Mapping the Induction of RpoS via High pH-Adapted Strains Obtained by Experimental Evolution

Escherichia coli are dynamic bacteria that possess the ability to grow and survive across a wide range of external pH conditions. E. coli can grow reasonably well up to ~pH 9.0 and can survive (but not grow) up to ~pH 9.8. We conducted a high pH laboratory evolution experiment in which we grew E. coli at the upper limits of alkali growth in buffered LB media supplemented with potassium. The pH of the culture media began at pH 9.0, but after 464 generations the pH was increased to pH 9.2, then to pH 9.25 after 1,187 generations, and finally to pH 9.3 after 1,901 generations. After 2,187 generations, the experiment was terminated, and eight strains from four populations were purified and sequenced. All populations demonstrated an increase in the endpoint optical density after 22 hours of growth in comparison to the ancestral strain, yet only four out of the eight evolved strains exhibited an increase in growth rate. In order to better understand the increase in fitness on a genetic basis, mutations in the eight evolved strains were predicted using the breseq computational pipeline. We hypothesize that mutations to sigma factor rpoS may be in part responsible for adaptation given that similar mutations occurred independently across multiple populations. One of the ways E. coli is able to thrive under stressful conditions is by upregulating RpoS, a stress response regulon responsible for the regulation of approximately 10% of its genome. Although much is known about the signaling induction of RpoS under nutrient starvation, acid stress, and other stressful conditions, little is known about the signaling induction of rpoS under high pH stress. We have found that RpoS is induced during growth in high external pH and is stabilized by a combination of the antiadaptors, IraM, IraP, and IraD

Strains used in this study.

RpoS is an alternative sigma factor needed for the induction of the general stress response in many gammaproteobacteria. Tight regulation of RpoS levels and activity is required for bacterial growth and survival under stress. In Escherichia coli, various stresses lead to higher levels of RpoS due to increased translation and decreased degradation. During non-stress conditions, RpoS is unstable, because the adaptor protein RssB delivers RpoS to the ClpXP protease. RpoS degradation is prevented during stress by the sequestration of RssB by anti-adaptors, each of which is induced in response to specific stresses. Here, we examined how the stabilization of RpoS is reversed during recovery of the cell from stress. We found that RpoS degradation quickly resumes after recovery from phosphate starvation, carbon starvation, and when transitioning from stationary phase back to exponential phase. This process is in part mediated by the anti-adaptor IraP, known to promote RpoS stabilization during phosphate starvation via the sequestration of adaptor RssB. The rapid recovery from phosphate starvation is dependent upon a feedback loop in which RpoS transcription of rssB, encoding the adaptor protein, plays a critical role. Crl, an activator of RpoS that specifically binds to and stabilizes the complex between the RNA polymerase and RpoS, is also required for the feedback loop to function efficiently, highlighting a critical role for Crl in restoring RpoS basal levels.</div

RpoS-Lac recovery from phosphate starvation depends on RpoS.

A) The RpoS-Lac fusion protein is inactive for transcription. The plasmid pSB23 bearing a transcriptional fusion between the RpoS-dependent promoter PgadB and mCherry (full description of the fusions and method of measurement described in S4 Fig) was introduced in strains containing RpoS-Lac in the presence (SG30013) and absence of RpoS (INH28). The loss of the mCherry signal upon deletion of rpoS confirms that the fusion protein is not able to activate an RpoS-dependent promoter. B) Western blot showing RpoS and RpoS-Lac stabilization and degradation during phosphate starvation and recovery in the strains containing the RpoS-Lac translational fusion in the presence of RpoS (strain SG30013), and in the absence of RpoS (INH28). The protocol is as in Fig 1, with chloramphenicol added to stop translation. C) Quantitation of RpoS and RpoS-Lac half-life during phosphate starvation from Western Blot as shown in S2B Fig (n = 3). (D) Quantitation of RpoS and RpoS-Lac half-life during recovery from phosphate starvation, from Western Blot as shown in S2B Fig (n = 3). (PDF)</p

Primers used in this study.

RpoS is an alternative sigma factor needed for the induction of the general stress response in many gammaproteobacteria. Tight regulation of RpoS levels and activity is required for bacterial growth and survival under stress. In Escherichia coli, various stresses lead to higher levels of RpoS due to increased translation and decreased degradation. During non-stress conditions, RpoS is unstable, because the adaptor protein RssB delivers RpoS to the ClpXP protease. RpoS degradation is prevented during stress by the sequestration of RssB by anti-adaptors, each of which is induced in response to specific stresses. Here, we examined how the stabilization of RpoS is reversed during recovery of the cell from stress. We found that RpoS degradation quickly resumes after recovery from phosphate starvation, carbon starvation, and when transitioning from stationary phase back to exponential phase. This process is in part mediated by the anti-adaptor IraP, known to promote RpoS stabilization during phosphate starvation via the sequestration of adaptor RssB. The rapid recovery from phosphate starvation is dependent upon a feedback loop in which RpoS transcription of rssB, encoding the adaptor protein, plays a critical role. Crl, an activator of RpoS that specifically binds to and stabilizes the complex between the RNA polymerase and RpoS, is also required for the feedback loop to function efficiently, highlighting a critical role for Crl in restoring RpoS basal levels.</div

Model for RpoS degradation after phosphate starvation.

This figure was created using clipart from BioRender.com.</p

Impact of Rsd and 6SRNA on RpoS degradation after phosphate starvation.

Primary data for Fig 8. A) Western blot against RpoS and the loading control EF-Tu showing RpoS stabilization during phosphate starvation in MG1655, ΔiraP (SB151), ΔssrS::kan (SB470) and Δrsd (SB505) strains. Samples were taken and treated as described in Fig 1A, chloramphenicol was added to stop translation. B) Western blot against RpoS and the loading control EF-Tu showing RpoS degradation during recovery from phosphate starvation in MG1655, ΔiraP (SB151), ΔssrS::kan (SB470) and Δrsd (SB505) strains. Samples were taken and treated as described in Fig 1A, chloramphenicol was added to stop translation. (PDF)</p

The phosphorylation status of RssB does not impact RpoS recovery from phosphate starvation.

A) The unphosphorylatable RssB-D58 alleles D58P and D58A and the phosphomimic allele D58E were tested for RpoS activity by following the expression of the transcriptional fusion between gadBp and mCherry expressed on a vector (pSB23) in the strains MG1655, ΔrpoS::tet (AB165), ΔrssB::tet (SB94), rssB-D58E (SB192), rssB-D58A (SB190) and rssB-D58P (SB198). B) RpoS degradation after phosphate starvation of MG1655, rssB-D58E (SB192), rssB-D58A (SB190) and rssB-D58P (SB198). Shown are means with SD, n = 2. Example western blots are shown in S8 Fig.</p

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. (PDF)</p