Spatial organization of the flow of genetic information in bacteria

Abstract

Eukaryotic cells spatially organize mRNA processes such as translation and mRNA decay. Much less is clear in bacterial cells where the spatial distribution of mature mRNA remains ambiguous. Using a sensitive method based on quantitative fluorescence in situ hybridization, we show here that in Caulobacter crescentus and Escherichia coli, chromosomally expressed mRNAs largely display limited dispersion from their site of transcription during their lifetime. We estimate apparent diffusion coefficients at least two orders of magnitude lower than expected for freely diffusing mRNA, and provide evidence in C. crescentus that this mRNA localization restricts ribosomal mobility. Furthermore, C. crescentus RNase E appears associated with the DNA independently of its mRNA substrates. Collectively, our findings show that bacteria can spatially organize translation and, potentially, mRNA decay by using the chromosome layout as a template. This chromosome-centric organization has important implications for cellular physiology and for our understanding of gene expression in bacteria.

Fig. 1

groESL mRNAs remain confined within subcellular regions. a, Visualization of groESL mRNAs in wild-type cells by RNA FISH using a groEL-Cy3 LNA probe. b, Visualization of groESL-lacO₁₂₀ mRNAs in CJW2966 cells using a lacO-Cy3 LNA probe. Note that the contrast of the lacO-Cy3 signal is scaled differently from (a) as it was significantly brighter.

Fig. 2

groESL and creS mRNAs largely remain at the site of birth for their entire lifespan. a, Representative FISH intensity profiles of groESL-lacO₁₂₀ mRNA (using lacO-Cy3 probe) along the cell length in individual CJW2966 cells. The red dashed line represents the background fluorescence. b, Co-visualization of groESL-lacO₁₂₀ gene locus and mRNA in CJW2969 cells. c, Schematic of the full width at half maximum (FWHM) values obtained from intensity profiles along cell length (left); histograms of FWHM values of groESL-lacO₁₂₀ mRNA (blue) and DNA (green) signals using RNA FISH and the lacO-Cy3 probe or DNA FISH and the lacO-Rev-FITC probe, respectively (right). d, Same as in (a) for creS-lacO₁₂₀ mRNAs in CJW2967 cells. e, Co-visualization of creS-lacO₁₂₀ mRNA and tetO₂₄₀- tagged DNA origins in CJW3102 cells.

Fig. 3

Endogenous LacZ-encoding mRNAs display diffraction-limited dispersion from sites of transcription in E. coli. a, RNA-FISH of wild-type MG1655 E. coli cells using 48 Cy3-labeled DNA probes complementary to the lacZ mRNA sequence after 20 min of IPTG induction. b, Representative FISH intensity profiles of lacZ mRNA signal in individual MG1655 cells. The red dashed line represents the background fluorescence from non-induced cells. c, Visualization of background fluorescence in non-induced MG1655 cells using conditions as in (a). d, Representative fluorescence intensity profiles of individual non-induced cells. The red dashed line represents background fluorescence from non-induced cells. e, Histogram of FWHM values of lacZ mRNA signals from IPTG-induced cells. f, Co-visualization of a chromosomal tetO₂₅₀ array (inserted into the cynX locus adjacent to the lac operon) and the lacZ mRNA signal in DL2875 E. coli cells.

Fig. 4

Dispersion of groESL-lacO₁₂₀ mRNA. a, Spatial distribution profiles of the 6.3-kb groESL-lacO₁₂₀ mRNA distribution profiles in a 3-μm virtual cell calculated with Eq.[6] (Supplementary Information), assuming that groESL-lacO₁₂₀ mRNA is freely diffusible and either ribosome-free (blue) or saturated with ribosomes (red). The dotted line delineates the source of mRNA (site of transcription). b, RNA-FISH image of lacO-Cy3 hybridized-groESL-lacO₁₂₀ mRNAs in CJW2966 cells after 15 min at 42°C. c, Representative lacO-Cy3 hybridized-groESL-lacO₁₂₀ mRNA intensity profiles of individual, heat-shocked CJW2966 cells. The blue dots are the experimental data and the black line is the best fit using the two-source (right) or one-source (left) model (see Supplementary Information). The red dashed line corresponds to the background fluorescence. d, Histogram of FWHM values of groESL-lacO₁₂₀ mRNA from fluorescence intensity profiles of CJW2966 cells grown at 30°C (blue) or after heat shock (42°C for 15 min; red).

Fig. 5

mRNA limits diffusion of translating ribosomes. a, Co-visualization of L1-GFP and DNA (DAPI) in CJW3365 cells. b, Fluorescence loss in photobleaching experiment. A 3.3-s laser pulse was used to bleach a small region of CJW3365 cells producing L1-GFP, either without (left) or with (right) rifampicin pre-treatment.

Fig. 6

RNase E colocalizes with the DNA in C. crescentus. a, Fluorescence and corresponding phase micrographs of CJW3100 cells producing RNase E-mGFP. b, Covisualization of RNase E-mGFP and DNA (DAPI) in CJW3099 cells grown at the restrictive temperature (37°C) for 4 h to produce large DNA-free regions. c, Same as (b) except after rifampicin pre-treatment.