posted on 2021-09-22, 19:07authored byEmanuel
G. Worst, Marc Finkler, Marc Schenkelberger, Ömer Kurt, Volkhard Helms, Vincent Noireaux, 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 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.