Rapid and robust signaling in the CsrA cascade via RNA-protein interactions and feedback regulation

Abstract

Bacterial survival requires the rapid propagation of signals through gene networks during stress, but how this is achieved is not well understood. This study systematically characterizes the signaling dynamics of a cascade of RNA-protein interactions in the CsrA system, which regulates stress responses and biofilm formation in Escherichia coli. Noncoding RNAs are at the center of the CsrA system; target mRNAs are bound by CsrA proteins that inhibit their translation, CsrA proteins are sequestered by CsrB noncoding RNAs, and the degradation of CsrB RNAs is increased by CsrD proteins. Here, we show using in vivo experiments and quantitative modeling that the CsrA system integrates three strategies to achieve rapid and robust signaling. These strategies include: (i) the sequestration of stable proteins by noncoding RNAs, which rapidly inactivates protein activity; (ii) the degradation of stable noncoding RNAs, which enables their rapid removal; and (iii) a negative-feedback loop created by CsrA repression of CsrD production, which reduces the time for the system to achieve steady state. We also demonstrate that sequestration in the CsrA system results in signaling that is robust to growth rates because it does not rely on the slow dilution of molecules via cell division; therefore, signaling can occur even during growth arrest induced by starvation or antibiotic treatment.

Keywords: gene circuit; reverse engineering; synthetic biology; systems biology.

Fig. 1.

CsrA system. (A) Simplified schematic of the synthetic CsrA system. (B) Mechanistic description of the synthetic CsrA system (see main text).

Fig. 2.

CsrA signaling: Stable signaling molecules can cause delays. Error bars are SEM of duplicate measurements. (A) Experimental schematic. (B) Target expression turned on directly by turning on glgC-gfp transcription (IPTG added) or indirectly by turning off csrA transcription (aTc removed). (C) Target expression turned off directly by turning off glgC-gfp transcription (IPTG removed) or indirectly by turning on csrA transcription (aTc added). (D) GlgC-GFP expression as a function of percentage maximum csrA transcription (calibrated using PLlacO-1:st7:gfp; Fig. S2Q). Target expression was also measured in strains without csrA (HL5594; cyan dashed line) or native (wt) csrA (HL5562 and HL5596; purple dashed line indicates both as the data overlay). The gray shading indicates the range over which the CsrA concentration has a significant effect on target expression (“regulatory range”). The open and closed circles are 100% and 0% of maximum [CsrA], respectively.

Fig. 3.

CsrB signaling: Sequestration can bypass downstream delays. Error bars are SEM of duplicate measurements. (A) Experimental schematic. (B) Target expression turned on directly by turning on glgC-gfp transcription (IPTG added) or indirectly by turning on csrB transcription (aTc added). (C) Target expression turned off directly by turning off glgC-gfp transcription (IPTG removed) or indirectly by turning off csrB transcription (aTc removed). (D) GlgC-GFP expression as a function of percentage maximum csrB transcription (calibrated using PLtetO-1:st7:gfp; Fig. S2R). The gray shading indicates the regulatory range for CsrB. The open and closed circles are 100% and 0% of maximum [CsrB], respectively. *Incomplete silencing of GlgC-GFP expression occurs if the total [CsrA] is less than the total [target mRNA] or if there is “leaky” CsrB expression.

Fig. 4.

CsrD signaling: Degradation can prevent downstream delays. Error bars are SEM of duplicate measurements. (A) Experimental schematic. (B) Target expression turned on directly by turning on glgC-gfp transcription (IPTG added) or indirectly by turning off csrD transcription (aTc removed). *Left and right y axis correspond to turning on glgC-gfp (gray) and turning off csrD (magenta), respectively. (C) Target expression turned off directly by turning off glgC-gfp transcription (IPTG removed) or indirectly by turning on csrD transcription (aTc added). (D) GlgC-GFP expression as a function of percentage maximum csrD transcription (calibrated using PLlacO-1:st7:gfp; Fig. S2Q). The gray shading indicates the regulatory range for CsrD. The open and closed circles are 100% and 0% of the maximum [CsrD], respectively.

Fig. 5.

Robust signaling during stress. Error bars are SEM of two or more measurements. (A) Experimental schematic. (B–E) Experiments performed using circuits shown in Figs. 2A and 3A without stress treatment (B), in M9 with no carbohydrate (C), in LB with 5 µg/mL trimethoprim (D), or in LB with 200 µg/mL novobiocin (E). In HL4860, csrA transcription was kept off (control) or turned off at t = 0. In HL4845, csrB transcription was kept on (control) or turned on at t = 0. Fluorescence levels were normalized to their respective control at each time point to correct for general effects of stress. The normalized values were rescaled to the initial measurement to determine the fold change in expression (“relative expression”).

Fig. 6.

Feedback in the native CsrA system. Error bars are SEM of duplicate measurements. (A) Experimental schematic with synthetic (black) and native genes with reported feedback loops (blue). The “synthetic cascade,” which was a benchmark for comparison, was composed of synthetic csrA and csrB. Normalized fluorescence was determined by dividing each value by the fluorescence value in a control with csrB transcribed constitutively; the resulting ratio was rescaled so the start and end points were 0 and 1, respectively (Fig. S2). (B–F) Comparison of systems with synthetic and native genes where synthetic csrB was induced at t = 0. (B) Cascade with native csrA, csrB, csrC, and csrD, and synthetic csrB (gold) versus synthetic cascade (black). (C) Cascade with native csrA and csrD (orange) versus synthetic cascade (black). (D) Cascade with native csrA (red) versus synthetic cascade (black). (E) Cascade with native csrD (blue) versus synthetic cascade (black). Native csrD is modeled at low (light blue) and high (dark blue) CsrA levels. (F) Cascade with and without synthetic csrD expression (magenta and black, respectively). (G) Comparison of cascade with native csrA, csrB, csrC and csrD, and synthetic csrB (gold) versus synthetic cascade (black) where synthetic csrB was turned off at t = 0.