Difference between revisions of "Part:BBa K554000"

Latest revision as of 05:43, 11 September 2024

SoxS promoter

SoxS is a promoter regulated by the SoxR transcription factor, which is activated by NO. E. coli can naturally respond to redox signals through a superoxide stress system composed by SoxR gene, SoxS promoter and the genes associated with SoxS promoter. Thus, in the presence of NO, SoxR activates transcription of the gene regulated by SoxS promoter (Hidalgo et al, 1998). The SoxS promoter is used by [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil UNICAMP-EMSE Brazil team] in the [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_2:_NO_sensor.2FIL-10_producer NO sensor device/ IL-10 producer] ("Device 2", which senses NO levels and responds by producing and secreting IL-10.

Sequence and Features

Usage and Biology

• This promoter is used by [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil UNICAMP-EMSE Brazil team] in the [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project/Device2 NO sensor device] ("Device 2", which senses NO levels and responds by producing and secreting IL-10. This part is shown in the red box in the following schema:

Three-dimensional structure representation

You can find below a tridimensional structure of Escherichia coli SoxR protein bound to SoxS promoter (DNA structure) (retrieved from PDB 2zhg, Watanabe et al. 2008), with both p35 (chain A) and p40 (chain B) chains. This is a jmol applet, in which you can interactively see the protein format:

MIT_MAHE 2020

Usage and Biology

The oxidation state of the iron-sulfur clusters in SoxR regulates the transcriptional activity of the protein: while reduced SoxR does not affect transcription, oxidized SoxR dramatically enhances the transcription rate of soxS, a gene that codes for a second transcriptional activator. The SoxS protein is a member of the AraC/XylS family of transcriptional regulators.

It is a pleiotropic regulator that binds many sites across the genome and plays an important role in antibiotic resistance through its influence on efflux activity. It also impacts biofilm formation, quorum sensing, pathogenicity and motility.

It has been shown that there is a rapid increase in transcription of soxS following exposure to antibiotics (and other inducer substrates), but that repression is rapidly reinstated following removal of the stimuli. The pool of pre-produced transcriptional regulators is degraded by proteases including Lon, and this “resetting” in impaired in lon deficient mutants (Griffith et al., 2004; Ricci et al., 2014). This ability to quickly produce, but then degrade SoxS allows for a fine-tuned, fast response to environmental stimuli to maximize bacterial fitness when under stress.

As well as controlling membrane permeability through efflux pump and outer membrane porin expression, SoxS is important for initiating transcription of genes to reduce superoxide and nitric oxide stress in the cell. When efflux is disrupted, cells respond with overexpression of this transcriptional regulator, though it is not understood by which mechanism this is regulated.

Binding of Fe-SoxR to the wild-type soxS promoter improved the subsequent binding of RNAP nearly 5-fold as determined by densitometric analysis.

As well as AcrAB-TolC, SoxS can also regulate the expression of other efflux pumps, such as the RND pump AcrEF (Bailey et al., 2010) and a member of the multidrug and toxic compound extrusion (MATE) family, mdtK (Sun et al., 2011) in response to environmental stress.

Increased expression of marA, ramA, and soxS up regulates efflux activity to allow detoxification of the cell. However, this also results in trade-offs in other phenotypes, such as impaired growth rates, biofilm formation and virulence.

ZQT-Nanjing 2024

Nitric-oxide-inducible Promoter SoxR/SoxS Characterization

To optimize the induction conditions, we decided to characterize the NO-inducible promoter SoxR/SoxS to determine the optimal inducer concentration and induction time with the plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7.We extracted the correctly sequenced plasmid and transformed it into Escherichia coli Nissle 1917(EcN) for expression. Due to the potential danger of NO, we used sodium nitroprusside (SNP) as the NO donor for induction.

Figure 1 NO-inducible plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7

We started by setting different final concentrations of SNP for the promoter-inducing condition optimization. Firstly, single colonies were picked and initially cultured in LB medium containing the resistance gene Kana, then expanded until the OD₆₀₀ value of the bacterial liquid reached approximately 1.0. Subsequently, the bacterial liquid was aliquoted into multiple test tubes and different concentrations of SNP (0, 50, 100, 200, 300, 400, 500, 800, 1000µM) were added for induction at 37°C for 10 hours. To determine the optimal concentration, the experiment analyzed the effects of different concentrations by measuring the fluorescence intensity of the reporter gene eGFP with a microplate reader and the OD₆₀₀ value of the bacterial liquid. As shown in Figure 2, the experimental results were analyzed using GraphPad Prism software, which indicated that the fluorescence intensity reached the highest value at 100µM, therefore, 100µM was chosen as the optimal SNP concentration.

Figure 2 the FL/OD₆₀₀ content under different SNP induction concentrations

Next, we set different induction times for exploration. Initially, single colonies were picked and cultured in LB medium containing the resistance gene Kana, then expanded until the OD₆₀₀ value of the bacterial liquid reached approximately 1.0. Samples were then taken at different induction time points (such as 0, 3, 5, 7, 10, 14, 24 hours), and after induction, protein samples were processed. The protein expression was observed through SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The final results, as shown in Figure 3, indicate that the protein expression level gradually increased with the increase in induction time. However, to determine the expression time with the highest speed, a microplate reader was used to further analyze the fluorescence reporter gene, with the results shown in Figure 4.

Figure 3 SDS-PAGE Gel electrophoresis for AMP expression under different expression time. From right to left are the results of induction for 0, 3, 5, 7, 10, 14, and 24 hours.

Figure 4 Different induction times correspond to the content of FL/OD₆₀₀.

Based on the above optimization conditions, the optimal induction concentration for the pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7 plasmid in EcN is 100µM, with the optimal induction time being 10 hours.

References

Hidalgo E, Leautaud V, Demple B (1998) The redox-regulated SoxR protein acts from a single DNA site as a repressor and an allosteric activator. The EMBO Journal 17(9)2629–2636 [http://www.ncbi.nlm.nih.gov/pubmed/?term=9564045%20 Link to Pubmed]

Watanabe S, Kita A, Kobayashi K, Miki K. Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA.Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4121-6. Epub 2008 Mar 11. [http://www.ncbi.nlm.nih.gov/pubmed/18334645 Link to PubMed]

Holden, E. R., & Webber, M. A. (2020). MarA, RamA, and SoxS as Mediators of the Stress Response: Survival at a Cost. Frontiers in microbiology, 11, 828. https://doi.org/10.3389/fmicb.2020.00828

Pomposiello, P. J., & Demple, B. (2000). Identification of SoxS-regulated genes in Salmonella enterica serovar typhimurium. Journal of bacteriology, 182(1), 23–29. https://doi.org/10.1128/jb.182.1.23-29.2000

Nunoshiba, T., Hidalgo, E., Amábile Cuevas, C. F., & Demple, B. (1992). Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene. Journal of bacteriology, 174(19), 6054–6060. https://doi.org/10.1128/jb.174.19.6054-6060.1992

Structure: