Indole signaling is an important cross-species communication pathway in the mammalian gut. In bacteria, upon induction by tryptophan, the molecular sensor (tnaC) controls indole biosynthesis by precisely coordinating dynamics of the corresponding macromolecular machineries during its transcription and translation. Our understanding of this regulatory program is still limited owing to its rapid dynamic nature. To address this shortcoming, we adopted a massively parallel profiling method to quantify the responses of 1,450 synthetic tnaC variants in the presence of three concentrations of tryptophan in living bacterial cells. The resultant dataset enabled us to comprehensively probe the key intermediate states of macromolecular machineries during the transcription and translation of tnaC. We also used modeling to provide a systems-level understanding of how these critical states collectively shape the output of this regulatory program quantitatively. A similar methodology will likely apply to other poorly understood dynamics-dependent cis-regulatory elements.
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NGS raw data from FACS–seq have been deposited in the NCBI Sequence Read Archive under BioProject PRJNA503322. Any other data or materials related to this work are available from the corresponding author upon request.
The Python script used to process raw FACS–seq data and perform reliability test simulation can be accessed at https://github.com/wtmbiohacker/FACS_NGS.git. The custom Python script used to perform the modeling of macromolecular coordination during the tnaC sensor response can be accessed at https://github.com/wtmbiohacker/tnaC_kinetics_modeling. We also provide a comprehensive user guide to enable reuse of this code.
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We would like to thank C. Pan for support in FACS experiment. We thank C. Guan and H. Feng from the C. Zhang and H. Xing laboratories for their assistance in treatment of raw NGS data and phylogenetic analysis. We also would like to thank J. Zhang for critical discussion about the dynamic process, Q. Hu for critical discussion of structural biology and J. Liu for comments on the manuscript. This work was supported by the National Natural Science Foundation of China (NSFC21676156, to C.Z.), Tsinghua University Initiative Scientific Research Program (20161080108, to C.Z.), the National Natural Science Foundation of China (NSFC21627812, to X.-H.X.), a postdoctoral innovation support plan from the China Postdoctoral Science Foundation (to T.W.) and a postdoctoral fellowship from the Tsinghua–Peking Joint Center for Life Sciences (to T.W.).
The authors declare no competing interests.
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Supplementary Tables 1–9, Supplementary Figs. 1–29, Supplementary Note
The result of FACS-seq about the response of each sensor variant (mean value, u)
The result of FACS-seq about the response of each sensor variant (noise, σ)
The nucleotide sequences of the 1,450 synthetic tnaC variants used in this work
The nucleotide sequences of all collected tnaC genes from gut bacteria
The mRNA folding energy values of tnaC variants with mutations covering codon 2~9 (n = 444)
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Wang, T., Zheng, X., Ji, H. et al. Dynamics of transcription–translation coordination tune bacterial indole signaling. Nat Chem Biol 16, 440–449 (2020). https://doi.org/10.1038/s41589-019-0430-3