An Hfq-dependent post-transcriptional mechanism fine tunes RecB expression in Escherichia coli

Irina Kalita author has email address
Ira Alexandra Iosub
Lorna McLaren
Louise Goossens
Sander Granneman
Meriem El Karoui author has email address

Centre for Engineering Biology, University of Edinburgh, Edinburgh, United Kingdom
Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
Max Planck Institute for Terrestrial Microbiology & Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany
Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Edinburgh, United Kingdom
The Francis Crick Institute, London, United Kingdom

https://doi.org/10.7554/eLife.94918.2

Open access
Copyright information

Figures and data

recB mRNAs are low abundant, short-lived and constitutively expressed.
A: Schematic description of the recBCD locus, its location on the E. coli chromosome and the corresponding mRNAs. B: Examples of fluorescence and bright-field images of recB mRNA FISH experiments in wild-type (WT) and ΔrecB strains. Scale bars represent 2 µm. C: Total recB mRNA distribution quantified with smFISH and presented in molecule numbers per cell. The histogram represents the average across three replicated experiments; error bars reflect the maximum difference between the repeats. Total number of cells, included in the distribution, is 15,638. D: recB mRNA degradation rate measured in a time-course experiment where transcription initiation was inhibited with rifampicin. Mean mRNA counts, calculated from the total mRNA distributions for each time point, were normalized to the mean mRNA number at time t = 0 and represented in the natural logarithmic scale. Vertical error bars represent the standard error of the mean (s.e.m.); horizontal errors are given by experimental uncertainty on time measurements. Shaded area shows the time interval used for fitting. The red line is the fitted linear function, −γ_mt, where γ_m is the recB mRNA degradation rate. The final degradation rate was calculated as the average between two replicated time-course experiments (Table S7). E: recB mRNA molecule numbers per cell from the experiments in Fig. 1C shown as a function of cell area. The black circles represent the data binned by cell size and averaged in each group (mean +/-s.e.m.). The solid line connects the averages across three neighbouring groups. Based on the mean mRNA numbers, all cells were separated into three sub-populations: newborns, cells in transition and adults. F: Experimental data from Fig. 1C conditioned on cell size (<3.0 µm², newborns; total cell number is 2,180) fitted by a negative binomial distribution, NB(r, p). The Kullback–Leibler divergence between experimental and fitted distributions is D_KL = 0.002.

RecB proteins are low abundant, long-lived and show an evidence of noise reduction.
A: Schematic of RecB-HaloTag fusion at the endogenous E. coli chromosomal locus of the recB gene. RecB-HaloTag is conjugated to the Janelia Fluor 549 dye (JF549). B: Examples of fluorescence and bright-field Halo-labelling images for the strain with the RecB-Halo fusion and its parental (no HaloTag) strain. Both samples were labelled with JF549 dye and the images are shown in the same intensity range. Scale bars represent 2 µm. Zoom-in: An example of a cell with five RecBHalo molecules (several single RecBHalo molecules are shown with light-green arrows). C: Total RecB protein distribution quantified with Halo-labelling and presented in molecules per cell. The histogram represents the average of three replicated experiments; error bars reflect the maximum difference between the repeats. Total number of analysed cells is 10,964. Estimation of false positives in no HaloTag control resulted in ∼0.3 molecules/cell. D: RecB removal rate measured in a pulse-chase Halo-labelling experiment where the dye was removed at time t = 0. Mean protein counts, calculated from the total protein distributions for each time point, were normalized to the mean protein number at time t = 0 and represented in the natural logarithmic scale. Shaded area shows the time interval used for fitting. The red line is the fitted linear function, −γ_pt, where γ_p is the RecB removal rate. The final removal rate was calculated as the average between two replicated pulse-chase experiments (Table S7). E: Comparison between the experimental RecB molecule distribution from Fig. 2C conditioned on cell size (in grey) and the results of Gillespie’s simulations (SSA) for a two-stage model of RecB expression (in red). Parameters, used in simulations, are listed in Table S7. The Kullback–Leibler divergence between the distributions is D_KL = 0.15. F: Comparison of the coefficients of variation, , for experimental (Data), simulated (SSA) data and analytical prediction (Theory). The experimental and simulated data from Fig. 2E were used; error bars represent standard deviations across three replicates; the black circles show the mean in each experiment. Theoretical prediction was calculated using Eq. (1).

