Membrane-associated σ factors disrupt rRNA operon clustering in Escherichia coli
Fig 4
Expression of rpoH disrupts rrn clustering as tracked by Mu transposition.
(A) Frequency of transposition of Mu located in the vicinity of rrnD (Bin 73) after one round of transposition. The number of insertions has been normalized to the read depth of each bin but not normalized to copy number. In the asynchronous population, many cells have partially replicated chromosomes [8]. The initial Mu position is indicated by a red triangle. The Escherichia coli genome was partitioned into 100 equally sized bins, so each bin is ~46 kb. Starting bin number and chromosomal regions are indicated on top. Each vertical bar represents the average normalized transposition frequency of three biological replicates at the indicated bin, expressed as a percentage (with the highest transposition frequency being set to 100%). Gray error bars are the standard deviation. Color of bars correspond to regions of the E. coli chromosome annotated up top [8]. (B) Same as (A) but with the rpoH vector. (C) Same as (B) but with induction of rpoH. (D) Heat map of the data from A through C. In the no-vector control, the highest insertion frequency (yellow bar) occurs in Bin 94 (close to oriC which resides in Bin 90). (E–J) Transposition frequency from mlaF (rrnD) into the indicated rrn operon-containing bin under conditions described in A through C. The individual data points and associated standard deviations are shown. Statistical significance was determined with Student t test (two-tailed), *p < 0.05. ns: not statistically significant. (K) Model for σD-mediated rrn clustering. σD-bound RNAP (dark green) is responsible for clustering rrns (blue circles) at the membrane either directly or through other unknown factor(s) (gray hexagon). GroEL/ES (red oval) represses the activity of σH (orange) preventing it from competing with σD for core RNAP. σH is typically localized to the inner membrane and degraded. Upon heat stress, σH levels rise, and GroEL/ES, being diverted to un-folded substrates (pink oval), is unable to repress σH activity. Elevated σH levels from a plasmid, phenocopy the heat response. σD-bound RNAP complexes are displaced through competition with increased σH, and σH-bound RNAP now transcribes genes in σH regulon (green circles), lowering the total amount of RNAP transcribing rrn operons at the membrane, destabilizing clustering. Data used for plotting Fig 4 graphs can be found in the Supplemental “S1 Data” file under appropriate figure headings. Created in BioRender. Ho, K. (2025) https://BioRender.com/d36h492.