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 ∆recB 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 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, − γmt, where γm is the recB mRNA degradation rate. The final degradation rate was calculated as the average between two replicated time-course experiments (Table 7). 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 DKL = 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 RecBHalo 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, − γpt, where γp is the RecB removal rate. The final removal rate was calculated as the average between two replicated pulse-chase experiments (Table 7). 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 7. The Kullback–Leibler divergence between the distributions is DKL = 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 upon double-strand break (DSB) induction.

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 cp and the mean mRNA concentration cm. 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 ∆hfq 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 ∆hfq 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 ∆hfq. Total number of analysed cells are 3,324 (WT) and 2,185 (∆hfq). Insert: Box plot shows significant difference (P value = 0.0007) between the average RecB concentrations in wild-type and ∆hfq cells verified by two-sample t-test. D: Examples of fluorescence and bright-field images of recB mRNA FISH experiments in ∆hfq 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 ∆hfq 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 ∆hfq. Total number of analysed cells are 7,270 (WT) and 5,486 (∆hfq). 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 ∆hfq cells. Average translational efficiency (for each strain) was calculated as the ratio between the mean protein concentration cp and the mean mRNA concentration cm 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 ∆hfq (shown in red). The histogram represents the average of two replicated experiments. RecB concentration histograms in wild type and ∆hfq 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 ∆hfq 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 ∆hfq 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, ∆hfq 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 cp and the mean mRNA concentration cm 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 ∆hfq and recB-5’UTR* is non-significant (P value > 0.05).

E. coli strains used in the study.

Plasmids used in the study.

Primers used for strain and plasmid construction.

The sequence of the gBlock used for the construction of the recB-5’UTR strain.

Oligos used for RT-qPCR quantification.

Sequences of recB RNA FISH probes labelled with TAMRA dye.

Parameters of a two-stage model of RecB expression.

smFISH image analysis performed by Spätzcell.

a: Peak height intensity profiles for spots detected in the wild-type and ∆recB samples. The dashed line indicates the intensity threshold (99.9%) that separates a specific signal from the background. b: Integrated spot intensity histogram for foci detected in the wild-type sample. The data, binned and averaged in groups, are shown in red circles. The data were fitted by the sum of two Gaussian distributions corresponding to one and two mRNA molecule(s) per focus. The grey lines correspond to single Gaussian distributions, while the black solid curve is the sum of these two Gaussians. The red dashed line indicates the intensity equivalent corresponding to the total integrated intensity of FISH probes (in average) bound to a single mRNA (One mRNA).

Sensitivity of the smFISH protocol allows for quantification of low-abundant recB mRNAs.

Top: Fluorescence signal, detected in the samples with recB over-expression from an arabinose-inducible plasmid (pIK02), is presented as a function of arabinose concentration (green-dashed line). The intensity of the fluorescence signal in the wild-type and ∆recB strains is indicated with blue-dashed and red-dashed lines, respectively. More than 350 cells for each induction condition were taken for the analysis. Bottom: Representative fluorescence images of the samples where recB expression was induced with 10−3%, 10−4% or 10−5% of arabinose. All fluorescence images are shown in the same intensity range. The yellow line segment represents 1 μm.

Independent transcription from recB gene copies.

recB mRNA distributions for newborns (left) and adults (right). The experimental data from Fig. 1C was conditioned on cell size (<3.0 μm2 for newborns) and (>3.5 μm2 for adults). Total number of cells: 2,180 (newborns) and 6,413 (adults). The recB mRNA statistics for newborns is well described by a negative binomial distribution, NB(r, p) (Fig. 1F). The sum of two independent variables, each of which is distributed as NB(r, p), is then described by a negative binomial distribution NB(2r, p). Thus, the adults data was compared to the predicted distribution, NB(2r, p), based on (i) the assumption of independent transcription from recB gene copies and (ii) the fitting parameters inferred on the newborns. The results were verified with Gillespie’s simulations (SSA). The Kullback–Leibler divergence between the experimental and simulated distributions for newborns and adults is DKL = 0.003 and DKL = 0.007, respectively.

Image analysis of Halo-labelling experiments performed by a modified version of Spätzcell.

Peak height intensity profiles for spots detected in the strain with RecB-HaloTag fusion (RecBHalo sample) and its parental strain (no HaloTag control). The dashed line indicates a chosen intensity threshold in order to separate a specific signal from the background.

Analysis of recB mRNA and RecB protein concentrations and growth rate measurements upon DSB induction.

a: recB mRNA concentration for perturbed (blue) and unperturbed (grey) samples represented as a function of cell area. The data from Fig. 3D were binned by cell size and averaged in each group (mean ± s.e.m.). The solid lines connect the averages while the dashed lines and shaded areas show mean ± s.e.m. calculated across all cells in each sample. b: RecB protein concentration for perturbed (blue) and unperturbed (grey) samples shown as a function of cell area. The data from Fig. 3E were binned by cell size and averaged in each group (mean ± s.e.m.). The solid lines connect the averages while the dashed lines and shaded areas show mean ± s.e.m. calculated across all cells in each sample. c: Growth curves obtained with optical density measurements (OD600). Cell cultures were grown with 4 ng/ml (blue) or without (grey) ciprofloxacin. The red arrow indicates the time when ciprofloxacin was added to the cipro+ sample. The exponential phase of growth (OD600 ∼ [0.08-0.4]) was used for growth rate estimation. The growth rates, averaged across three replicated experiments, are shown in the legend. Error bars on the bar chart represent 95% confidence intervals.

