PhoU interaction with the PhoR PAS domain is required for repression of the pho regulon and Salmonella virulence, but not for polyphosphate accumulation

Seungwoo Baek; Soomin Choi; Yoontak Han; Eunna Choi; Shinae Park; Jung-Shin Lee; Eun-Jin Lee

doi:10.71150/jm.2505013

Articles

Page Path: HOME > J. Microbiol > Volume 63(9); 2025 > Article

Full article PhoU interaction with the PhoR PAS domain is required for repression of the pho regulon and Salmonella virulence, but not for polyphosphate accumulation: Seungwoo Baek^1,†, Soomin Choi^1,†, Yoontak Han^1,†, Eunna Choi¹, Shinae Park², Jung-Shin Lee², Eun-Jin Lee^1,*; Journal of Microbiology 2025;63(9):e2505013.
DOI: https://doi.org/10.71150/jm.2505013
Published online: September 30, 2025

¹Department of Life Sciences, School of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea

²Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University, Chuncheon 24341, Republic of Korea

*Correspondence Eun-Jin Lee eunjinlee@korea.ac.kr

†These authors contributed equally to this work

• Received: May 29, 2025 • Revised: July 13, 2025 • Accepted: July 21, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

49 Views
2 Download

Full Article

Download PDF

ABSTRACT
Introduction
Materials and Methods
Results
Discussion
Notes
Supplementary Information
References

ABSTRACT

The pho regulon plays a critical role in maintaining phosphate homeostasis in bacteria, with the PhoU protein functioning as a regulator that bridges the PhoB/PhoR two-component system and the PstSCAB₂ phosphate transporter. While PhoU is known to suppress PhoR autophosphorylation under high phosphate conditions via interaction with its PAS domain, its broader regulatory functions remain elusive. Here, we investigated the role of the PhoU Ala147 residue in Salmonella enterica serovar Typhimurium using a phoU^A147E substitution mutant. Bacterial two-hybrid and immunoprecipitation assays confirmed that Ala147 is essential for PhoU-PhoR PAS domain interaction, and its substitution leads to derepression of pho regulon genes, even in high phosphate conditions. This disruption impaired Salmonella survival inside macrophages and mouse virulence, demonstrating the importance of PhoU-PhoR interaction in Salmonella pathogenesis. However, unlike the phoU deletion mutant, the phoU^A147E mutant does not exhibit growth defects or polyphosphate accumulation, indicating that the PhoU-PhoR interaction is not involved in these phenotypes. Our findings reveal PhoU as a multifaceted regulator, coordinating phosphate uptake and pho regulon expression through distinct molecular interactions, and provide new insights into its role in bacterial physiology and virulence.
Keywords: phosphate transport, mouse virulence, polyphosphate, histidine kinase

Introduction

The pho regulon plays an important role in regulating homeostasis of inorganic phosphate (Pi), one of the essential molecules in biology (Hsieh and Wanner, 2010). The core components of this regulon include phoB/phoR, pstSCAB, phoE, and phoU, which encode the PhoB/PhoR two-component system, the PstSCAB₂ high-affinity phosphate transporter, the PhoE outermembrane porin, and the PhoU regulatory protein, respectively (Fig. 1). The PhoB/PhoR two-component system controls the signalling pathway of the pho regulon and is highly conserved in both Gram-negative and Gram-positive bacteria (Glover et al., 2007; Hsieh and Wanner, 2010; Lamarche et al., 2008). In this system, PhoR is a membrane-bound histidine kinase that senses phosphate availability and activates its response regulator, PhoB. PhoR contains three functional domains: a Per-Arnt-Sim (PAS) domain, a dimerization and histidine phosphotransfer (DHp) domain, and a catalytic active/ATP-binding (CA) domain (Lamarche et al., 2008). In a phosphate-limiting condition, PhoR auto-phosphorylates a histidine residue within its DHp domain and activates PhoB by transferring a phosphoryl group (Ellison and McCleary, 2000; Hsieh and Wanner, 2010; Makino et al., 1996). When the PhoB response regulator is phosphorylated, it binds to the promoter region known as the Pho box and activates PhoB-dependent gene expression (Bachhawat et al., 2005; Gao and Stock, 2015; Makino et al., 1988). By contrast, in a high phosphate condition, the expression of the pho regulon is repressed to maintain Pi homeostasis.

In this phosphate signalling, PhoU primarily functions as a negative regulator, as indicated by the observation that phoU deletion increases PhoR autophosphorylation and the expression of pho regulon genes regardless of phosphate levels (de Almeida et al., 2015; Lubin et al., 2016; Steed and Wanner, 1993; Wang et al., 2013). In addition, phoU seems to function as a molecular bridge in phosphate signalling pathway. Unlike other sensor kinases that directly recognize and respond to the environmental changes through periplasmic domains, PhoR does not possess a periplasmic domain possibly sensing extracellular phosphate concentrations. Instead, it is proposed that PhoR kinase activity is tightly linked to the activity of the PstSCAB₂ transporter (Piepenbreier et al., 2017). PhoU seems to be central in this regulation, because PhoU physically binds both the PAS domain of PhoR and PstB of PstSCAB₂ transporter (Gardner et al., 2014), suggesting that PhoU functions as a molecular bridge connecting the PhoB/PhoR two-component system and the PstSCAB₂ phosphate transporter (Gardner et al., 2014, 2015) (Figs. 1 and S1E). PhoU binding to the PhoR PAS domain is believed to be essential for suppressing PhoR kinase activity in high phosphate conditions, based on the observation that phoU deletion derepresses phoB-dependent gene expression in both high and low phosphate conditions (de Almeida et al., 2015; Lubin et al., 2016; Steed and Wanner, 1993; Wang et al., 2013). Similarly, it is assumed that activation of PhoR kinase in low phosphate is mediated by PhoU’s detachment from the PAS domain of PhoR; however, this mechanism remains unclear.

Recently, PhoU was proposed to function as a dual regulator in phosphate signalling. In Salmonella enterica serovar Typhimurium, PhoU interacts with PhoR via not only the PAS domain but also the CA domain (Choi et al., 2022). Amino acid substitution studies have shown that PhoU interaction through the CA domain is required for activating the expression of phosphate transport genes in low Mg²⁺ medium, an infection-relevant condition. Given that the PhoU-PhoR PAS domain interaction is involved in repression of phosphate signalling in a high phosphate condition, the PhoU protein appears to modulate phosphate signalling differently in high phosphate and low Mg²⁺ conditions via distinct interactions. Therefore, PhoU’s role in controlling PhoR histidine kinase activity needs to be dissected more carefully. Additionally, PhoU likely serves as a link between the phosphate-sensing PhoB/PhoR two-component system and the potassium-sensing KdpE/KdpD two-component system (Schramke et al., 2017), as PhoU enables the KdpE-dependent expression of potassium transporter genes in the absence of the KdpD histidine kinase, in response to phosphorylation initiated by the PhoR histidine kinase (Schramke et al., 2017).

