Part:BBa_K5332001:Design
Reactive oxygen species promoter
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Brief introduction of katG
KatG, a gene encoding hydroperoxidase in bacteria, is regulated by a variety of signaling molecules. At present, most studies focus on the influence of its mutation on bacterial resistance and its upstream pathway. Hydrogen peroxide is one of the reactive oxygen species (ROS). In aerobic breathing organisms, it is transferred from a single electron to oxygen to form a superoxide (O2-), which is then catalyzed by superoxide dismutase. There are genes in Escherichia coli that can directly respond to excess H2O2, such as oxyR, which encodes that oxyR regulator can bind to katG promoter after oxidation to promote katG expression and transcription.
Contents
The reason for choosing the promoter of gen katG
Expression of katG during proliferation of Escherichia coli
All the RNA mentioned in this paper was extracted by thermal phenol method, and the data source of MPCR was obtained by RT-MPCR.
The Strain UC574 (arg56 nad113 araD81) derived from E. coli K-12 was considered as the parental wild type.) After overnight culture in LB broth, part of the medium was inoculated in fresh medium diluted and cultured on a shaking table at 150rpm at 37℃. After 1.5, 2, 3, 5, 7, and 12h after inoculation, the same amount of bacterial liquid was taken out and cooled to 0℃ rapidly, and the growth of E. coli was detected by OD600 measurement, and the RNA content of katG and the activity of catalase of the product were determined. In the figure, the dark column indicates catalase activity, and the white column indicates the RNA content expressed by katG.
With the proliferation of E. coli, the concentration of H2O2 produced in the medium will also gradually increase due to the aerobic respiration of the bacteria. Therefore, the RNA content expressed by katG and catalase activity also increased during the rise of OD600 measurement curve. When the number of bacteria became stable, the RNA content decreased and the catalase activity remained unchanged (Fig.1). This suggests that the promoter of katG can accurately respond to the regulation of ROS stress response pathway.
Fig.1. Peroxidase activity during course of growth of wild-type bacteria in nutrient LB medium.
Comparison of katG promoter responsiveness with other genes
Since the oxyR regulator also regulates dps, gorA, and ahpCF genes, the following is a comparison by detecting the expression of four genes in the same H2O2 environment at the same time.
Wild-type E. coli cultured overnight in M9 base medium were diluted in fresh medium and incubated in a shaking bed at 37°C and 150 rpm. OD600 values were measured at regular intervals. When the OD600 value reached 0.2, half of the culture was taken out and H2O2 was added to make a solution with a final concentration of 10 M. The other half of the culture served as a control group. Samples were collected immediately after addition of H2O2 (within 1min) and after exposure for 5, 10, 15, and 20 min. Samples were frozen with liquid nitrogen, RNA was purified and measured. The fluorescence signal of each PCR product was compared with that of the reference control gene gapA, and the value of the treated sample was divided by the corresponding control value as the data source in the figure. All genes were analyzed, but only those that indicated a statistically significant (P < 0.05) increase was observed over a given time period.
When H2O2 was added, all four genes were expressed, katG and dps were the most, while gorA and ahpCF were less expressed. Subsequently, the expression of all genes decreased with the passage of time, and only a small amount of katG was still expressed at the 20th minute. Compared with other genes, the promoter of katG can not only rapidly respond to upstream signals and cause high gene expression, but also has higher sensitivity when H2O2 content decreases over time (Fig.2).
Fig.2. The expression of four different genes in response to the ROS.
To observe and compare intuitively, we tested the corresponding strength of the promoters of katG, dps, and gorA in the process of IBD occurrence. Using mathematical modeling, we can obtain the response activity strength of promoters from the katG, dps, and gorA in the high ROS environment caused by IBD over time, as shown in Fig.3.
Compared with the dps and gorA promoters, the activity strength of the katG promoter is much higher, and the response time is also obviously longer. This further verifies that the katG promoter has good application prospects for regulating the target gene under the condition of ROS as upstream signal molecules.
Fig.3. The promotor activity within the IBD progression.
Source
From the promoter sequence of katG.
References
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2 Storz G, Tartaglia LA, Ames BN. The OxyR regulon. Antonie Van Leeuwenhoek. 1990 Oct;58(3):157-61. doi: 10.1007/BF00548927. PMID: 2256675.
3 Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol. 2001 Mar;19(3):109-14. doi: 10.1016/s0167-7799(00)01542-0. PMID: 11179804.
4 Tao K. In vivo oxidation-reduction kinetics of OxyR, the transcriptional activator for an oxidative stress-inducible regulon in Escherichia coli. FEBS Lett. 1999 Aug 20;457(1):90-2. doi: 10.1016/s0014-5793(99)01013-3. PMID: 10486570.
5 Michán C, Manchado M, Dorado G, Pueyo C. In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress. J Bacteriol. 1999 May;181(9):2759-64. doi: 10.1128/JB.181.9.2759-2764.1999. PMID: 10217765; PMCID: PMC93716.