Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid

Abstract

Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

Keywords: antibiotics; nucleoid exclusion; single-molecule imaging; single-molecule tracking; transcription-translation coupling.

Fig. 1.

Tracking of individual ribosomal subunits in living E. coli cells. The cells were imaged at 50 Hz for 5 min on agarose pads with a laser excitation exposure time of 5 ms. The geometries of the imaged cells were determined from the positions of the individual ribosomal subunits. The lengths of the imaged cells were determined to be between 1.8 and 2.9 µm. Similar results are obtained if the geometries of the imaged cells are determined from out-of-focus bright-field images (SI Appendix, Fig. S9). (A) Distributions of apparent diffusion coefficients of individual large (Left) and small (Right) ribosomal subunits. The distributions in the untreated cells (red bars) are fitted with the corresponding distributions in rifampicin-treated cells (blue outlines) in the regions indicated by solid cyan lines. Each distribution corresponds to >1,000 trajectories from eight cells. (B) Trajectories of individual ribosomal subunits. Trajectories of free and mRNA-bound subunits are plotted separately. (C) Distributions of relative short-axis positions of bound and free ribosomal subunits in the cylindrical parts of eight cells (solid red curves) fitted with a model for nucleoid-excluded particles (dashed blue curves). Each distribution corresponds to >1,000 positions. The relative exclusion radius (r_e) is 0 for an evenly distributed particle (dashed green curves) and 1 for a membrane-bound particle.

Fig. 2.

High-throughput imaging and tracking of individual HU proteins and small ribosomal subunits in living E. coli cells in microfluidic devices. (A) Phase-contrast (Center Left), bright-field (Center Right), and fluorescence (Right) images of a microcolony in a microfluidic device (Left). The microfluidic device contains 51 cell traps with dimensions of 40 × 40 × 0.9 µm³. Each trap contains ∼200 E. coli cells that grow in a monolayer. The traps are surrounded by 10-µm-deep flow channels that provide the cells with fresh medium for exponential growth and flush out cells that grow out of the traps. The cell contours are determined from the phase-contrast image. The bright-field and phase-contrast images are used to map the positions of the HU proteins and ribosomal subunits to internal cell coordinates. (B) Spatial distributions of HU proteins (Left) and small bound (Center) and free (Right) ribosomal subunits in living E. coli cells at different stages of the cell-division cycle. Each microcolony was imaged at 50 Hz for 32 s with a laser excitation exposure time of 5 ms. The probability density maps were constructed by generating histograms of normalized positions for three different cell length intervals. The cell length intervals are 1.5–2.5 µm, 2.5–3.5 µm, and 3.5–4.5 µm. Each probability density map corresponds to >5,000 positions from >500 cells.

Fig. 3.

Mean square displacements of mRNA-bound (Left) and free (Right) ribosomal subunits in living E. coli cells. The cells were imaged at 10 Hz for 5 min on agarose pads with a laser excitation exposure time of 5 ms. The mean square displacements of the bound subunits are also included in the right plot as a reference. Each experimental curve corresponds to >70 trajectories from 16 cells. The simulated mean square displacements (dashed curves) were obtained by simulating 10,000 normal diffusion trajectories in a sphere with a radius of 300 (green curve) or 800 (cyan curve) nm. The diffusion coefficient was set to 0.055 (green curve) or 0.40 (cyan curve) μm²·s⁻¹. The error bars represent SEMs.

Fig. 4.

Tracking of individual HU proteins in untreated (Left) and erythromycin-treated (Right) E. coli cells. The cells were imaged at 50 Hz for 5 min on agarose pads with a laser excitation exposure time of 5 ms. The geometries of the imaged cells were determined from out-of-focus bright-field images. The lengths of the imaged cells were determined to be between 2.0 and 3.2 µm. (A) Distributions of apparent diffusion coefficients of individual HU proteins. Each distribution corresponds to >900 trajectories from eight cells. (B) Trajectories of individual HU proteins. (C) Distributions of relative short-axis positions of HU proteins in the cylindrical parts of eight cells (solid red curves) fitted with a model for nucleoid-associated particles (dashed blue curves). Each distribution corresponds to >10,000 positions. r_n is the relative nucleoid radius.

Fig. 5.

Tracking of individual ribosomal particles in erythromycin-treated E. coli cells. The cells were imaged at 50 Hz for 5 min on agarose pads with a laser excitation exposure time of 5 ms. The geometries of the imaged cells were determined from the positions of the individual ribosomal subunits. The lengths of the imaged cells were determined to be between 1.8 and 2.9 µm. Similar results are obtained if the geometries of the imaged cells are determined from out-of-focus bright-field images (SI Appendix, Fig. S10). (A) Distributions of apparent diffusion coefficients of individual ribosomal particles. (Left) Large ribosomal subunits. (Right) Small ribosomal subunits. The distributions in the erythromycin-treated cells (red bars) are fitted with the corresponding distributions in rifampicin-treated cells (blue outlines) in the regions indicated by solid cyan lines. Each distribution corresponds to >1,000 trajectories from eight cells. (B) Trajectories of individual ribosomal particles. Trajectories of free and mRNA-bound particles are plotted separately. (C) Distributions of relative short-axis positions of bound and free ribosomal particles in the cylindrical parts of eight cells (solid red curves) fitted with a model for nucleoid-excluded particles (dashed blue curves). Each distribution corresponds to >1,000 positions. The relative exclusion radius (r_e) is 0 for an evenly distributed particle (dashed green curves) and 1 for a membrane-bound particle.