Recent studies have further established that PhoU is a multifaceted regulator involved in bacterial signalling and physiology (Baek and Lee, 2024). Interestingly, phoU mutations lead to the accumulation of polyphosphate (polyP) granules in several bacterial species (de Almeida et al., 2015; Li and Zhang, 2007; Lubin et al., 2016; Morohoshi et al., 2002). Although both polyphosphate accumulation and derepression of the pho regulon are linked to phoU deletion, these phenotypes do not always co-occur. In Caulobacter crescentus, for example, PhoU has been reported to promote polyphosphate accumulation without repressing pho regulon signalling in high-phosphate conditions (Lubin et al., 2016). Polyphosphate accumulation is also associated with various phenotypes in other bacteria. In Staphylococcus epidermidis and Mycobacterium marinum, phoU deletion leads to polyphosphate accumulation and exhibits impaired stress resistance and persister formation (Wang et al., 2013, 2017). Moreover, in Streptomyces, phoU deletion accumulates polyphosphate and decreases secondary metabolite biosynthesis.

Although PhoU has been implicated in regulating diverse cellular functions as described above, the mechanism by which a single protein governs such a wide range of phenotypes remains unclear. In particular, how PhoU interacts with and influences other proteins is not well understood. To address this, we investigated how a specific amino acid substitution in PhoU disrupting the PhoR PAS domain interaction contributes to its regulatory functions. By comparing the phenotypes of a phoU deletion mutant and a PhoU A147E-substituted mutant, we found that not all ΔphoU-associated phenotypes result from disruption of the PhoU-PhoR PAS domain interaction. This genetic dissection revealed that PhoU-PhoR PAS domain interaction is involved in repressing the pho regulon in high phosphate conditions and contributes to Salmonella pathogenesis. However, the same interaction is not involved in maintaining normal growth rate or preventing polyphosphate accumulation, suggesting that these phenotypes result from different molecular mechanisms.

Materials and Methods

Bacterial strains, plasmids, primers, and culture conditions

The bacterial strains and plasmids used in this study are listed in Table S1. All Salmonella enterica serovar Typhimurium strains are derived from the wild-type strain 14028s and were constructed by phage P22-mediated transductions. DNA oligonucleotides are also listed in Table S1. Bacteria were grown at 37°C in Luria-Bertani broth (LB), and N-minimal media (pH 7.7) supplemented with 0.1% casamino acids, 38 mM glycerol, and the indicated concentrations of MgCl₂. For low-phosphate N-minimal media, 10 mM KH₂PO₄ in the N-minimal media was replaced by 0.01 mM KH₂PO₄. Escherichia coli DH5α was used as the host for the preparation of plasmid DNA, and BTH101 lacking the cya gene was used as the host for the bacterial two-hybrid system. Ampicillin was used at 50 μg/ml, chloramphenicol at 25 μg/ml, kanamycin at 50 μg/ml, or tetracycline at 10 μg/ml. IPTG (isopropyl β-D-1-thiogalactopyranoside) was used at 0.25 mM, L-arabinose at 1 mM, and X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) at 80 μM.

Plasmid construction

For the plasmid pBAD33-phoU^A147E-His was constructed as follows: a PCR fragment corresponding to the phoU^A147E gene was generated by PCR with the primer pairs KHU767/KHU768 using SM323 genomic DNA as a template. The amplified DNA fragment was digested with XbaI and HindIII and cloned into pBAD33 digested with the same enzymes.

Construction of phoU^A147E strain with a C-terminal Myc-tagged version of the phoR gene at its native chromosomal locus

Salmonella phoU^A147E strain with a C-terminal myc-tag fused to the phoR gene was generated by the PCR-based tandem epitope tagging system (Cho et al., 2006). A Km^R cassette for the phoR-8×myc gene was PCR amplified from plasmid pBOP508 using KU452 and KU453 primers. The resulting PCR product was integrated into the SM323 chromosome to generate SM443 (phoU^A147E, phoR-8×myc::Km^R). The SM457 (phoU^A147E, phoR-8×myc) strain was generated by removing the Km^R cassette from SM443 using plasmid pCP20 as described (Datsenko and Wanner, 2000).

Bacterial two hybrid (BACTH) assay

To assess protein (or peptide)-protein interactions in vivo, a bacterial two-hybrid (BACTH) assay was conducted as described (Karimova et al., 1998). The Escherichia coli BTH101 strain was co-transformed with derivatives of the pUT18, pUT18C, and pKT25 plasmids. The strains were grown overnight at 37°C in LB supplemented with ampicillin (50 μg/ml) and kanamycin (50 μg/ml). Then, 4 μl of cells was spotted on LB agar plate with 500 μM IPTG, 100 μM ampicillin, 100 μM kanamycin, and 80 μM X-Gal for 48 h at 30°C.

Immunoprecipitation assay

The interaction between the PhoU and PhoR proteins was investigated in wild-type Salmonella expressing the phoU gene from an arabinose-inducible plasmid (pBAD33-phoU-His and pBAD33-phoU^A147E-His) and a C-terminally gfp-tagged phoR gene or its derivatives (pTGFP-phoR), or the empty vector (pTGFP) from the constitutive P_lac promoter.

Cells were grown overnight in N-minimal medium containing 10 mM Mg²⁺, then diluted 1:100 into 15 ml of fresh N-minimal medium containing 10 mM Mg²⁺ and incubated for 3 h. Cells were washed and resuspended in 15 ml of N-minimal medium containing 0.5 mM Mg²⁺, 1 mM L-arabinose and 0.25 mM IPTG, then incubated for an additional 1 h. After normalizing cell density by measuring OD₆₀₀, crude extracts were prepared by sonication in Tris-buffered saline (TBS). For the pull-down assay using anti-GFP antibodies, 50 μl of crude extract was reserved as input. The remaining 400 μl was incubated with 25 μl of GFP-Trap^®_A beads (Chromotek) for 1 h at 4°C on a nutator. The beads were washed twice with TBS and collected by centrifugation. Bound proteins were eluted with SDS sample buffer. The protein extracts were separated on 12% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and analyzed by Western blot. Membranes were incubated overnight at 4°C with primary antibodies against His-tag (rabbit, 1:5,000; Rockland^TM, 600-401-382S) and GFP (rabbit, 1:2,500; Rockland^TM, 600-401-215). After washing, blots were incubated for 1 h with HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000; ThermoFisher, 31460). Detection was performed using the SuperSignal^® West Femto Maximum Sensitivity Substrate (ThermoFisher).

Western blot analysis

Cells were cultured for 5 h in 15 ml of N-minimal medium supplemented with either 10 mM or 0.01 mM inorganic phosphate (Pi). After incubation, cell density was normalized by measuring OD₆₀₀. Crude extracts were prepared by sonication in Tris-buffered saline (TBS). Proteins were separated on a 12% SDS–polyacrylamide gel and transferred to nitrocellulose membranes. Fur and Myc-tagged proteins were detected using anti-Fur (rabbit, 1:5,000) and anti-Myc (mouse, 1:5,000; MBL, M192-3) primary antibodies. Membranes were incubated overnight at 4°C with these primary antibodies. Subsequently, the blots were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-rabbit IgG (ThermoFisher, 31460) or anti-mouse IgG (Sigma-Aldrich, NA931V), each at a 1:10,000 dilution. Signal was detected using the SuperSignal^® West Femto Maximum Sensitivity Substrate (ThermoFisher, 34095).

