Constitutive activation of the Escherichia coli Pho regulon upregulates rpoS translation in an Hfq-dependent fashion.

The preferred source of phosphorus for Escherichia coli is inorganic phosphate (Pi), and E. coli uses several transporters to import Pi into the cytoplasm. These are the two low-affinity Pi transporters PitA and PitB (8, 10) and the high-affinity transporter Pst. The Pst transporter is a complex composed of the periplasmic Pi-binding protein PstS, the integral inner membrane proteins PstA and PstC, and the ATP-binding protein PstB (for a review see reference 32), and it serves as the cell's gauge for the extracellular availability of Pi as follows. The Pst transport system is associated with the PhoR histidine kinase/phosphatase, which controls the phosphorylation state of the response regulator PhoB. The functional status of the Pst transport system is sensed through an unknown mechanism by PhoR, so that if Pst can easily transport Pi into the cytoplasm (because of high extracellular Pi concentration), PhoR acts as a PhoB phosphatase. Consequently, unphosphorylated PhoB cannot activate the transcription of the Pho regulon, a group of genes that encode proteins involved in scavenging and metabolizing phosphorus sources other than Pi. On the contrary, low extracellular Pi concentrations reduce transport by Pst, which increases the kinase activity of PhoR, resulting in the accumulation of phosphorylated PhoB (PhoB-P). Likewise, loss-of-function mutations in the pst genes lead to the constitutive phosphorylation of PhoB, regardless of the external Pi concentration. Once phosphorylated, PhoB-P transcriptionally activates the Pho regulon, and an effort to scavenge phosphorus begins. However, because phosphorus is limiting in nature, even the induction of the Pho regulon might not be enough to ensure cell survival. Therefore, to protect itself during Pi starvation, E. coli triggers a developmental program known as stationary phase (5). Stationary phase is a general stress response that leads to dramatic changes in the protein profile and cellular composition and metabolism, which increase the cell's resistance to many different harmful conditions (for a review see reference 9). The central regulator of stationary-phase transcription is the sigma factor RpoS (or σs), whose concentration in the cell is tightly regulated at the levels of transcription, translation, and protein stability (9). Although recent studies have identified new factors involved in the transcriptional control of rpoS (9), much more is known about the mechanisms controlling its translation and stability. During exponential growth, rpoS translation is maintained at very low levels by the inhibitory action of a cis-acting element present in the rpoS mRNA. The rpoS message has an unusually long 5′ untranslated region (UTR) that contains a region capable of binding to the ribosome-binding site (rbs) and the initiation region for rpoS translation (for a review see reference 7). As a result of this RNA-RNA interaction, access to the rbs by the ribosome is blocked, so rpoS translation is reduced. This inhibitory binding can be modulated by the action of several regulatory noncoding small RNAs (sRNAs) and the RNA chaperone Hfq. Specifically, the DsrA and RprA sRNAs positively regulate rpoS translation by pairing with the upstream element of the 5′ UTR that occludes the rbs, while the OxyS sRNA negatively regulates rpoS translation through an unknown mechanism (7, 9, 16, 18, 19, 35). All three sRNAs require the RNA chaperone, Hfq (19, 30, 35, 36), to regulate rpoS translation, but how Hfq promotes interactions between these different sRNAs and rpoS is not clear. Another mechanism for the cell to increase RpoS levels is by preventing its degradation. Besides reduced rpoS synthesis, degradation of RpoS by the ClpXP proteolytic complex ensures that the RpoS content in the cell is very low during exponential growth (26). The regulatory mechanism controlling this proteolysis involves SprE (also named RssB), an orphan response regulator (20, 22). In vitro studies have shown that SprE catalytically delivers RpoS to the ClpXP protease (11, 37), but it still remains unknown how SprE's activity is regulated. Understanding the regulation of this proteolysis is further complicated by the existence of a negative feedback loop; namely, transcription of sprE is RpoS dependent (25). Here we show that mutations that disrupt genes encoding the Pst transporter cause accumulation of RpoS during exponential growth. As described above, mutations that disrupt this Pi transporter lead to the constitutive activation of the Pho regulon. Like the activation of the Pho regulon, the upregulation of RpoS levels also requires PhoBR. Using lacZ reporter fusions, we determined that the mutation in pstS increases rpoS translation. Because this regulation requires Hfq, we propose that there is involvement of a PhoBR-regulated sRNA.