Fig 1.
Distance between rrnA and rrnD operons under various stress conditions.
(A) Scheme for rrn operon tagging. Two different par sites (parSpMT and parSP1) were placed upstream of rrnA and rrnD, respectively, in the parent strain MG1655. These sites were visualized by co-expression of their cognate fluorescent ParB proteins (pMT1 GFP-ParB and P1 CFP-ParB). (B) Violin plots of the distance between rrnA-rrnD under indicated stress conditions (see Methods for details and S1 Table for strains and plasmids). The median distance (nm) between labeled loci is given below each graph. Between 400 and 900 foci are shown for each sample. The solid line indicates the median distance, and the top and bottom dashed lines indicate the third and first quartile, respectively. We note that due to the large number of foci observed, a small change in the median distance is considered statistically significant (p < 0.001) under Mann–Whitney test. The data shown are for one of three biological replicates. We arbitrarily considered a 2.5-fold change of median distance to be significant. We also note that since we have values that are 0, the border tries to encompass these data points, giving a false impression of negative distances. Data used for plotting this, and all other graphs can be found in the Supplemental “S1 Data” file under appropriate figure headings. (C) A representative image of rrnA-rrnD foci without added stress; many such images were used to generate data in (B). GFP and CFP were false-colored and enhanced for better visualization. One cell is outlined, with an arrow pointing to a merged GFP/CFP focus. The foci are edited in post-processing as a perfect circle to provide better contrast and visualization. Most cells appear to contain two copies of the rrn operons, indicating that this region of the chromosome is replicated. (D) Representative image of rrnA-rrnD de-clustering with heat stress. Panel labels as in C. Colored arrows indicate focus from either GFP-field (green) or CFP (red). We note that the number of rrnA-GFP foci is reduced to 1 focus per cell. See Methods for the code used to analyze fluorescent foci in this, and all similar figures. Created in BioRender. Ho, K. (2025) https://BioRender.com/p78s829.
Fig 2.
σH promotes de-clustering of rrnA-rrnD independent of its transcriptional activity.
(A) Distance between rrnA-D with ectopic expression of rpoH from pTc (+). Induction was carried out for one hour prior to microscopy. In the fourth plot from left, rpoD is cloned downstream of TetR, from a constitutively expressed promoter, divergent from pTc. All other descriptions as in Fig 1. (B) Transcriptional activity of ectopically expressed rpoH. Miller assay was carried out as described in Methods. Student t’s test was performed pairwise to determine statistical significance (two-tailed, ns: not statistically significant, *p < 0.05, **p < 0.01, ****p < 0.0001). (C) Distance between rrnA-D with ectopic expression of indicated rpoH mutants, compared to empty vector control. (D) Transcriptional activity of ectopically expressed rpoH mutants, as described in B. Data used for plotting Fig 2 graphs can be found in the Supplemental “S1 Data” file under appropriate figure headings. Created in BioRender. Ho, K. (2025) https://BioRender.com/k54q156.
Fig 3.
Disruption of rrn clustering by heat stress and by FecI.
(A) Effect of heat stress on clustering of indicated rrn pairs. Other descriptions as in Fig 1. (B) Schematic summary of data in A. Each of the 7 rrn operons was visited at least once. (C) Response to rpoH induction of tested rrn pairs. +/− symbols are self-explanatory. (D) Effect of induced ectopic expression of five σ factors on rrnA-D distance, all placed under control of pTc with the same RBS. Data used for plotting Fig 3 graphs can be found in the Supplemental “S1 Data” file under appropriate figure headings. Created in BioRender. Ho, K. (2025) https://BioRender.com/g63t231.
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.