Inorganic phosphate (Pi) is one of the essential molecules required for many biological processes, including synthesis of ATP and phospholipids. However, Pi transport is often coupled to transport of counterions, possibly due to the toxicity that arises when cytoplasmic Pi levels exceed a certain threshold within the bacterium [1]. An illustrative example in Escherichia coli involves the identification of the pitA gene through transposon mutagenesis, which encodes a constitutively expressed phosphate transporter [2]. Notably, the pitA mutant exhibited increased resistance to Zn2+ and accumulated lower levels of intracellular Zn2+ ions compared with the wild-type [2], indicating a potential coordination between Zn2+ and Pi transport through a possible Zn:Pi cotransport via PitA. The toxicity mediated by ion imbalance can disrupt enzymatic reactions 1, 3, highlighting the importance of sensing and responding to Pi concentration in biological processes.

In bacteria, the PhoB/PhoR two-component system controls Pi homeostasis in response to available Pi concentrations. This system has been extensively studied in E. coli, and homologous proteins have been identified in diverse species, spanning from Gram-negative to Gram-positive bacteria 4, 5, 6. PhoR, a membrane-bound histidine kinase, activates the response regulator PhoB. Within its cytoplasmic region, PhoR contains three distinct domains: a Per-Arnt-Sim (PAS) domain, a dimerization and histidine phosphotransfer domain, and a catalytic-active/ATP-binding (CA) domain [7]. In phosphate-scarce environments, PhoR undergoes autophosphorylation and subsequently activates PhoB by transferring a phosphoryl group 4, 8, 9. Once phosphorylated, PhoB binds to specific promoter regions known as Pho boxes, regulating the expression of PhoB-dependent genes 10, 11, 12. The PhoB-dependent genes include the PhoB/PhoR two-component system itself, the PhoU regulator, and the PstSCAB high-affinity phosphate transporter, which imports Pi under phosphate-limiting conditions [7] (Figure 1a).

Interestingly, unlike other histidine kinases in a two-component system, PhoR lacks a periplasmic domain responsible for sensing phosphate concentrations. Instead, the kinase activity of PhoR is tightly linked to the transport activity of the PstSCAB transporter 13, 14. A key player in this regulation is PhoU, encoded by the last gene in the pstSCAB-phoU operon 15, 16, which connects between the PhoB/PhoR two-component system and the PstSCAB phosphate transporter. Previous studies have suggested that PhoU acts as a negative regulator of phosphate signaling, as evidenced by elevated expression levels of pho regulon genes and PhoR autophosphorylation in the phoU mutant, independently of phosphate concentrations 17, 18, 19•, 20. Additionally, PhoU physically interacts with both the PhoR PAS domain and the PstB ATPase 15, 21••, which can be supported by structural modeling (Figure 1, Figure 1b and c). Together, these indicate PhoU’s role in inhibiting PhoR autophosphorylation, ultimately suppressing the pho regulon.

Nevertheless, recent studies have presented evidence that challenges the notion of PhoU functioning as a simple negative regulator in phosphate signaling. In fact, PhoU has been found to be necessary for activating PhoR histidine kinase, through its interaction with the CA domain of PhoR 21••, 22. This discovery adds complexity to PhoU's role and suggests multiple functions beyond being a negative regulator of phosphate signaling. As accumulating evidence points to additional roles for PhoU, this review aims to highlight the diverse functions of PhoU in bacterial regulation and discuss the physiological significance of PhoU-mediated regulation.