RecB proteins are low abundant, long-lived and show an evidence of noise reduction.
A: Schematic of RecB-HaloTag fusion at the endogenous E. coli chromosomal locus of the recB gene. RecB-HaloTag is conjugated to the Janelia Fluor 549 dye (JF549). B: Examples of fluorescence and bright-field Halo-labelling images for the strain with the RecB-Halo fusion and its parental (no HaloTag) strain. Both samples were labelled with JF549 dye and the images are shown in the same intensity range. Scale bars represent 2 µm. Zoom-in: An example of a cell with five RecBHalo molecules (several single RecBHalo molecules are shown with light-green arrows). C: Total RecB protein distribution quantified with Halo-labelling and presented in molecules per cell. The histogram represents the average of three replicated experiments; error bars reflect the maximum difference between the repeats. Total number of analysed cells is 10,964. Estimation of false positives in no HaloTag control resulted in ∼0.3 molecules/cell. D: RecB removal rate measured in a pulse-chase Halo-labelling experiment where the dye was removed at time t = 0. Mean protein counts, calculated from the total protein distributions for each time point, were normalized to the mean protein number at time t = 0 and represented in the natural logarithmic scale. Shaded area shows the time interval used for fitting. The red line is the fitted linear function, −γ_pt, where γ_p is the RecB removal rate. The final removal rate was calculated as the average between two replicated pulse-chase experiments (Table S7). E: Comparison between the experimental RecB molecule distribution from Fig. 2C conditioned on cell size (in grey) and the results of Gillespie’s simulations (SSA) for a two-stage model of RecB expression (in red). Parameters, used in simulations, are listed in Table S7. The Kullback–Leibler divergence between the distributions is D_KL = 0.15. F: Comparison of the coefficients of variation, , for experimental (Data), simulated (SSA) data and analytical prediction (Theory). The experimental and simulated data from Fig. 2E were used; error bars represent standard deviations across three replicates; the black circles show the mean in each experiment. Theoretical prediction was calculated using Eq. (1).

Translational efficiency of RecB is increased under DNA damage conditions.
A: Schematic of the experimental workflow. DSBs were induced with 4 ng/ml of ciprofloxacin for two hours, and protein (mRNA) quantification was performed with Halo-labelling (smFISH). Mean protein and mRNA concentrations were calculated from the distributions, and the average protein-to-mRNA ratio (translational efficiency) was estimated. B: Examples of bright-field images for unperturbed (Cipro⁻) and perturbed (Cipro⁺) conditions. Scale bars represent 2 µm. C: Box plot with cell area distributions for perturbed (blue) and unperturbed (grey) samples. The medians of cell area in each sample are 1.9 µm² (Cipro⁻) and 3.6 µm² (Cipro⁺). D: recB mRNA concentration distributions quantified with smFISH in intact (grey) and damaged (blue) samples. The histograms represent the average of three replicated experiments. The medians of the recB mRNA concentrations are shown by dashed lines: 0.28 mol/µm² (Cipro⁻) and 0.12 mol/µm² (Cipro⁺). Total numbers of analysed cells are 11,700 (Cipro⁻) and 6,510 (Cipro⁺). Insert: Box plot shows significant difference between the average recB mRNA concentrations (P value = 0.0023, two-sample t-test). E: RecB concentration distributions quantified with Halo-labelling in unperturbed and perturbed conditions. The histograms represent the average of three replicated experiments. The medians are shown by dashed lines: 2.12 mol/µm² (Cipro⁻) and 2.28 mol/µm² (Cipro⁺). Total number of analysed cells are 1,534 (Cipro⁻) and 683 (Cipro⁺). The difference between the average RecB concentrations was insignificant (P value = 0.36, two-sample t-test). F: The average number of proteins produced per one mRNA in intact and damaged conditions. Average translational efficiency for each condition was calculated as the ratio between the mean protein concentration c_p and the mean mRNA concentration c_m. The error bars indicate the standard deviation of the data; statistical significance between the conditions was calculated with two-sample t-test (P value = 0.0001).

RecB expression is regulated by Hfq protein in vivo.
A: Genome browser track showing Hfq binding to recC-ptrA-recB-recD mRNAs. The coverage is normalized to reads per million (RPM). The major peaks of interest are highlighted by red dashed boxes. B: Examples of fluorescence and bright-field images of RecB quantification experiments in Δhfq and wild-type strains. Yellow outlines indicate rough positions of bacterial cells in the fluorescence channel. Scale bars represent 2µm. Both fluorescence images are shown in the same intensity range while different background modulation was applied in the zoom-in figures (for better spots visualization). C: RecB concentration distributions quantified with Halo-labelling in wild-type cells and the Δhfq mutant. The histograms represent the average of five replicated experiments. The medians are shown by dashed lines: 2.12 mol/µm² for WT and 2.68 mol/µm² for Δhfq. Total number of analysed cells are 3,324 (WT) and 2,185 (Δhfq). Insert: Box plot shows significant difference (P value = 0.0007) between the average RecB concentrations in wild-type and Δhfq cells verified by two-sample t-test. D: Examples of fluorescence and bright-field images of recB mRNA FISH experiments in Δhfq and wild-type cells. Yellow outlines indicate rough positions of bacterial cells in the fluorescence channel. Scale bars represent 2 µm. E: recB mRNA concentration distributions quantified with smFISH in the Δhfq mutant and wild-type cells. The histograms represent the average of three replicated experiments. The medians are shown by dashed lines: 0.28 mol/µm² for WT and 0.21 mol/µm² for Δhfq. Total number of analysed cells are 7,270 (WT) and 5,486 (Δhfq). The insignificant difference between the average recB mRNA concentrations in both strains was verified by two-sample t-test (P value = 0.11). F: RecB translational efficiency in wild-type and Δhfq cells. Average translational efficiency (for each strain) was calculated as the ratio between the mean protein concentration c_p and the mean mRNA concentration c_m across replicated experiments. The error bars indicate the standard deviation of the data; statistical significance between the strains was calculated with two-sample t-test (P value = 0.014). G: The difference between theoretically predicted and experimentally measured coefficient of variation (squared). The theoretical values were computed according to Eq. (1). Error bars represent s.e.m. calculated from the replicated experiments. Statistical significance between the samples was calculated with two-sample t-test (P value = 0.0088).