Real-time quantitative reverse transcription PCR (qRT-PCR)

Bacteria were cultured for 5 h in 15 ml of N-minimal medium containing either 10 mM or 0.01 mM inorganic phosphate (Pi). Total RNA was extracted using the RNeasy Kit (QIAGEN) following the manufacturer’s instructions. The purified RNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies). Complementary DNA (cDNA) was synthesized using the PrimeScript^TM RT reagent Kit (TaKaRa). The mRNA levels of phoU and phoE were quantified by real-time PCR using SYBR Green PCR Master Mix (TOYOBO) and specific primers (phoU: KHQ097/KHQ098, phoE: KHQ015/KHQ016). Reactions were performed on a StepOnePlus^TM Real-Time PCR system (Applied Biosystems, USA). Transcript levels were calculated based on a standard curve generated from Salmonella enterica serovar Typhimurium 14028s genomic DNA of known concentration (Bong et al., 2024). Expression levels were normalized to 16S ribosomal RNA using primers 6970 and 6971.

Structural modeling of the PhoR–PhoU–PstSCAB₂ complex

Structural predictions of the PhoR-PhoU-PstB within the PhoR–PhoU–PstSCAB₂ complex were generated using AlphaFold3. Amino acid sequences of Salmonella enterica serovar Typhimurium 14028s PhoU (UniProt: A0A0F6B922), PhoR (A0A0F6AXL9), and PstB (A0A0F6B923) were retrieved from the UniProt database. Protein complex structures were predicted using the AlphaFold-Multimer implementation, which accounts for protein-protein interaction interfaces (Abramson et al., 2024). Modeling was performed using default parameters, and the highest-ranked prediction was selected based on the predicted TM-score (pTM = 0.71) and the inter-chain predicted TM-score (ipTM = 0.69). This model was then used for interface analysis. Visualization and annotation of the structural models, including color-coding of individual proteins and highlighting of the PhoU Ala147 residue, were performed using PyMOL (Schrödinger, LLC).

Membrane vesicle preparation

Cells were grown in N-minimal medium containing 10 mM phosphate (high Pi) until cells reached an OD₆₀₀ of 0.5. Cells were normalized based on OD₆₀₀ measurements. Crude extracts were prepared in TBS buffer by sonication, and the supernatants were centrifuged for 1 h at 32,000 × g (LaboGene, 1730R). The resulting pellets were resuspended in 50 μl of TBS buffer. The protein concentration in the prepared membrane fractions was determined using a NanoDrop spectrophotometer (Thermo Fisher).

Measuring autophosphorylation of PhoR histidine kinase

The autophosphorylation of PhoR histidine kinase was measured as previously described (Choi et al., 2019). Membrane vesicles (50 µg) prepared from strains expressing either wild-type PhoU or the PhoU A147E variant were incubated in 100 µl of TBS buffer containing 1 mM MgCl₂ at room temperature. The reaction was initiated by adding [γ-³²P] ATP (10 µCi; PerkinElmer), and 10 µl aliquots were collected at the indicated time points. Each aliquot was immediately mixed with 10 µl of 5×SDS loading buffer (Biosesang) to stop the reaction. Samples were incubated at 70°C for 5 min and electrophoresed on a 12% SDS-polyacrylamide gel. Following electrophoresis, gels were dried on Whatman filter paper using a Model 583 gel dryer (Bio-Rad) and analyzed for phosphorylated PhoR using a Typhoon phosphorimager (GE Healthcare). Phosphorylated PhoR bands were identified using samples prepared from wild-type and phoR mutant Salmonella grown for 5 h in N-minimal medium containing 0.01 mM Pi, a condition known to induce the PhoB/PhoR two-component system.

Confocal laser scanning microscopy

Cells were grown overnight in N-minimal media containing 10 mM Mg²⁺. A 1:100 dilution of the overnight culture was inoculated into 15 ml of fresh N-minimal medium containing 0.01 mM Mg²⁺ and incubated for 5 h. Bacterial cells were harvested by centrifugation and washed three times with McIlvaine buffer (0.2 M Na₂HPO₄ [16.47 ml], 0.1 M citric acid [3.53 ml], pH 7.0). The cells were stained with 50 μg/ml DAPI solution (Sigma-Aldrich, F6057) for 30 min in the dark, followed by a brief wash with UltraPure water. For intracellular polyphosphate quantification, the stained bacteria were transferred to a 96-well plate, and fluorescence was measured using a Synergy microplate reader with excitation at 415 nm and emission at 560 nm. For visualization of polyphosphate granules, DAPI-stained cells were additionally labeled with FM4-64 (Invitrogen, T3166) at a final concentration of 20 μg/ml for 10 min at room temperature. After a brief wash, the stained bacteria were placed onto poly-L-lysine-coated glass slides, fixed, and mounted with Anti-Fade Fluorescence Mounting Medium (Abcam, ab104135). A coverslip (Marienfeld) was placed on top, and the sealed with nail polish. Confocal images were acquired using a Leica SP8X confocal laser scanning microscope (CLSM) with a 100× oil immersion objective. The excitation/emission detection settings were as follows: nucleus (405 nm/420 nm), FM4-64 (515 nm/640 nm), and polyphosphate (405 nm/570 nm).

Macrophage survival assay

Intracellular survival assays were performed using the J774A.1 murine macrophage-like cell line. A total of 5 × 10⁵ macrophages were seeded in 24-well plates containing Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C with 5% CO₂. Overnight-grown bacteria were added to the macrophages at a multiplicity of infection (MOI) of 10. Plates were centrifuged at 1,000 rpm for 5 min at room temperature and incubated for an additional 30 min to facilitate bacterial uptake. Extracellular bacteria were removed by washing the wells three times with phosphate-buffered saline (PBS), followed by incubation in DMEM supplemented with 10% FBS and 120 μg/ml gentamicin for 1 h to kill remaining extracellular bacteria. To determine intracellular bacterial counts at 1 h post-infection, macrophages were lysed with PBS containing 0.1% Triton X-100, and serial dilutions of the lysates were plated on Luria-Bertani (LB) agar. For 21 h time points, the medium was replaced after the initial 1 h with fresh DMEM containing 12 μg/ml gentamicin, and incubation continued at 37°C. After 21 h, cells were lysed as above, and viable intracellular bacteria were quantified by plating. Percentage survival was calculated by dividing colony-forming units (CFUs) recovered at 21 h by those recovered at 1 h. All experiments were performed in duplicate and repeated independently at least three times.

Mouse virulence assay

Six- to eight-week-old female C3H/HeN mice were inoculated intraperitoneally with approximately 10³ colony-forming units (CFU) of Salmonella strains. Mouse survival was monitored for 21 days. Virulence assays were repeated three times with consistent outcomes, and the presented data represent groups of five mice per strain. All animals were housed in a temperature- and humidity-controlled facility with a 12 h light/12 h dark cycle. All experimental procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (approval no. KW-210728-1).

