rssB transcription and RssB protein induction during phosphate starvation and recovery.

A) P1 and P2 rssB promoter sequences and fusion schematics. 1597pb upstream of the rssB coding gene and the first 24 bp of the rssB gene were fused to mCherry. The start point for the P1 promoter is located 1135pb upstream of the first rssB codon and 139 nt upstream of the first rssA codon; P2 is located 64 bp upstream of the first rssB gene codon. Note that the position of the P2 start was determined from dRNA-seq data determined by Thomason et al [46] and differs from the start site previously identified for P2. B) Western Blot of RssB levels during phosphate starvation and after phosphate was added back (phosphate recovery) in MG1655 (WT). C) Western Blot of RssB levels during phosphate starvation (1–60’ samples) and one hour after phosphate was added back (120’ sample) in WT and ΔrpoS (AB165) strains. D) IraP-SPA levels during glucose starvation, phosphate starvation (0, 10, 30 and 60-minute samples for each), exponential phase (OD₆₀₀ = 0.5) and stationary phase (OD₆₀₀ = 1.4). Western blot against FLAG-tag of samples from a strain in which a SPA tag was inserted at the C-terminus of IraP in the chromosome (strain SB212) and against the loading control GroEL. Phosphate and glucose starvation follow the protocol as described in Figs 1 and 2. Note that we were unable to detect untagged IraP by western blot. E) Stability of IraP-SPA protein during and after phosphate starvation. Chase experiment in the strain SB212 containing iraP-SPA at the iraP locus and Western Blot against Flag-tag detecting the SPA tag of IraP. Samples were taken from cells undergoing phosphate starvation; chloramphenicol was added to start the chase as described in Figs 1 and 2. F) Effect of IraP-SPA tag on RpoS stabilization and recovery. RpoS chase during and after phosphate starvation in strains WT (MG1655) and SB212 containing iraP-SPA at the iraP locus, following the protocol as described in Fig 1A. G) RpoS chase during phosphate starvation in strains WT (MG1655) and SB212 containing iraP-SPA at the iraP locus, following the protocol as described in Fig 1A.

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