Part:BBa_K3823006

CstR

A transcription factor sensing hydrogen sulfide

Introduction

CstR is a CsoR-like sulfur transferase repressor found in Staphylococcus aureus^[1], reported to react with HS_nH and form sulfhydrated proteins, which can turn on their regulated genes^[2] . Compared with other HS_nH sensors, it shows significant response to HS_nH induction. And it shows no response to H₂O₂, a structural analogue of HSSH and a common intracellular metabolite(Figure 1.).^[3]

Figure 1. Test of reported HS_nH-sensitive TFs in E. coli. (A) Schematic representation of FisR, CstR, and BigR testing plasmids. These plasmids were transformed into E. coli BL21. (B) CstR and BigR based plasmids responded to HS_nH induction in E. coli, while the FisR based one did not; 200 μM HS_nH was incubated with 2 mL of mid log phase cells (OD_{600 nm = 0.5) for 2 h (37 °C, 225 rpm shaking). mKate fluorescence was detected by flow cytometry. (C) BigR based plasmid responded to H2O2 (1 mM) and CstR based plasmid did not. (D) CstR based plasmid only responded to S8 and HSnH.^[3]}

Results

1. To figure out how CstR works by sensing H₂S, we characterized the function of CstR with Pcstr using pTrchis2A-CstR-Pcstr-mKate-CpSQR(donated by Professor Liu from Shandong University). The expression level regulated by Pcstr at different concentrations of S^2- was shown by the fluorescence intensity of mKate(/OD_{600 nm}).

At the beginning, bacteria cultured in 5 mL were treated with 0, 6.5, 13.0, 26.0 and 39.0 mg/L Na₂S (0, 20, 40, 80 and 120 mg/L Na₂S·9H₂O) respectively, and then the fluorescence intensity of mKate was measured with a microplate reader(Figure 2.).

Figure 2. (A)The fluorescence intensity variation relative to the concentration of Na₂S; (B)The photo of the bacteria solution treated by gradient concentrations of Na₂S;(C)The curve of fluorescence intensity relative to time; (D)The curve of OD_{600 nm} relative to time

It can be seen that the presence of S^2- at different concentrations has no significant effect on the growth of bacteria. However, with the increase of S^2-, the fluorescence intensity increases at first and then decreases, indicating that when S^2- is too high, the lifting effect on CstR inhibition is weakened. We hope to find out an appropriate concentration range in which the strength of Pcstr is positively correlated with the concentration of S^2-, which tells us that we need to reduce the concentration gradient and concentration range for further characterization.

We further used 0, 3.25, 6.50, 9.75, 13.00, 16.25 mg/L Na₂S (0, 10, 20, 30, 40 and 50 mg/L Na₂S·9H₂O) to treat 5 mL bacterial solution and then measured the fluorescence intensity of mKate using a microplate reader(Figure 3.).

Figure 3. The fluorescence intensity variation relative to the concentration of Na₂S with a smaller range

It can be seen from the figure that in a smaller concentration range, there is a positive correlation between fluorescence intensity and Na₂S concentration, visible to naked eyes, which implies that we can use data within this range to regulate Pcstr by giving different concentration H₂S.

2. Also, to get this part for our plasmid construction, we successfully amplified CstR-Pcstr(Figure 3A), which was proved correct by sequencing and we has cloned it into our backbone to construct CstR-Pcstr-MazE-Dre(Figure 3B),which was also proved correct by sequencing.

Figure 4. Agarose electrophoresis

References

• [1]Grossoehme, N., et al., Control of Copper Resistance and Inorganic Sulfur Metabolism by Paralogous Regulators in Staphylococcus aureus. Journal of Biological Chemistry, 2011. 286(15): p. 13522-13531.
• [2]Giedroc, D.P., A new player in bacterial sulfide-inducible transcriptional regulation. Mol Microbiol, 2017. 105(3): p. 347-352.
• [3]Liu, H., et al., Synthetic Gene Circuits Enable Escherichia coli To Use Endogenous H(2)S as a Signaling Molecule for Quorum Sensing. ACS Synth Biol, 2019. 8(9): p. 2113-2120.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 133
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 133
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 110
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 133
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 133
  
• 1000
  COMPATIBLE WITH RFC[1000]