A negative feedback loop is critical for recovery of RpoS after stress in Escherichia coli
Fig 1
RpoS degradation quickly resumes after phosphate starvation.
A) Experimental protocol. RpoS levels and degradation during and after phosphate starvation were determined as outlined here. Cells were grown in MOPS minimal medium containing 0.2% glucose and 2mM KPO4 until mid-exponential phase (OD600 ≈ 0.3). The medium was then filtered, and cells were resuspended in MOPS medium without phosphate and incubated for one hour (starvation). RpoS degradation during phosphate starvation was monitored by adding chloramphenicol and aliquots were taken right before (0 minute) and at appropriate time points afterwards (chase). RpoS recovery was measured in two ways. After one hour of starvation, PO4 was restored to a portion of the culture (recovery), while another portion was further incubated under the starvation condition. Samples were taken from both cultures and analyzed either by monitoring RpoS levels with time (RpoS accumulation) or by determining RpoS half-life (RpoS degradation) by a chase after adding chloramphenicol 2 minutes after phosphate was restored and taking aliquots at different time points. After TCA precipitation, samples were loaded onto an SDS-PAGE gel and a Western Blot against RpoS, using EF-Tu as a loading control. Note that in this work, an antibiotic chase to block new protein synthesis is used to follow the half-life of proteins, rather than a radioactive or other label followed by a chase. One difference for such an antibiotic chase, as compared to a pulse-chase, will be that the protein followed during the chase is not necessarily newly synthesized, but both should provide similar estimates of degradation. This figure was created using clipart from BioRender.com. B) Western blot showing RpoS degradation (chase) during phosphate starvation in WT (MG1655) and ΔiraP (SB151) as described in Fig 1A. C) Western blot against RpoS and the loading control EF-Tu showing RpoS degradation (chase) during recovery from phosphate starvation in WT (MG1655) and ΔiraP (SB151) strains following the protocol as described in Fig 1A. D) RpoS levels in WT (MG1655) during and after phosphate starvation, with (chase) and without (accumulation) chloramphenicol, measuring RpoS degradation and overall RpoS levels, respectively (quantitation of WT data from experiments as shown in Fig 1B and 1C for the RpoS chase data and S1A Fig for the RpoS accumulation data). Shown are means with SD, n = 3. E) Effect of IraP on RpoS stabilization during and recovery after phosphate starvation. Quantification of RpoS degradation (chase) during and after phosphate starvation in WT (MG1655) and ΔiraP (SB151) strains (quantitation of Fig 1B and 1C; n > 3; note that data for WT is the same as in Fig 1D).