Dynamic expression of the translational machinery during Bacillus subtilis life cycle at a single cell level

Abstract

The ability of bacteria to responsively regulate the expression of translation components is crucial for rapid adaptation to fluctuating environments. Utilizing Bacillus subtilis (B. subtilis) as a model organism, we followed the dynamics of the translational machinery at a single cell resolution during growth and differentiation. By comprehensive monitoring the activity of the major rrn promoters and ribosomal protein production, we revealed diverse dynamics between cells grown in rich and poor medium, with the most prominent dissimilarities exhibited during deep stationary phase. Further, the variability pattern of translational activity varied among the cells, being affected by nutrient availability. We have monitored for the first time translational dynamics during the developmental process of sporulation within the two distinct cellular compartments of forespore and mother-cell. Our study uncovers a transient forespore specific increase in expression of translational components. Finally, the contribution of each rrn promoter throughout the bacterium life cycle was found to be relatively constant, implying that differential expression is not the main purpose for the existence of multiple rrn genes. Instead, we propose that coordination of the rrn operons serves as a strategy to rapidly fine tune translational activities in a synchronized fashion to achieve an optimal translation level for a given condition.

Figure 1. Expression of ribosomal components is dynamic during vegetative growth.

(A–B) Strains carrying P_rrnA-gfp (AR13) (A-rrnA), P_rrnE-gfp (AR16) (A-rrnE), P_rplA-gfp (AR25) (B-P_rplA), rplA-gfp (AR5) (B-RplA) were grown in rich medium (CH). Samples were taken at the indicated time points [hrs] and the GFP signal monitored using fluorescence microscopy. All fluorescence images were normalized to the same intensity range. Of note, the localization of RplA-GFP was consistent with previous results . Scale bar corresponds to 1 µm. (C) Strains carrying P_rrn-gfp (rrnO, A, B, D, E, I, J), P_rplA-gfp or rplA-gfp were grown in rich medium (CH). Samples were taken at the indicated time points [hrs] and the GFP signal monitored using fluorescence microscopy. The data represent the average of three independent biological repeats. Fluorescence from at least 60 cells was measured and averaged for each time point, and is shown in arbitrary units (a.u.) (see Materials and Methods). (D) Variability of GFP intensity among single cells carrying the indicated P_rrn-gfp reporter at the different time points described in (C). Coefficient of Variability (CV) was calculated as SD divided by mean (see Materials and Methods). (E) Strains carrying P_rrn-gfp (rrnO, A, B, D, E, I, J), P_rplA-gfp or rplA-gfp were grown in minimal (S7) medium and samples processed as in (C). (F) Variability of GFP intensity among single cells carrying the indicated P_rrn-gfp reporter at the different time points described in (E). Coefficient of Variability (CV) was calculated as SD divided by mean (see Materials and Methods).

Figure 2. Bimodal rrn activity during stationary phase.

Cells carrying the P_rrnO-gfp (AR17) were grown in poor medium (S7) (A) or rich medium (CH) (B). Shown are GFP fluorescence images acquired from stationary phase cells (upper panels). Normalized fluorescence distribution of at least 100 individual cells (lower panels) is scored for each phase and is shown in arbitrary units (a.u.). Arrowheads highlight cells displaying either low (A) or high (B) rrn promoter activity, compared to the average level. Scale bars correspond to 1 µm.

Figure 3. Translational machinery is differentially expressed within mother-cell and forespore.

(A–B) Strains carrying P_rrnA-gfp (AR13) (A) or rplA-gfp (AR5) (B) were induced to sporulate. Samples were taken at the indicated time points [hrs] and the GFP signal monitored using fluorescence microscopy. FM4–64 membrane stained cells (0 and 2 hrs) and phase contrast images (5–8 hrs) are shown in the upper panels and corresponding GFP fluorescence images (0–8 hrs) in the lower panels. Arrowheads designate the position of forespores. Scale bar corresponds to 1 µm. (C–D) Strains carrying P_rrn-gfp (rrnO, A, B, D, E, I, J), P_rplA-gfp or rplA-gfp were induced to sporulate. Samples were taken at the indicated time points [hrs] and the GFP signal from predivisional cells (0–2 hrs) and from mother-cell (C) and forespore (D) monitored using fluorescence microscopy. The data represent the average of 2 independent biological repeats. Fluorescence from at least 100 cells was measured and averaged for each time point and is shown in arbitrary units (a.u.) (see Materials and Methods).

Figure 4. Expression of translational components increases transiently within the forespore.

Strains carrying gltX-gfp (AR11), rnpA-gfp (AR10), thrS-gfp (AR9), rplA-gfp (AR5), or spoIIIG::cat, rplA-gfp (AR20) were induced to sporulate and samples taken at the indicated time points [hrs]. Shown are phase contrast images (left panels) and corresponding GFP fluorescence images (right panels). Arrowheads designate the position of forespores. Scale bar corresponds to 1 µm.

Figure 5. Expression of translational machinery components is biphasic during germination and outgrowth.

(A–B) Strains carrying P_rrnA-gfp (AR13) (A-rrnA), P_rrnE-gfp (AR16) (A-rrnE), P_rplA-gfp (AR25) (B-P_rplA), rplA-gfp (AR5) (B-RplA) were germinated in rich LB medium and tracked by time lapse fluorescence microscopy. GFP fluorescence images acquired at the indicated time points [min] are shown. Of note, the localization of RplA-GFP changed during germination from a diffuse dispersion pattern (0 min) into a distinct focus (30 min). Scale bar corresponds to 1 µm. (C) Spores of strains carrying P_rrn-gfp (rrnO, A, B, D, E, I, J), P_rplA-gfp or rplA-gfp were germinated in rich LB medium and tracked by time lapse fluorescence microscopy. GFP fluorescence images were acquired and analyzed at the indicated time points [min]. The data is representative of one out of three independent biological repeats. Fluorescence from at least 50 cells was measured and averaged for each time point and is shown in arbitrary units (a.u.) (see Materials and Methods). Scale bar corresponds to 1 µm.

Figure 6. Contribution of each rrn promoter to overall translational activity.

The contribution of each rrn promoter during different phases of B. subtilis life cycle is represented as an average percentage, with the sum activity of the seven tested promoters taken as 100%.