Team/CINVESTAV-IPN-UNAM MX/oxigenresponse.htm

As we can see, in figure 1A the PpsR promoter system, in R. sphaeroides, shows a high signal (17.66%) of GFP expression, it implies that constitutive proteins from this bacteria were able to activate our system. The growth conditions were aerobic/darkness, although oxidized PpsR binds its target promoter, it is known that AppA can avoid the binding affinity of PpsR in the dark probably by the interference of an AppA-(PpsR)2 complex (Kim, 2006). In the case of AppA/PpsR complete system, we have a high GFP expression due to activity of the extra AppA and PpsR enzymes that were introduced.

The blue columns show the low GFP expression with both PrrA promoter and PrrA/PrrB complete system; in aerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this activated PrrA binds to its promoter region as a transcriptional repressor (Bauer, 2003).  All the results show an equivalent result with the images that were obtained by fluorescence microscope.

Figure 2A shows that in R. sphaeroides, the AppA/PpsR system promoted the GFP expression, it is possible because reduced PpsR is unable to bind its promoter and AppA is a flavin with a photoreceptor, thus under light, AppA is unable to forma a complex with PpsR and we can see GFP expression in the bacterial population.

In opposite way, PrrA/PrrB system shows less GFP expression, it is probably due to the low growth rate in R. sphaeroides, under photosynthetic conditions (anaerobic/light), both systems were tested in one and a half days of growing, and maybe it was not enough time to have a culture in exponential phase of growing. PrrA/PrrB is supposed to be functional because in anaerobic conditions PrrB autophospholylate and can transfer the phosphate group to PrrA, when PrrA receive the phosphate group, it can bind its target promoter and the transcription is possible (Bauer, 2008).

AppA/PpsR system can be sense to signals: oxygen and light, in figure 3A, the GFP expression is high with PpsR promoter because we have the natural R. sphaeroides AppA/PpsR system working on our promoter. In anaerobic conditions the reduced PpsR has low affinity to his target sequence and AppA is forming the complex ApA-(PpsR)2, (Bauer, 2001).  We can see it  with  the  level of GFP expression 21.21%, however, when we introduce the complete system the GFP expression is much less, probably because we are introducing extra AppA and PpsR enzymes that can interfere with the constitutive system. PrrA/PrrB show GFP expression higher in PrrA promoter than in complete system, these results probably have the same problem, that bacterial cultures were not in exponential phase of growing or that the complete system can be interfering with the constitutive.