound induces on native Pseudomonas aeruginosa.
To characterize the promotor, we cloned the 960 bp long DNA part before GFP. As a template, we used the BioBrick BBa_E0240. This is a GFP-tri-part consisting of the ribosome binding site BBa_B0032, GFP with the number BBa_E0040 and the double terminator BBa_B0010 und BBa_B0012.
We transformed the plasmid pSB1C3::PchR::BBa_E0240 in Escherichia coli DH5a cells. For the expression tests the cells were cultivated to an OD [595 nm]= 0,372. Then a 96-well plate (black plate with transparent ground) was loaded with the cells and different concentrations of NPch-Fe-, NPch-Ga-, Pch-Fe- und Pch-Ga-complexed siderophores. Loading scheme:
Figure 1: The Pipetting scheme of the 96-well plate for measurement of the OD[595 nm] and the Fluorescence of GFP.
Over the next eight hours the plate was incubated at 37°C and 220 rpm and we measured the absorption at 595 nm and the fluorescence of the GFP, (excitation wavelenght = 475 nm and emission wavelength = 509 nm) in a plate reader every 18 min.
Growth and fluorescence of the cells induced with NPch-Fe-complex over the entire time:
Figure 2: The OD[595 nm] measurement of the NPch-Fe-complex induced cells over the time.
Figure 3: The fluorescence measurement of the NPch-Fe-complex induced cells over the time.
Growth and fluorescence of the cells induced with NPch-Ga-complex over the entire time:
Figure 4: The OD[595 nm] measurement of the NPch-Ga-complex induced cells over the time.
Figure 5: The fluorescence measurement of the NPch-Ga-complex induced cells over the time.
Growth and fluorescence of the cells induced with Pch-Fe-complex over the entire time:
Figure 6: The OD[595 nm] measurement of the Pch-Fe-complex induced cells over the time.
Figure 7: The fluorescence measurement of the Pch-Fe-complex induced cells over the time.
Growth and fluorescence of the cells induced with Pch-Ga-complex over the entire time:
Figure 8: The OD[595 nm] measurement of the Pch-Ga-complex induced cells over the time.
Figure 9: The fluorescence measurement of the Pch-Ga-complex induced cells over the time.
The growth curves for the cells induced with the siderophore-gallium-complex show an almost periodically drop of the absorption. Therefore, the assumption is that the induction led to a periodic cell death. The periodic cell death explains the fluctuation of the absorption. We suppose that the cells take up the gallium-siderophore-complex and die due to the sudden release of the cytotoxic gallium.
The fluorescence was divided though the absorption to get a relation of the fluorescence and the cell growth. To remove the leakiness out of the equation we only measured the cells that were transformed with the plasmid but not induced with siderophores (called “blank” in the following).
Fluorescence against absorption of the cells induced with the NPch-Fe-complex.
Figure 12: The fluorescence was divided though the absorption to get a relation of the fluorescence and the cell growth measurement of the NPch-Fe-complex induced cells over the time.
The graph shows that all values for a NPche-Fe-complex concentration of 0.195 µM bis 50 µM lie above the blank value and prove therefore an induction of the promotor. Lower concentrations induce weaker than the higher concentrations.
Fluorescence against absorption of the cells induced with the NPch-Ga-complex.
Figure 13: The fluorescence was divided though the absorption to get a relation of the fluorescence and the cell growth measurement of the NPch-Ga-complex induced cells over the time.
The graph shows an induction of the promoter as all values for the NPch-Ga-concentrations of 1.563 µM to 50 µM lie above the blank value. Lower concentrations induce weaker than higher concentrations.
Fluorescence against absorption of the cells induced with the Pch-Fe-complex.
Figure 14: The fluorescence was divided though the absorption to get a relation of the fluorescence and the cell growth measurement of the Pch-Fe-complex induced cells over the time.
The graph shows an induction of the promotor as the values for all Pch-Fe-complex concentrations between 6.25 µM and 50 µM lie above the blank value. Lower concentrations induce weaker than higher concentrations.
Fluorescence against absorption of the cells induced with the Pch-Ga-complex.
Figure 15: The fluorescence was divided though the absorption to get a relation of the fluorescence and the cell growth measurement of the Pch-Ga-complex induced cells over the time.
The graph shows that only the values for the Pch-Ga-complex concentration of 50 µM lie above the blank value and induce the promotor. Lower concentrations induce weaker than higher concentrations.
The graphs of the fluorescence against the absorption show a more effective induction of the promotor for the NPch-siderophore-complexes than for the Pch-siderophore-complexes. Also, the iron-complexed siderophores provoke a stronger induction than the gallium-complexed siderophores, as the for the induction needed iron-siderophore-complex concentrations of 12.5 µM, 25 µM und 50 µM lie above the gallium-siderophore-complex concentration of 50 µM. The relevance of the cell death could not be further quantified. The NPch-siderophor is more effective than the Pch-siderophore as the signals of the corresponding concentrations are significantly stronger for the NPch-siderophore than the Pch-siderophore.
Furthermore, the receptor must be leaky as even the non-induced cells showed a fluorescence that is partly even slightly higher or almost equal to values for cells induced with a low concentration of a siderophor-complex. Also, the graphs show that there seems to be a saturation of the promotor, as the curve flattens after a certain time. This can be attributed to the fact that there is only a certain number of receptors that is depending on the number of cells and therefore limits the GFP production and consequently sets the intensity of the fluorescence.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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