Results

The Ala147 residue of Salmonella PhoU is required for interaction with PhoR histidine kinase

In E. coli, PhoU binds to the PAS domain of PhoR, leading to the suppression of pho regulon gene expression under high phosphate conditions (Gardner et al., 2014; Muda et al., 1992). The Ala147 and Arg 148 residues in PhoU were proposed to be critical for this interaction in the previous genetic analysis and protein modeling in E. coli (Gardner et al., 2015; Muda et al., 1992; Torriani and Rothman, 1961). In addition, amino acid sequence alignment showed that these residues are conserved in PhoU from E. coli and Salmonella enterica (Choi et al., 2022). To determine whether these residues in Salmonella PhoU are similarly involved in binding PhoR histidine kinase, we performed a bacterial two-hybrid assay using a strain coexpressing T18C-fused wild-type PhoU or the PhoU A147E variant and PhoR-T25 (Fig. 2A). Coexpression of T18C-fused wild-type PhoU and PhoR-T25 exhibited a blue color on LB medium containing X-gal, indicating a physical interaction between PhoU and PhoR (Fig. 2B). In contrast, the PhoU A147E variant did not exhibit a color (Fig. 2B), demonstrating that the Ala147 residue in Salmonella PhoU is essential for PhoR interaction.

Protein modeling suggests that PhoU interacts with the PAS domain of PhoR and is in close proximity to the base of PstB in the PstSCAB₂ phosphate transporter (Fig. S1). In this model, the PhoU Ala147 residue is oriented toward the PAS domain of PhoR but does not appear to contact PstB. As predicted, the PhoU A147E substitution weakened the interaction with PhoR (Fig. S1C and S1E) but did not affect the interaction with PstB (Fig. S1D and S1F). As a control, we chose the PhoU A147K substitution, which was previously reported to have no effect on the interaction (Choi et al., 2022). Since the phoU R148A substitution also maintained the interactions with PhoR and PstB, the R148 residue appears to be less important for the PhoU-PhoR PAS domain interaction, unlike in E. coli (Fig. S1C and S1D) (Gardner et al., 2015).

The PhoU-PhoR interaction was further confirmed by immunoprecipitation. A C-terminally GFP-tagged PhoR protein successfully immunoprecipitated C-terminally His-tagged wild-type PhoU (Fig. 2C and 2D). Similarly to the two-hybrid assay, the same PhoR protein failed to immunoprecipitate the PhoU A147E variant (Fig. 2C and 2D), indicating that PhoU Ala147 residue is necessary for PhoR interaction.

The phoU^A147E substitution derepresses mRNA and protein levels of pho genes in high phosphate

We then investigated the consequence of the PhoU A147E substitution, which disrupts PhoR interaction. Because PhoU primarily functions as a negative regulator of PhoR kinase activity in high phosphate, we measured the mRNA levels of phoE, a PhoB-dependent gene encoding the PhoE phosphoporin, in both high- and low-phosphate media. In wild-type, phoE mRNA was undetectable in high phosphate but was strongly induced in low phosphate (Fig. 3A). In contrast, the phoU A147E substitution strongly elevated phoE mRNA levels in both high and low phosphate media (Fig. 3A). A similar expression pattern was detected in the phoU deletion mutant, indicating that the disruption of the PhoU-PhoR interaction via the A147E substitution results in constitutive phoE expression in high phosphate media. The phoU gene itself is also controlled by the PhoB/PhoR two-component system. Thus, phoU mRNA levels in the wild-type and the phoU A147E mutant were comparable to those of phoE, whereas no phoU transcript was detected in the phoU deletion mutant (Fig. 3B).

In contrast, the phoU A147E substitution did not affect expression patterns of the phoE and phoU genes in low Mg²⁺ (Fig. 3A–3C), suggesting the phoU A147E substitution affects phosphate signaling but not low Mg²⁺-mediated signaling. As a control, mRNA levels of mgtC encoding a virulence protein were highly induced in all above strains in low Mg²⁺ (Fig. 3C) (Alix and Blanc-Potard, 2007; Lee and Lee, 2015).

Additionally, when we measured PhoR protein levels using a strain with a C-terminally myc-tagged phoR gene, the phoU A147E substitution elevated PhoR protein levels in both high and low phosphate media (Fig. 3D and 3E). The elevated expression of phoE and phoU mRNA or PhoR protein levels of the phoU A147E mutant in high phosphate may result from the fact that the A147E substitution disrupts both the PhoU-PhoR PAS domain interaction and the associated suppression of PhoR autophosphorylation activity. Indeed, when we measured the rate of PhoR autophosphorylation in membrane vesicles prepared from the wild-type and phoU A147E mutant, the A147E substitution increased the rate of PhoR autophosphorylation compared to the wild-type in high phosphate (Fig. 3F and 3G). Collectively, these data demonstrate that the PhoU Ala147 residue is required for its interaction with the PhoR histidine kinase and for the repression of PhoR autophosphorylation and PhoB-dependent gene expression in high phosphate.

PhoU Ala147-mediated interaction with PhoR is required for Salmonella virulence

Maintaining phosphate (Pi) homeostasis is essential for Salmonella virulence, as Pi concentrations are proposed to be low within the Salmonella-containing vacuole during infection (García-del Portillo et al., 2008; Röder et al., 2021). In agreement with this notion, the phoU deletion mutant exhibited attenuated virulence in Salmonella (Choi et al., 2022).

Given that the phoU A147E mutant shows similar derepression of pho regulon genes as the phoU deletion mutant, we wondered whether the phoU A147E substitution also affects Salmonella pathogenesis. When we infected wild-type Salmonella strain into J774A.1 macrophage-like cells, intracellular replication of wild-type Salmonella increased approximately 8-fold by 21 h post-infection (Fig. 4A). In contrast, the phoU A147E substitution mutant showed decreased replication, reaching only ~20% of wild-type levels (Fig. 4A). This decrease in macrophage replication was similar to that of the phoU deletion mutant, indicating that the macrophage survival phenotype of the phoU A147E mutant resemble that of the phoU deletion mutant, consistent with its failure to repress pho regulon expression in high phosphate.

Consistent with macrophage survival, when we injected ~3,000 CFU of Salmonella strains into C3H/HeN mice, the phoU A147E substitution resulted in a complete loss of mouse virulence, with 100% survival of infected mice, compared to only 20% survival in mice infected with wild-type Salmonella (Fig. 4B). Similarly, the phoU deletion mutant also exhibited a defect in mouse virulence, with 80% of mice surviving 20 days post-infection (Fig. 4B). Together, these results demonstrate that both the phoU A147E substitution and phoU deletion impair intramacrophage survival and attenuate mouse virulence. In conclusion, these findings suggest that the interaction between PhoU Ala147 and the PAS domain of PhoR is essential for Salmonella virulence and for repression of pho regulon expression in high phosphate.

The phoU Ala147-to-Glu substitution does not exhibit a growth defect regardless of phosphate concentration

However, not all phenotypes of the phoU deletion mutant were recapitulated by the phoU A147E substitution mutant. First, the phoU deletion mutant forms small colonies on LB solid agar but the colony size of the phoU A147E mutant was similar to that of wild-type. When we measured a growth curve in N-minimal media containing high phosphate, the phoU A147E substitution mutant grew similarly to wild-type, whereas the phoU deletion mutant exhibited delayed growth in the same media (Fig. 5A). In N-minimal media containing low phosphate, the growth defect of the phoU deletion mutant was exaggerated (Fig. 5B). However, even in this condition, the phoU A147E substitution mutant grew similarly to wild-type, indicating that disruption of the PhoU-PhoR PAS domain interaction derepresses pho regulon gene expression in high phosphate but does not lead to a growth defect (Fig. 5B). This represents the first report of a phoU mutation that uncouples the growth defect phenotype from the derepression of pho gene expression in high phosphate.