Main Hfq binding peak within ptrA gene identified in the Hfq-CRAC experiment in E. coli [78].

The Hfq binding peak located within ptrA-recB-recD mRNA presented in raw counts (Hits). The vertical dashed line and black box indicate a translation initiation site (ATG) and Shine-Dalgarno sequence (SD), respectively. The black vertical arrows show the nucleotides where direct Hfq cross-linking was detected in the CRAC data. Hfq-binding motifs, A-R(A/G)-N(any nucleotide) [73, 79], are highlighted in the red boxes while the cluster of Hfq binding motifs is underlined.

Cell size analysis, RT-qPCR quantification and growth rate measurements in the ∆hfq mutant.

a: Examples of bright-field images for wild-type and ∆hfq strains. Scale bars represent 2 μm. b: Box plots represent cell area distributions for wild-type and ∆hfq samples. c: ptrA, recB, recC and recD transcripts quantified by RT-qPCR in the ∆hfq mutant and normalized to the corresponding expression levels in wild-type cells. The data represent averages and standard deviations across three replicates for each gene. rrfD was used as a reference gene. d: Growth curves obtained by optical density measurements (OD600) in ∆hfq (orange) and wild type (grey). The strains were grown in the medium supplemented with glucose and amino acids. The exponential phase of growth (OD600 ∼ [0.08-0.4]), shown in the logarithmic scale in the insert, was used for growth rate calculation. The growth rates, averaged across three replicated experiments, are shown in the legend. Error bars on the bar chart represent 95% confidence intervals.

Controls for Hfq complementation experiment.

a: RecB protein concentration distributions quantified in the ∆hfq mutant carrying pQE80L (blue) or pQE-Hfq (red) plasmid. The histograms represent the average across two replicated experiments per each condition. The RecB concentration histograms for wild type and ∆hfq mutant are plotted as references in grey and orange, respectively. The dashed lines represent the mean RecB concentration in each condition. Significance was evaluated with two-sample t-test (P values: 0.045(*) for ∆hfq and ∆hfq+pQE-Hfq; 0.71(ns) for ∆hfq and ∆hfq+pQE80L). b: hfq RNA levels quantified by RT-qPCR in ∆hfq cells carrying pQE-Hfq plasmid and normalized to the hfq RNA level in wild-type cells. c: ptrA, recB, recC and recD transcripts quantified by RT-qPCR in ∆hfq carrying pQE80L (left) or pQE-Hfq (right) plasmid and normalized to the corresponding expression levels in ∆hfq and ∆hfq+pQE80L, respectively. d: A 5-fold serial dilution assay of ∆hfq strain carrying pQE80L or pQE-Hfq plasmid. Cells were plated onto LB plates without or with 6 ng/ml of ciprofloxacin.

Controls for ChiX over-expression experiment.

a: RecB protein concentration distributions quantified in wild-type cells carrying pZA21-ChiX (green), pZA21 (blue) or pZA21-CyaR (purple) plasmid. The histograms represent the average across two replicated experiments per each condition. The RecB concentration histograms for wild type and the ∆hfq mutant are plotted as references in grey and orange, respectively. The dashed lines represent the mean RecB concentration in each condition. Significance was evaluated with two-sample t-test (P values: 0.021(*) for WT and WT+pZA21-ChiX; 0.42(ns) for WT and WT+pZA21; 0.37(ns) for WT and WT+pZA21-CyaR). b: chiX and cyaR RNA levels quantified by RT-qPCR in wild-type cells carrying pZA21-ChiX (left) or pZA21-CyaR (right) over-expression plasmid. The results were normalized to the corresponding expression in the cells carrying the backbone plasmid (pZA21). c: ptrA, recB, recC and recD transcripts quantified by RT-qPCR in wild-type cells carrying pZA21-ChiX (left), pZA21 (middle) or pZA21-CyaR (right) plasmid. RNA levels were normalized to the corresponding expression levels in wild type.

RecB protein and mRNA quantification in ∆chiX.

a: RecB protein concentration distribution quantified in ∆chiX cells. The histogram represents the average across two replicated experiments. The RecB concentration histograms for wild type and the ∆hfq mutant are plotted as references in grey and orange, respectively. The dashed lines represent the mean RecB concentration in each condition. Significance was evaluated with two-sample t-test (P value for WT and ∆chiX is 0.96(ns)). b: ptrA, recB, recD and recC transcripts quantified by RT-qPCR in chiX mutants and normalized to the corresponding expression levels in the wild-type sample.

Toxicity of RecBCD overexpression upon DSB induction.

Viability assays performed for the wild type, wild type carrying RecBCD over-expression plasmid, pDWS2 [133], and ∆recB. Cells were plated onto LB plates supplemented with ampi-cillin and either without or with 8/10/12/16 ng/ml of ciproflaxacin. The average survival factors were calculated for at least three replicated experiments while the error bars indicate standard estimation of the mean.