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Pap final 23.07.20.pdf (2.39 MB)

A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA in an All-E. Coli Cell-Free Expression System

preprint
submitted on 23.12.2020, 09:47 and posted on 24.12.2020, 06:08 by Emanuel Worst, oemer Kurt, Marc Finkler, Marc Schenkelberger, Vincent Noireaux, Volkhard Helms, Albrecht Ott

Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary, alternate DNA methylation of only two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study the function of pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. Based on our observations we suggest that Lrp and the conformation of the self-complementary regulatory DNA play a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for engineering stable, hereditary and strong expression control based on methylation.

Funding

SFB 1027:  Physical Modelling of Non-Equilibrium Processes in Biological Systems

Deutsche Forschungsgemeinschaft

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History

Email Address of Submitting Author

albrecht.ott@physik.uni-saarland.de

Institution

Universität des Saarlandes

Country

Germany

ORCID For Submitting Author

0000-0003-0481-2658

Declaration of Conflict of Interest

None

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Initial Version

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