The phoU Ala147-to-Glu substitution does not lead to in vivo polyphosphate accumulation

Polyphosphate (polyP) is a linear polymer of phosphate units linked by high-energy phosphoanhydride bonds. In bacteria, polyP is involved in diverse physiological processes, such as energy storage, stress responses, antibiotic resistance, and virulence (Kornberg et al., 1999; Rao et al., 2009). Previous studies have reported the accumulation of polyphosphate in phoU mutants in many bacterial species (de Almeida et al., 2015; Li and Zhang, 2007; Lubin et al., 2016; Morohoshi et al., 2002). To access whether the phoU A147E substitution affects polyphosphate accumulation in Salmonella, we used confocal laser scanning microscopy (CLSM) to visualize polyP granules. As expected, the phoU mutant displayed strong signals from polyP granule staining compared to the wild-type strain (Fig. 6). However, the phoU A147E substitution mutant did not exhibit detectable polyphosphate granule signals, even lower than that of the wild-type (Fig. 6). These data indicate that the phoU A147E substitution does not lead to polyphosphate accumulation in Salmonella. Collectively, disruption of PhoR PAS-PhoU interaction via PhoU A147E substitution results in derepression of pho regulon gene expression and attenuation of Salmonella virulence, but does not affect phenotypes such as growth defects or polyphosphate accumulation.

Discussion

Using bacterial two-hybrid assay and immunoprecipitation (Fig. 2), we identified that PhoU interacts with the PAS domain of PhoR via Ala147 in Salmonella, consistent with a prior study conducted in E. coli (Gardner et al., 2015). The introduction of the phoU Ala147-to-Glu substitution in Salmonella disrupts this interaction, leading to increased phoE mRNA levels even under high phosphate conditions (Fig. 3). This phenotype supports the role of that PhoU as a negative regulator of the phoU regulon via binding to the PhoR PAS domain. Additionally, the increased protein level of PhoR in phoU Ala147-to-Glu mutant under high phosphate conditions further highlights the importance of Ala147 in repressing pho regulon gene expression (Fig. 3). Together, the constitutive expression in phoE and elevated PhoR levels due to the Ala147-to-Glu substitution suggest that PhoU binding to the PhoR PAS domain via Ala147 suppresses pho regulon gene expression, likely by inhibiting auto-phosphorylation activity of PhoR. In this aspect, the phoU A147E substitution phenocopies the phoU deletion mutant, indicating that disruption of the PhoU-PhoR PAS domain interaction is sufficient to explain phenotypes of the phoU deletion mutant. In agreement with this notion, the phoU A147E mutant completely attenuated Salmonella virulence in mice, similar to the phoU deletion mutant (Fig. 4).

Interestingly, however, not all phenotypes of the phoU deletion mutant were recapitulated in the phoU A147E substitution mutant. While the phoU deletion mutant showed a growth defect and accumulated polyphosphate granules, the A147E substitution did not impair growth or induce polyP accumulation in Salmonella (Figs. 5 and 6). This suggests that the growth defect and polyphosphate accumulation phenotypes in the phoU mutant are not linked to the PhoU-PhoR PAS interaction, and that the phenotypes associated with phoU deletion can be genetically dissected into distinct groups.

Previous studies have shown that PhoU directly interacts with PstB in the PstSCAB₂ transporter (Gardner et al., 2014), and this interaction may inhibit phosphate uptake (diCenzo et al., 2017). Our structure modeling of the PhoR-PhoU-PstSCAB₂ complex, along with two-hybrid assays between PhoU and PstB (Fig. S1E), support this interaction. Although disruption of the PhoU-PhoR PAS domain interaction elevates pho regulon gene expression and the production of pho regulon components such as the PstSCAB₂ transporter and PhoU, the interaction between PhoU and PstB is still maintained. Thus, this is expected to suppress the activity of the PstSCAB₂ transporter in both high and low phosphate conditions. In agreement with this idea, it was previously reported that the E. coli phoU35 mutant – which was later identified to carry the same substitution in phoU – did not show an altered rate of Pi incorporation compared to the wild-type in phosphate-depleted conditions (Gardner et al., 2015; Muda et al., 1992). Because the phoU A147E substitution maintains the PhoU-PstB interaction and does not result in polyphosphate accumulation, this suggests that relieving suppression of phosphate uptake activity might contribute to the polyphosphate accumulation phenotype. Interestingly, the phoU A147E mutant exhibited even higher levels of phoE mRNA and PhoR protein than the phoU deletion mutant (Fig. 3). Since the A147E substitution differs from the phoU deletion in that it disrupts the PhoU-PhoR interaction while maintaining the PhoU-PstB interaction within the PhoR-PhoU-PstSCAB₂ complex, these differences in expression levels appear to result from altered feedback regulation: loss of PhoR interaction prevents suppression of PhoR kinase activity or switching to phosphatase activity, while maintenance of the PstB interaction may limit phosphate uptake in high phosphate conditions. These mechanisms remain to be further elucidated.

Our previous work demonstrated that Salmonella’s PhoU can interact with PhoR histidine kinase at two distinct sites: the PAS and CA domains (Choi et al., 2022). The interaction with the PhoR CA domain, mediated by Arg184 residue of PhoU, is required for the activation of PhoR in low Mg²⁺ (Choi et al., 2022), a condition physiologically relevant during host infection. Thus, the PhoU-PhoR CA domain interaction is necessary for Salmonella virulence (Choi et al., 2022). Here, we show that the substitution of Ala147 with Glu in PhoU, which disrupts the PhoU-PhoR PAS interaction, also attenuates virulence in mouse, suggesting that this interaction likewise contributes to Salmonella pathogenicity (Fig. 4). Notably, the phoU A147E substitution does not impair bacterial growth in low Mg²⁺, a condition commonly used to model infection-relevant conditions, providing an example that uncouples growth in low Mg²⁺ from in vivo virulence. Since both PhoU-PhoR interactions-one inhibitory (PAS) and one activating (CA)-are required for full virulence, this highlights the importance of fine-tuning pho regulon expression in Salmonella pathogenesis.

This study provides offers a new perspective on the physiological roles of PhoU in bacteria. The increase in antibiotic and secondary metabolite production observed in Streptomyces (Martin-Martin et al., 2017; Tang et al., 2022) appears to be linked to the PhoB-PstB interaction, as polyphosphate (polyP) accumulation is a key regulatory factor. Similarly, the reduced persister formation and antibiotic resistance in the phoU deletion mutant (Wang et al., 2013, 2017), which are associated with intracellular phosphate levels, suggest that these phenotypes are mediated by PhoU’s interaction with the PstSCAB₂ transporter. Furthermore, in Caulobacter crescentus, polyphosphate accumulation occurs independently of the PhoB/PhoR system (Lubin et al., 2016), likely due to mis-regulation of the Pst system. In conclusion, targeted amino acid substitutions, rather than complete deletion of phoU, allows dissection of the complex regulatory roles of PhoU in pho regulon control and phosphate homeostasis. These findings highlight the complexity of PhoU-mediated regulation and PhoU’s role in coordinating multiple physiological pathways in bacteria.