RecB expression is regulated by Hfq protein in vivo.
A: Genome browser track showing Hfq binding to recC-ptrA-recB-recD mRNAs. The coverage is normalized to reads per million (RPM). The major peaks of interest are highlighted by red dashed boxes. B: Examples of fluorescence and bright-field images of RecB quantification experiments in Δhfq and wild-type strains. Yellow outlines indicate rough positions of bacterial cells in the fluorescence channel. Scale bars represent 2µm. Both fluorescence images are shown in the same intensity range while different background modulation was applied in the zoom-in figures (for better spots visualization). C: RecB concentration distributions quantified with Halo-labelling in wild-type cells and the Δhfq mutant. The histograms represent the average of five replicated experiments. The medians are shown by dashed lines: 2.12 mol/µm² for WT and 2.68 mol/µm² for Δhfq. Total number of analysed cells are 3,324 (WT) and 2,185 (Δhfq). Insert: Box plot shows significant difference (P value = 0.0007) between the average RecB concentrations in wild-type and Δhfq cells verified by two-sample t-test. D: Examples of fluorescence and bright-field images of recB mRNA FISH experiments in Δhfq and wild-type cells. Yellow outlines indicate rough positions of bacterial cells in the fluorescence channel. Scale bars represent 2 µm. E: recB mRNA concentration distributions quantified with smFISH in the Δhfq mutant and wild-type cells. The histograms represent the average of three replicated experiments. The medians are shown by dashed lines: 0.28 mol/µm² for WT and 0.21 mol/µm² for Δhfq. Total number of analysed cells are 7,270 (WT) and 5,486 (Δhfq). The insignificant difference between the average recB mRNA concentrations in both strains was verified by two-sample t-test (P value = 0.11). F: RecB translational efficiency in wild-type and Δhfq cells. Average translational efficiency (for each strain) was calculated as the ratio between the mean protein concentration c_p and the mean mRNA concentration c_m across replicated experiments. The error bars indicate the standard deviation of the data; statistical significance between the strains was calculated with two-sample t-test (P value = 0.014). G: The difference between theoretically predicted and experimentally measured coefficient of variation (squared). The theoretical values were computed according to Eq. (1). Error bars represent s.e.m. calculated from the replicated experiments. Statistical significance between the samples was calculated with two-sample t-test (P value = 0.0088).

Specific alteration of RecB translation by Hfq in vivo.
A: A model of Hfq downregulating RecB translation by blocking the ribosome binding site of the ptrA-recB mRNA. B: Partial complementation of RecB expression to the wild-type level demonstrated by expression of a functional Hfq protein from a multicopy plasmid (pQE-Hfq) in Δhfq (shown in red). The histogram represents the average of two replicated experiments. RecB concentration histograms in wild type and Δhfq mutant from Fig. 4C are shown for relative comparison. Dashed lines represent the average RecB concentration in each condition. C: An increase in RecB protein production caused by sequestering of Hfq proteins with highly abundant small RNA ChiX (shown in green). The histogram represents the average of two replicated experiments. RecB concentration histograms in wild type and Δhfq mutant from Fig. 4C are shown for relative comparison. Dashed lines represent the average RecB concentration in each condition. D: recB mRNA concentration distribution quantified with smFISH in the strain with the deletion of the main Hfq binding site recB-5’UTR* (shown in blue). The histogram represents the average of three replicated experiments. The recB mRNA distribution in the wild-type cells is shown for relative comparison. The medians are indicated by dashed lines. An approximate location of the removed sequence is schematically shown in the insert (red star). E: RecB protein concentration distribution quantified with RecBHalo-labelling in recB-5’UTR* strain (shown in blue). The histogram represents the average of three replicated experiments. RecB concentration histograms in wild type and Δhfq mutant from Fig. 4C are shown for relative comparison. Dashed lines represent the average RecB concentration in each condition. F: Translational efficiency of RecB in wild type, Δhfq mutant and the strain with the deletion of the main Hfq binding site, recB-5’UTR*. Average translational efficiency was calculated as the ratio between the mean protein concentration c_p and the mean mRNA concentration c_m across replicated experiments. The error bars indicate the standard deviation of the data; statistical significance between the samples was calculated with two-sample t-test. The P value for WT and recB-5’UTR* is 0.0007; while the difference between Δhfq and recB-5’UTR* is non-significant (P value > 0.05).

Sign up for email alerts