Crl-dependent RpoS feedback loop.

A) Bacterial two-hybrid experiment showing the interaction between RpoS and Crl. A plasmid expressing the T25 domain of adenylate cyclase was fused to the rpoS coding gene at its 5’ end and a plasmid expressing the T18 domain was fused to the wild-type crl or crl-R51A coding gene at its 5’ end. Western Blot detecting the T18 domain of the adenylate cyclase (below graph) shows similar production of T18-Crl and T18-Crl-R51A in the cya⁺ MG1655 strain. B) Fluorescence over time of the translational rssB fusion between mCherry contained on the pSB37 plasmid in WT, ΔrpoS (AB165), ΔrssB (SB94), Δcrl (SB147) and crl-R51A (SB148) strains, grown from exponential to stationary phase in a microplate reader that measured mCherry fluorescence and OD600 every 20 minutes. C) Western blot against RpoS showing RpoS and RpoS-Lac recovery accumulation from phosphate starvation in WT, iraP::kan and crl::kan strains. rpoS⁺ strains: SG300013 (crl+ iraP⁺), SB179 (iraP^-) and SB180 (crl^-). ΔrpoS strains: INH28 (crl+), SB175 (iraP^-) and SB176 (crl^-). ΔrpoS rssA2::cm strains: SB150 (crl⁺), SB173 (iraP^-) and SB174 (crl^-). D) Western blot showing RpoS and RpoS-Lac accumulation during phosphate starvation in the crl⁺ iraP⁺ strains used in C. Protocol is as in Fig 1. E) Relative RpoS-Lac levels from the Western Blot in S6D Fig at 0’ time points (pre-phosphate starvation). Values are represented as percentage relative to RpoS-Lac levels in the rpoS⁺ strain, set to 100.

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