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

Emanuel G. Worst, Marc Finkler, Marc Schenkelberger, Ömer Kurt, Volkhard Helms, Vincent Noireaux, Albrecht Ott

Research output: Contribution to journalArticlepeer-review

Abstract

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 DNA methylation of either one of 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 molecular functions of the pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. On the basis of our observations we suggest that besides Lrp, the conformation of the self-complementary regulatory DNA plays 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 mimicking stable, hereditary, and strong expression control based on methylation. The conformation of the regulatory DNA corresponds to a Holliday junction. Gene expression must be expected to respond if opposite arms of the junction are drawn outward.

Original languageEnglish (US)
Pages (from-to)2725-2739
Number of pages15
JournalACS Synthetic Biology
Volume10
Issue number10
DOIs
StatePublished - Oct 15 2021

Bibliographical note

Funding Information:
We acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG) within the collaborative research center (CRC/SFB 1027) as well as financial support from the Human Frontiers Science Program Organization (HFSP, RGP0037/2015). We thank Julia Flesch and Lucina Kainka for their assistance with the amplification of DNA templates. We thank Po-Hsien for fruitful discussions.

Publisher Copyright:
© 2021 American Chemical Society.

Keywords

  • E. coli
  • Lrp
  • cell-free expression
  • methylation patterns
  • pap switch
  • pyelonephritis associated pili

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't

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