Acknowledgments

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) [NRF-2022R1A2B5B02002256, NRF-2022R1A4A1025913, NRF-RS-2025-00561488 to E.-J.L.]; and the Ministry of Education [NRF-RS-2024-00350890, RS-2024-00461215 to S.C.] and a grant from Korea University.

Author Contributions

E.-J.L. designed the research, analyzed the data, and wrote the manuscript; S.B. performed the experiments and wrote the manuscript; S.C. and Y.H. performed experiments; E.C. performed initial experiments; S.P. and J.-S.L. performed mouse experiments.

Conflict of Interest

The authors declare no competing interests.

Ethical Statements

All experimental procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (approval no. KW-210728-1).

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.71150/jm.2505013.

Table S1.

Bacterial strains, plasmids, and oligonucleotides used in this study

jm-2505013-Supplementary-Table-S1.pdf

Fig. S1.

PhoU Ala147 is not required for interaction with PstB. (A–B) Schematic representation of the bacterial two-hybrid assay used to assess interactions between PhoR or PstB and PhoU or its variants. (C–D) Escherichia coli BTH101 strains were co-transformed with plasmid pairs: pKT25 derivatives expressing a C-terminal fusion of the cyaA T25 fragment to phoR (C) or pstB (D), and pUT18 or pUT18C derivatives expressing N-terminal fusions of the cyaA T18 fragment to wild-type phoU or C-terminal fusions to phoU^A147E, phoU^A147K, or phoU^R148A. Cells containing pUT18C served as a negative control. Cells co-expressing pUT18-mgtC and pKT25-mgtR served as a positive control. Cultures were spotted onto LB agar plates containing 80 μM X-Gal and 0.5 mM IPTG and incubated at 30°C for 48 h. Blue colonies indicate positive interactions. (E–F) Alphafold-assisted modeling of PhoR-PhoU-Pst complex. PhoU (UniProt: A0A0F6B922) and its interacting partners PhoR (A0A0F6AXL9) and PstB (A0A0F6B923) in Salmonella Typhimurium 14028s, were used to generate complex prediction. PhoU is shown in green, with Ala147 highlighted in red. PhoR and PstB are shown in pink and sky blue, respectively.

jm-2505013-Supplementary-Fig-S1.pdf

Fig. 1.

Regulatory role of PhoU in Salmonella. Under low-phosphate conditions, the histidine kinase PhoR undergoes autophosphorylation at a conserved histidine residue in its DHp domain and subsequently transfers the phosphate to the response regulator PhoB. Phosphorylated PhoB binds to promoter regions and activates the expression of the pho regulon, which includes the PhoB/PhoR two-component system itself, the high-affinity phosphate transporter PstSCAB₂, and the negative regulator PhoU. PhoU interacts with both PhoR and the ATPase subunit PstB of the PstSCAB₂ transporter to inhibit PhoR kinase activity under high-phosphate conditions. The Ala147 residue of PhoU mediates its interaction with the PAS domain of PhoR. Substitution of Ala147 with glutamate (PhoU A147E) disrupts this interaction and constitutively activates pho regulon expression, independent of phosphate availability.

Fig. 2.

PhoU Ala147 is required for interaction with PhoR. (A) Schematic representation of the bacterial two-hybrid assay used to assess the interaction between PhoR and either wild-type PhoU or the PhoU^A147E mutant. (B) Escherichia coli BTH101 cells were co-transformed with plasmid combinations expressing N-terminal fusions of the cyaA T18 fragment to wild-type phoU (pUT18C-phoU) or the A147E variant (pUT18C-phoU^A147E), and C-terminal fusions of the cyaA T25 fragment to phoR (pKT25-phoR), or with the empty vector (pUT18C) as a negative control. Cells co-expressing pUT18-mgtC and pKT25-mgtR served as a positive control. Spotted cultures were incubated on LB agar plates supplemented with 0.5 mM IPTG and 80 μM X-Gal at 30°C for 48 h. Blue colonies indicate a positive protein–protein interaction. (C–D) The C-terminally GFP-tagged PhoR protein immunoprecipitates PhoU. (Left) Crude lysates prepared from wild-type Salmonella strains coexpressing PhoR-GFP and either PhoU-His or PhoU^A147E-His were analyzed by immunoblotting with anti-His (C) and anti-GFP (D) antibodies. (Right) Eluted fractions from the same strains were subjected to immunoprecipitation using anti-GFP antibody-coated beads and analyzed by immunoblotting with anti-His (C) and anti-GFP (D) antibodies.

Fig. 3.

The phoU^A147E substitution derepresses pho regulon gene expression under high-phosphate conditions. (A–C) Relative mRNA levels of phoE (A), phoU (B), and mgtC (C) were measured in Salmonella strains carrying wild-type phoU (14028s), the A147E mutant allele (SM323), or a ∆phoU deletion (SM101). Bacteria were grown for 5 h in N-minimal medium supplemented with 10 mM Pi and 10 mM Mg²⁺ (High Pi Mg²⁺), 10 mM Mg²⁺ and 0.01 mM Pi (Low Pi), or 10 mM Pi and 0.01 mM Mg²⁺ (Low Mg²⁺). Data represent means ± SD from three independent experiments (n = 3). Relative mRNA levels were calculated as (target RNA / rrsH RNA) × 10⁴. Statistical significance between wild-type and corresponding mutant strains was assessed using a paired two-tailed t-test; ns = not significant, p^* < 0.05, p^** < 0.01. (D–E) The phoU^A147E substitution derepresses PhoR protein levels under high-phosphate conditions. Western blot analysis of crude extracts from strains carrying wild-type phoU (SM454), the phoU^A147E mutation (SM457), or an untagged control strain (14028s), all in a background with C-terminally 8×Myc-tagged phoR, except for the no-tag control. Blots were probed with anti-Myc (D) to detect PhoR-8×Myc and with anti-Fur (E) as a loading control. Bacteria were grown for 5 h in N-minimal medium supplemented with either 10 mM or 0.01 mM inorganic phosphate (Pi), as described in 'Materials and Methods'. (F–G) Autophosphorylation assay of Salmonella strains carrying either wild-type phoU (F) or the phoU A147E substitution (G), performed with [γ-³²P] ATP at the indicated times (min). Bacteria were grown for 5 h in N-minimal medium supplemented with 10 mM Pi, and the membrane vesicles were prepared as described in 'Materials and Methods'.

Fig. 4.

PhoU Ala147-mediated interaction with PhoR is required for Salmonella virulence. (A) Substitution of Ala147 with Glu in PhoU reduces intracellular survival in J774A.1 macrophages. Wild-type Salmonella (14028s), a chromosomal phoU^A147E mutant (SM323), and a phoU deletion mutant (SM101) were used to infect macrophages, and bacterial counts were determined at 1 h (T1) and 21 h (T21) post-infection. Fold replication was calculated as [CFU at T21 / CFU at T1]. Data represent means ± SD from three independent infections. Statistical significance between strains was assessed using a paired two-tailed t-test; ns = not significant, p^* < 0.05. (B) The phoU^A147E mutation also decreases virulence in mice. Survival of C3H/HeN mice (n = 5 per group) inoculated intraperitoneally with ~10³ CFU of the strains listed above was monitored over 21 days. According to the log-rank test, there were significant differences among three groups (P < 0.0001).

