Time-dependent effects of transcription- and translation-halting drugs on the spatial distributions of the Escherichia coli chromosome and ribosomes

Abstract

Previously observed effects of rifampicin and chloramphenicol indicate that transcription and translation activity strongly affect the coarse spatial organization of the bacterial cytoplasm. Single-cell, time-resolved, quantitative imaging of chromosome and ribosome spatial distributions and ribosome diffusion in live Escherichia coli provides insight into the underlying mechanisms. Monte Carlo simulations of model DNA-ribosome mixtures support a novel nucleoid-ribosome mixing hypothesis. In normal conditions, 70S-polysomes and the chromosomal DNA segregate, while 30S and 50S ribosomal subunits are able to penetrate the nucleoids. Growth conditions and drug treatments determine the partitioning of ribosomes into 70S-polysomes versus free 30S and 50S subunits. Entropic and excluded volume effects then dictate the resulting chromosome and ribosome spatial distributions. Direct observation of radial contraction of the nucleoids 0-5 min after treatment with either transcription- or translation-halting drugs supports the hypothesis that simultaneous transcription, translation, and insertion of proteins into the membrane ('transertion') exerts an expanding force on the chromosomal DNA. Breaking of the DNA-RNA polymerase-mRNA-ribosome-membrane chain in either of two ways causes similar nucleoid contraction on a similar timescale. We suggest that chromosomal expansion due to transertion enables co-transcriptional translation throughout the nucleoids.

Figure 1

E. coli nucleoid morphology vs time from SYTOX Orange staining of a single cell. (A) Time course of phase contrast images. (B) Time course of SYTOX Orange-stained DNA images. (C) Length of the cell (L_cell) is determined from contour length of the central spine of the phase contrast image. (D) Normalized cell length vs time for 12 cells from the same field of view. The mean is plotted as a thick black line. The grey swath shows the extremes of cell-to-cell variability. The culture is not synchronized. Part of the cell-to-cell variability is due to the time-independent contributions of the two endcaps to total cell length, a larger fraction of L_cell for short cells than for long cells. (E) Fluorescence image of a cell stained with SYTOX Orange and imaged with the 561 nm laser, defining the nucleoid length (L_DNA) and width (W_DNA). (F) Width W_DNA (t) of the nucleoids vs time for the same 12 cells as in (D), normalized to W_DNA (t = 0). (G) Length of the nucleoids L_DNA (t) relative to the cell length L_cell (t) for the same 12 cells. Scale bars in (A), (C), and (E) are 1 μm. The mean behavior shown in panels D, F, and G reproduced across five separate experiments with at least 10 cells each.

Figure 2

Effect of rifampicin on nucleoid morphology and ribosome spatial distribution. (A) Snapshots of a SYTOX Orange-stained cell before and after injection of Rif (300 μg/mL) at t = 0. Times in minutes. The phase contrast image of the cell is shown at top. Scale bar =1 μm. (B) SYTOX Orange radial fluorescence intensity profile (along the short, y-axis) for three different times. (C) SYTOX Orange axial fluorescence intensity profile (along the x-axis) for three different times. (D) Snapshots of ribosomal 30S subunits labeled as S2-YFP from a different Rif-treated cell of similar length to that in (A). Scale bar =1 μm. (E) Mean normalized cell length vs time (thick orange line) and range of lengths (orange swath) for 36 Rif-treated cells. Grey line is mean for untreated cells. (F) Mean and range of normalized nucleoid width vs time for 11 Rif-treated cells (orange) from the same experiment. Grey is mean for untreated cells. The experiment was repeated five times and consistently yielded the same result. (G) Mean and range of normalized nucleoid length vs time for 11 Rif-treated cells (orange). Grey is mean for untreated cells. (H) Distribution of single-molecule diffusion constants D_i from 30S-mEos2 trajectories of untreated cells (grey) and Rif-treated cells (orange). D_i values are estimated from 6-step, 60 ms/frame trajectories with lag time τ = 180 ms. (I) Mean diffusion constant of the 30S-mEos2 label at different times after injection of Rif. Horizontal bars represent the duration of data acquisition; vertical bars represent estimated errors in initial slope of MSD plots. (J) Orange: Mean total intensity of SYTO RNAselect fluorescence from cells at different time lags after Rif injection. Grey: Control cells growing without Rif treatment. The mean behavior shown in panels E, F, and G reproduced across five separate experiments with at least 10 cells each.

Figure 3

(A) Model polysome of thirteen 70S beads (20-nm diameter) dissociates into 50S beads (17 nm diameter) and 30S beads (14 nm diameter). (B) Equilibrated distribution of DNA and polysomes within a 50-nm thick central slice showing DNA (top) and polysomes (middle) and the composite (bottom). (C) 50-nm thick central slice through a snapshot of the equilibrated distributions of DNA polymer (first image) in presence of 50S subunits (second) and 30S subunits (third), along with the composite (fourth). (D) Relative axial density of DNA beads (along the x-axis) in DNA-polysome simulation (green) and in DNA-30S-50S simulation (red). Dashed line shows distribution for a uniformly filled spherocylinder. (E) Relative axial density (along the x-axis) of polysome beads (green) in DNA-polysome simulation and of 50S (blue) and 30S (yellow) subunits in DNA-30S-50S simulation. Dashed line shows distribution for a uniformly filled spherocylinder.

Figure 4

Effect of translation inhibitors on nucleoid morphology and ribosome distribution. (A) Snapshots of SYTOX Orange-stained cell before and after injection of Cam (300 μg/mL) at t = 0. Times in minutes. The phase contrast image of the cell is shown at top. (B) Snapshots of ribosome (30S-YFP) distribution from another Cam-treated cell of comparable size. (C and D) Snapshots of one SYTOX Orange-stained cell and a different 30S-YFP labeled cells before and after injection of Ksg (5 mg/mL) at t = 0. Times in minutes. Normalized cell length (E), normalized nucleoid width (F) and relative nucleoid length (G) are plotted vs time for cells treated with Cam (300 μg/mL) and Ksg (5 mg/mL). The mean trend is the thick line. The colored swath shows the cell-to-cell variability. N_cell = 13 for Cam and 18 for Ksg. Mean of data from untreated cells is heavy grey line. Cells treated with Cam are Ksg are shown in red and blue, respectively. (H) Distribution of ribosome (30S-YFP) diffusion constants D_i for untreated cells (left, gray), cells treated with Cam for 15 min (middle, red), and cells treated with Ksg for 15 min (right, blue). D_i values are estimated from 6-step, 100 ms/frame trajectories with lag time τ = 300 ms. (I) Mean ribosome (30S-YFP) diffusion constant from MSD plots at different time intervals after injection of Cam (300 μg/mL) and Ksg (5 mg/mL). Heavy lines are from 5-point smoothing of data. Scale bars in parts (A) to (D) are 1 μm. The mean behavior shown in panels E, F, and G reproduced across five separate experiments with at least 10 cells each.

Figure 5

(A) Schematic of DNA-tethering to the membrane via transertion inside E. coli cell. (B) Schematic of nucleoid-ribosome mixing hypothesis. In normal growth condition 70S-polysomes dominate, and they are immiscible with the nucleoids due to excluded volume and entropic effects. This creates spatial segregation between the polysomes and nucleoid (top). Rif treatment causes dissociation of 70S-polysomes into free 30S and 50S subunits. The 30S and 50S subunits mix readily with the nucleoids and cause expansion of the nucleoid (bottom).