A negative feedback loop is critical for recovery of RpoS after stress in Escherichia coli
Fig 4
RssB is produced during phosphate starvation dependent upon RpoS.
A) The upstream region of rssB that includes both rssB promoters P1 and P2 was translationally fused to mCherry contained on a pQE80L-derived vector (pSB37, S4A Fig). The fusion includes 24 bp of rssB (encoding 8 amino acids), fused in frame to mCherry. Expression of mCherry was measured during growth in MOPS glucose minimal medium at 37°C in WT, ΔrpoS (AB165) and ΔrssB (SB94) strains carrying the fusion on plasmid pSB37. The RFU (relative fluorescence units) were calculated by dividing the fluorescence values by the OD600 at each time point. B) RssB levels measured in WT and ΔrpoS during and after phosphate starvation. The experiment was performed as described in Fig 1 with the exception that samples were probed for RssB in the Western blot (see S3 Fig for Western blots; n >2). RssB levels in MG1655 at the end of one hour of phosphate starvation were set at 100 and other samples are plotted compared to that. C) RssB and IraP-SPA tagged levels over the course of phosphate starvation and recovery. Strains MG1655 and SB212 (MG1655 containing iraP C-terminally tagged with SPA) were grown in MOPS minimal medium and subjected to phosphate starvation and recovery as described in Fig 1A. Samples were taken before starvation (0’), at 10, 30, and 60 minutes during starvation and after 30 and 60 minutes of recovery (corresponding to 90 and 120 minutes on the graph). Samples were subjected to a Western Blot using anti-Flag to detect the SPA C-terminal tag of IraP-SPA, and the bands were quantified. To compare RssB to IraP-SPA during starvation and recovery, levels of both proteins after 60’ of starvation were set at 100% (N = 3). D) IraP-SPA chase after one hour of phosphate starvation and phosphate recovery, following a similar procedure as described in Fig 1A. Western Blot using anti-Flag antibody was performed to detect IraP-SPA (quantitation from Western Blot as shown in S3E Fig). E) RpoS chase during recovery from phosphate starvation in strains WT (MG1655) and SB212 containing iraP-SPA at the iraP locus (quantitation from Western blot as shown in S3F Fig).