Fig. 5.

The phoU^A147E substitution does not impair bacterial growth under either high- or low-phosphate conditions. (A, B) Growth curves of Salmonella strains 14028s (wild-type), SM323 (phoU^A147E), and SM101 (ΔphoU). Bacteria were cultured in N-minimal medium containing 10 mM Pi (A) or 0.01 mM Pi (B) at 37°C for 8 h. Optical density at 600 nm (OD₆₀₀) was measured hourly.

Fig. 6.

The phoU^A147E substitution does not affect in vivo polyphosphate accumulation. Confocal laser scanning microscopy (CLSM) images of wild-type Salmonella (14028s), the chromosomal phoU^A147E mutant (SM323), and the phoU deletion mutant (SM101). Bacteria were grown for 5 h in N-minimal medium containing 0.01 mM Mg²⁺. Polyphosphate granules and nucleoids were stained with DAPI, and plasma membranes were stained with FM4-64, as described in ‘Materials and Methods.’ Merged fluorescence images are shown to visualize polyphosphate localization. polyP: polyphosphate; PM: plasma membrane.

References

Abramson J, Adler J, Dunger J, Evans R, Green T, et al. 2024. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 630: 493–500. Article PubMed PMC PDF
Alix E, Blanc-Potard AB. 2007. MgtC: a key player in intramacrophage survival. Trends Microbiol. 15: 252–256. Article PubMed
Bachhawat P, Swapna GV, Montelione GT, Stock AM. 2005. Mechanism of activation for transcription factor PhoB suggested by different modes of dimerization in the inactive and active states. Structure. 13: 1353–1363. Article PubMed PMC
Baek S, Lee EJ. 2024. PhoU: a multifaceted regulator in microbial signaling and homeostasis. Curr Opin Microbiol. 77: 102401.Article PubMed
Bong D, Sohn J, Lee SJV. 2024. Brief guide to RT-qPCR. Mol Cells. 47: 100141.Article PubMed PMC
Cho BK, Knight EM, Palsson BO. 2006. PCR-based tandem epitope tagging system for Escherichia coli genome engineering. Biotechniques. 40: 67–72. Article PubMed
Choi S, Choi E, Cho YJ, Nam D, Lee J, et al. 2019. The Salmonella virulence protein MgtC promotes phosphate uptake inside macrophages. Nat Commun. 10: 3326.Article PubMed PMC PDF
Choi S, Jeong G, Choi E, Lee EJ. 2022. A dual regulatory role of the PhoU protein in Salmonella Typhimurium. mBio. 13: e0081122. Article PubMed PMC PDF
Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 97: 6640–6645. Article PubMed PMC
de Almeida LG, Ortiz JH, Schneider RP, Spira B. 2015. phoU inactivation in Pseudomonas aeruginosa enhances accumulation of ppGpp and polyphosphate. Appl Environ Microbiol. 81: 3006–3015. Article PubMed PMC PDF
diCenzo GC, Sharthiya H, Nanda A, Zamani M, Finan TM. 2017. PhoU allows rapid adaptation to high phosphate concentrations by modulating PstSCAB transport rate in Sinorhizobium meliloti. J Bacteriol. 199: e00143–17. Article PubMed PMC PDF
Ellison DW, McCleary WR. 2000. The unphosphorylated receiver domain of PhoB silences the activity of its output domain. J Bacteriol. 182: 6592–6597. Article PubMed PMC PDF
Gao R, Stock AM. 2015. Temporal hierarchy of gene expression mediated by transcription factor binding affinity and activation dynamics. mBio. 6: e00686–15. Article PubMed PMC PDF
García-del Portillo F, Núñez-Hernández C, Eisman B, Ramos-Vivas J. 2008. Growth control in the Salmonella-containing vacuole. Curr Opin Microbiol. 11: 46–52. Article PubMed
Gardner SG, Johns KD, Tanner R, McCleary WR. 2014. The PhoU protein from Escherichia coli interacts with PhoR, PstB, and metals to form a phosphate-signaling complex at the membrane. J Bacteriol. 196: 1741–1752. Article PubMed PMC PDF
Gardner SG, Miller JB, Dean T, Robinson T, Erickson M, et al. 2015. Genetic analysis, structural modeling, and direct coupling analysis suggest a mechanism for phosphate signaling in Escherichia coli. BMC Genet. 16(Suppl 2): S2.Article PubMed PMC PDF
Glover RT, Kriakov J, Garforth SJ, Baughn AD, Jacobs WR Jr. 2007. The two-component regulatory system senX3-regX3 regulates phosphate-dependent gene expression in Mycobacterium smegmatis. J Bacteriol. 189: 5495–5503. Article PubMed PMC PDF
Hsieh YJ, Wanner BL. 2010. Global regulation by the seven-component Pi signaling system. Curr Opin Microbiol. 13: 198–203. Article PubMed PMC
Karimova G, Pidoux J, Ullmann A, Ladant D. 1998. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci USA. 95: 5752–5756. Article PubMed PMC
Kornberg A, Rao NN, Ault-Riché D. 1999. Inorganic polyphosphate: a molecule of many functions. Annu Rev Biochem. 68: 89–125. Article PubMed
Lamarche MG, Wanner BL, Crépin S, Harel J. 2008. The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis. FEMS Microbiol Rev. 32: 461–473. Article PubMed
Lee JW, Lee EJ. 2015. Regulation and function of the Salmonella MgtC virulence protein. J Microbiol. 53: 667–672. Article PubMed PDF
Li Y, Zhang Y. 2007. PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob Agents Chemother. 51: 2092–2099. Article PubMed PMC PDF
Lubin EA, Henry JT, Fiebig A, Crosson S, Laub MT. 2016. Identification of the PhoB regulon and role of PhoU in the phosphate starvation response of Caulobacter crescentus. J Bacteriol. 198: 187–200. Article PubMed PMC PDF
Makino K, Amemura M, Kawamoto T, Kimura S, Shinagawa H, et al. 1996. DNA binding of PhoB and its interaction with RNA polymerase. J Mol Biol. 259: 15–26. Article PubMed
Makino K, Shinagawa H, Amemura M, Kimura S, Nakata A, et al. 1988. Regulation of the phosphate regulon of Escherichia coli. Activation of pstS transcription by PhoB protein in vitro. J Mol Biol. 203: 85–95. Article PubMed
Martín-Martín S, Rodríguez-García A, Santos-Beneit F, Franco-Domínguez E, Sola-Landa A, et al. 2017. Self-control of the PHO regulon: the PhoP-dependent protein PhoU controls negatively expression of genes of PHO regulon in Streptomyces coelicolor. J Antibiot (Tokyo). 71: 113–122. Article PubMed PDF
Morohoshi T, Maruo T, Shirai Y, Kato J, Ikeda T, et al. 2002. Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803. Appl Environ Microbiol. 68: 4107–4110. Article PubMed PMC PDF
Muda M, Rao N, Torriani A. 1992. Role of PhoU in phosphate transport and alkaline phosphatase regulation. J Bacteriol. 174: 8057–8064. Article PubMed PMC PDF
Piepenbreier H, Fritz G, Gebhard S. 2017. Transporters as information processors in bacterial signalling pathways. Mol Microbiol. 104: 1–15. Article PubMed PDF
Rao NN, Gomez-Garcia MR, Kornberg A. 2009. Inorganic polyphosphate: essential for growth and survival. Annu Rev Biochem. 78: 605–647. Article PubMed
Röder J, Felgner P, Hensel M. 2021. Single-cell analyses reveal phosphate availability as critical factor for nutrition of Salmonella enterica within mammalian host cells. Cell Microbiol. 23: e13374. Article PubMed
Schramke H, Laermann V, Tegetmeyer HE, Brachmann A, Jung K, et al. 2017. Revisiting regulation of potassium homeostasis in Escherichia coli: the connection to phosphate limitation. Microbiologyopen. 6: e00438. Article PubMed PMC PDF
Steed PM, Wanner BL. 1993. Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon. J Bacteriol. 175: 6797–6809. Article PubMed PMC PDF
Tang J, Chen J, Liu Y, Hu J, Xia Z, et al. 2022. The global regulator PhoU positively controls growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona. Front Microbiol. 13: 904627.Article PubMed PMC
Torriani A, Rothman F. 1961. Mutants of Escherichia coli constitutive for alkaline phosphatase. J Bacteriol. 81: 835–836. Article PubMed PMC PDF
Wang X, Han H, Lv Z, Lin Z, Shang Y, et al. 2017. PhoU2 but not PhoU1 as an important regulator of biofilm formation and tolerance to multiple stresses by participating in various fundamental metabolic processes in Staphylococcus epidermidis. J Bacteriol. 199: e00219-17.Article PubMed PMC PDF
Wang C, Mao Y, Yu J, Zhu L, Li M, et al. 2013. PhoY2 of mycobacteria is required for metabolic homeostasis and stress response. J Bacteriol. 195: 243–252. Article PubMed PMC PDF

