In the early preparation of our project, we investigated the dissimilatory metabolic pathway and learned the remarkable nitrite reduction ability of E. coli utilizing nitrite reductase. Inspired by such discovery, our team decided to harness and amplify this ability to realize efficient nitrite monitoring. After referring to several published studies, the regulatory network of E. coli’s two nitrite reductase nirB and nrfA was constructed (Figure 1).
Figure 1. How nitrite reductases get regulated
Based on this regulatory network, we found several transcription factors, specifically, NarP, NarL, CRP, and FNR, are of great significance. In addition, previous research has shown that NarP has a predominant influence over NarL when regulating the expression of nitrite reductases. Therefore, we intended to up-regulate the expression level of NarP to realize the overexpression of NrfA and NirB. It is also worth noting that FNR and CRP are two oxygen-responsive transcription factors that are essential in switching E. coli from aerobic to anaerobic metabolism. In the presence of oxygen, the oxidation of the Fe-S cluster will inactivate the transcription factor, inhibiting the expression of the downstream genes. Therefore, our project has considered excluding oxygen in experimental design and product application.
Referring to our experiment results, the existence of oxygen is proven to be an essential variable that may impact the metabolic activity and the enzyme expression of the bacteria. However, we found it challenging to evaluate the strictness of the anaerobic condition. In other words, we could not specify a particular time point that marks the initiation of anaerobic metabolism. The induction of the expression level of nitrite reductases also requires the inducing of nitrite. Under these circumstances, having a better understanding of the conversion from aerobic to anaerobic metabolism is critical for our project to acquire the maximum amount of enzyme.
We therefore decided to construct a more comprehensive regulatory model with more precise presentation of the oxygen regulating pattern. To start with, we looked up the sequence of the promoter region of two nitrite reductases, which is shown in the following figure (Figure 2&3).
Figure 2. Promoter sequence of nrfA
Figure 3. Promoter sequence of nirB
Then, we searched through the internet to get the motif sequence of FNR and CRP, which are represented as follows:
Figure 4. Logo representation for FNR’s motif
Figure 5. Logo representation for CRP’s motif
We further scanned the motif sequence through the promoter region and filtered all the results with the p value larger or equals to 0.001. We therefore found several unidentified binding sites for FNR and CRP in the promoter region of both nrfA and nirB. The results are listed as follows:
Figure 6. Newly predicted motif binding site of CRP to nrfA
Figure 7. Newly predicted motif binding site of CRP to nirB
Figure 8. Newly predicted motif binding site of FNR to nrfA
Figure 9. Newly predicted motif binding site of FNR to nirB
Figures 10 & 11 show possible CRP and FNR binding sites near the promoter region of nrfA and nirB. We selected the sequences containing 700 base pairs (from -500 to +200) for both enzymes as the possible binding region. With a p-value less than 0.001, for CRP, there are 4 motif occurrences on the promoter of nrfA, 2 motif occurrences on the promoter of nirB; for FNR, there are 4 motif occurrences on the promoter of nrfA, 1 motif occurrence on the promoter of nirB. The detailed matched sequences are shown in the figures. If the matched sequence is from 5' to 3', the binding site is on the positive chain; otherwise, the binding site is on the negative chain. In conclusion, we successfully constructed a more comprehensive oxygen regulatory model.
Figure 10 CRP binding sites on nrfA and nirB
Figure 11 FNR binding sites on nrfA and nirB
After the regulatory pattern for two oxygen-responsive transcription factors is better solved, researchers could control the experiment condition more flexibly. On the one hand, RNAi or CRISPER could be introduced to block the binding of these transcription factors, allowing further research to be conducted in aerobic conditions and therefore simplify the experiment requirements to a great extent; on the other hand, transforming motif binding sites of either CRP or FNR to the bacteria could make the oxygen a threshold to control the expression of the target proteins, which increases the safety of the engineered organism as well as making the experiments more controllable. Moreover, our experiment explores several conditions to create a qualified anaerobic condition, which could be a reference for later research.
After generating matrices of the motifs of narP and narL , we scanned the motif sequence through the promoter region and filtered all the results with a p-value larger or equal to 0.001. Then we found 2 possible binding sites for narP on the promoter region of nrfA, 1 possible binding site for narP on the promoter region of nirB, 3 possible binding sites for narL on the promoter region of nrfA, and 2 possible binding sites for narL on the promoter region of nirB. The results are listed as follows:
Figure 12. Newly predicted motif binding site of narP to nrfA
Figure 13. Newly predicted motif binding site of narP to nirB
Figure 14. Newly predicted motif binding site of narL to nrfA
Figure 15. Newly predicted motif binding site of narL to nirB
Figure 16. NarP binding sites on nrfA and nirB
Figure 17. NarL binding sites on nrfA and nirB
At last, we gather all binding sites prediction results of 4 transcription factors mentioned above into the following figure. Then we surprisingly found that there are some overlaps between different transcription factors.
On the promoter region of nrfA, CRP can bind to the +151 to +172 region on the positive chain, narP can bind to the +156 to +171 region on the positive chain. This overlap shows the competition between CRP and narP: under aerobic conditions, CRP may be more likely to bind to this site, repressing the transcription of nrfA; under anaerobic conditions, narP may be more likely to bind to this site, enhancing the transcription of nrfA.
On the promoter region of nirB, narP can bind to the -352 to -337 and -343 to -328 regions on the positive chain, narL can bind to the -342 to -336 region on the negative chain. This overlap shows the importance of this region since narP and narL both can enhance the transcription of nirB. CRP can bind to the -51 to -30 region on the both positive and negative chains, FNR can bind to the -51 to -30 region on the positive chain. This overlap shows the competition between CRP and FNR: under aerobic conditions, CRP may be more likely to bind to this site, repressing the transcription of nirB; under anaerobic conditions, FNR may be more likely to bind to this site, enhancing the transcription of nirB.
Figure 18. Summary of 4 transcription factors’ binding sites on the promoter region of nrfA and nirB
Reference
Unden, G., & Schirawski, J. (2003). The oxygen-responsive transcriptional regulator FNR of Escherichia coli: the search for signals and reactions. Molecular Microbiology, 25(2), 205-210. Wang, H., & Gunsalus, R. P. (2000). The nrfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite. Journal of Bacteriology, 182(20), 5813-5822.