Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria
Gene regulation is one of the most ubiquitous processes in biology. However, while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here, we take a first step toward multipromoter dissection and show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect multiple bacterial promoters in a systematic way. We show this approach on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all cases, we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input–output relationships. Given the generality of the approach presented here, it opens up the possibility of quantitatively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.
© 2018 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). Edited by Curtis G. Callan Jr., Princeton University, Princeton, NJ, and approved April 6, 2018 (received for review December 19, 2017). We thank David Tirrell, Bradley Silverman, and Seth Lieblich for access to their Beckman Coulter MoFlo XDP cell sorter. Jost Vielmetter and Nina Budaeva provided access to their Cell Disruptor. We also thank Hernan Garcia, Manuel Razo-Mejia, Griffin Chure, Suzannah Beeler, Heun Jin Lee, Justin Bois, and Soichi Hirokawa for useful discussion. This work was supported by La Fondation Pierre-Gilles de Gennes; the Rosen Center at Caltech; NIH Grants DP1 OD000217 (Director's Pioneer Award), R01 GM085286, 1R35 GM118043-01 (Maximizing Investigators' Research Award), and 1S10RR029594-01A1; the Gordon and Betty Moore Foundation through Grant GBMF227; and the Beckman Institute. N.M.B. was supported by an HHMI International Student Research Fellowship. Author contributions: N.M.B., D.L.J., and R.P. designed research; N.M.B., S.L.B., D.L.J., M.J.S., A.M., S.H., and J.B.K. performed research; N.M.B., W.T.I., M.J.S., A.M., and J.B.K. analyzed data; N.M.B., S.L.B., and R.P. provided conceptualization; N.M.B., S.L.B., W.T.I., D.L.J., and J.B.K. provided methodology; N.M.B. provided investigation; N.M.B., W.T.I., D.L.J., M.J.S., and J.B.K provided software; N.M.B., W.T.I., M.J.S., and J.B.K. provided validation; S.H. and R.P. acquired funding; N.M.B., S.L.B., J.B.K., and R.P. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: Raw sequencing files have been deposited on the NCBI Sequence Read Archive, https://www.ncbi.nlm.nih.gov/sra (accession no. SRP121362) and Sort-Seq regulatory sequencing data from Escherichia coli has been deposited in NCBI BioSample, https://www.ncbi.nlm.nih.gov/biosample (accession no. SAMN07830099). Thermo RAW mass spectrometry files have been deposited in the jPOST Repository, https://repository.jpostdb.org (accession no. PXD007892). Files containing processed data and python code association with data analysis and plotting have been deposited on GitHub and Zenodo (available at https://www.github.com/RPGroup-PBoC/sortseq_belliveau and https://doi.org/10.5281/zenodo.1184169, respectively). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1722055115/-/DCSupplemental.
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