Part:BBa_K3350857

yhaJ (DNA-binding transcriptional activator)
Several transcriptional regulatory protein families are involved in the metabolism of aromatic compounds in bacteria, and have differences in both their structures and in the activation mechanisms. The LysR-type transcriptional regulators (LTTRs) are characterized by an N-terminal DNA-binding helix-turn-helix motif and a C-terminal co-inducer-binding domain. According to Tropel and V an Der Meer, all LTTRs involved in aromatic compounds degradation pathways can act as transcriptional activators with the co-inducers, which are either degraded compounds or intermediates in the degradation pathways  ^[1]. The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the yqjF (BBa_K1316002) promoter.

Usage and Biology

The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the yqjF (BBa_K1316002) promoter. We generated the yqjF3rd (BBa_K3350862) promoter that shows much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type yqjF promoter,and we overexpressed the yhaJ gene together with an yqjF3rd-containing reporter construct in the engineered bacteria, we could reduce the DNT detection threshold of the yqjF promoter from 25 mg/L to 0.25 mg/L, which is a 100-fold increase of the sensitivity.

Characterization and Measurement

We had optimized the yqjF promoter to obtain its new version yqjF3rd (BBa_K3350862). To further improve the DNT detection sensitivity, we planned to overexpression yhaJ. We generated the engineered bacteria with YhaJ overexpression and tested their response to different concentrations of DNT. As shown in Fig. 1, YhaJ overexpression could remarkably reduce the DNT detection threshold from 25 mg/L to 0.25 mg/L, which is a 100-fold increase of the sensitivity. For the mechanism underlying this significantly enhanced sensitivity, we predicted that 2,4,5-trihydroxytoluene (THT) molecules could have saturated the endogenous YhaJ proteins to activate their transcriptional activity; With overexpressed YhaJ, more THT would have chances to bind increasingly expressed YhaJ and cause their activation, consequently leading to robustly enhanced EGFP transcription even at relatively low amounts of DNT.

Fig. 1. Effects of YhaJ overexpression on DNT detection sensitivity of engineered bacteria.

References

[1] Palevsky, N., Shemer, B., Connolly, J. P., & Belkin, S. (2016). The Highly Conserved Escherichia coli Transcription Factor YhaJ Regulates Aromatic Compound Degradation. Frontiers in microbiology, 7, 1490.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]