Figure & Data

References

Citations

Citations to this article as recorded by

ePub Link

Cite this Article

Cite this Article: export Copy Download Format; Close

Download Citation

Download a citation file in RIS format that can be imported by all major citation management software, including EndNote, ProCite, RefWorks, and Reference Manager.

Format:

RIS — For EndNote, ProCite, RefWorks, and most other reference management software
BibTeX — For JabRef, BibDesk, and other BibTeX-specific software

Include:

Citation for the content below
Citation and abstract for the content below

PhoU interaction with the PhoR PAS domain is required for repression of the pho regulon and Salmonella virulence, but not for polyphosphate accumulation

J. Microbiol. 2025;63(9):e2505013 Published online September 30, 2025

DOI: https://doi.org/10.71150/jm.2505013

XML Download

Figure

PhoU interaction with the PhoR PAS domain is required for repression of the pho regulon and Salmonella virulence, but not for polyphosphate accumulation

Fig. 1. Regulatory role of PhoU in Salmonella. Under low-phosphate conditions, the histidine kinase PhoR undergoes autophosphorylation at a conserved histidine residue in its DHp domain and subsequently transfers the phosphate to the response regulator PhoB. Phosphorylated PhoB binds to promoter regions and activates the expression of the pho regulon, which includes the PhoB/PhoR two-component system itself, the high-affinity phosphate transporter PstSCAB2, and the negative regulator PhoU. PhoU interacts with both PhoR and the ATPase subunit PstB of the PstSCAB2 transporter to inhibit PhoR kinase activity under high-phosphate conditions. The Ala147 residue of PhoU mediates its interaction with the PAS domain of PhoR. Substitution of Ala147 with glutamate (PhoU A147E) disrupts this interaction and constitutively activates pho regulon expression, independent of phosphate availability.

Fig. 2. PhoU Ala147 is required for interaction with PhoR. (A) Schematic representation of the bacterial two-hybrid assay used to assess the interaction between PhoR and either wild-type PhoU or the PhoUA147E mutant. (B) Escherichia coli BTH101 cells were co-transformed with plasmid combinations expressing N-terminal fusions of the cyaA T18 fragment to wild-type phoU (pUT18C-phoU) or the A147E variant (pUT18C-phoUA147E), and C-terminal fusions of the cyaA T25 fragment to phoR (pKT25-phoR), or with the empty vector (pUT18C) as a negative control. Cells co-expressing pUT18-mgtC and pKT25-mgtR served as a positive control. Spotted cultures were incubated on LB agar plates supplemented with 0.5 mM IPTG and 80 μM X-Gal at 30°C for 48 h. Blue colonies indicate a positive protein–protein interaction. (C–D) The C-terminally GFP-tagged PhoR protein immunoprecipitates PhoU. (Left) Crude lysates prepared from wild-type Salmonella strains coexpressing PhoR-GFP and either PhoU-His or PhoUA147E-His were analyzed by immunoblotting with anti-His (C) and anti-GFP (D) antibodies. (Right) Eluted fractions from the same strains were subjected to immunoprecipitation using anti-GFP antibody-coated beads and analyzed by immunoblotting with anti-His (C) and anti-GFP (D) antibodies.

Fig. 3. The phoUA147E substitution derepresses pho regulon gene expression under high-phosphate conditions. (A–C) Relative mRNA levels of phoE (A), phoU (B), and mgtC (C) were measured in Salmonella strains carrying wild-type phoU (14028s), the A147E mutant allele (SM323), or a ∆phoU deletion (SM101). Bacteria were grown for 5 h in N-minimal medium supplemented with 10 mM Pi and 10 mM Mg2+ (High Pi Mg2+), 10 mM Mg2+ and 0.01 mM Pi (Low Pi), or 10 mM Pi and 0.01 mM Mg2+ (Low Mg2+). Data represent means ± SD from three independent experiments (n = 3). Relative mRNA levels were calculated as (target RNA / rrsH RNA) × 104. Statistical significance between wild-type and corresponding mutant strains was assessed using a paired two-tailed t-test; ns = not significant, p* < 0.05, p** < 0.01. (D–E) The phoUA147E substitution derepresses PhoR protein levels under high-phosphate conditions. Western blot analysis of crude extracts from strains carrying wild-type phoU (SM454), the phoUA147E mutation (SM457), or an untagged control strain (14028s), all in a background with C-terminally 8×Myc-tagged phoR, except for the no-tag control. Blots were probed with anti-Myc (D) to detect PhoR-8×Myc and with anti-Fur (E) as a loading control. Bacteria were grown for 5 h in N-minimal medium supplemented with either 10 mM or 0.01 mM inorganic phosphate (Pi), as described in 'Materials and Methods'. (F–G) Autophosphorylation assay of Salmonella strains carrying either wild-type phoU (F) or the phoU A147E substitution (G), performed with [γ-32P] ATP at the indicated times (min). Bacteria were grown for 5 h in N-minimal medium supplemented with 10 mM Pi, and the membrane vesicles were prepared as described in 'Materials and Methods'.

Fig. 4. PhoU Ala147-mediated interaction with PhoR is required for Salmonella virulence. (A) Substitution of Ala147 with Glu in PhoU reduces intracellular survival in J774A.1 macrophages. Wild-type Salmonella (14028s), a chromosomal phoUA147E mutant (SM323), and a phoU deletion mutant (SM101) were used to infect macrophages, and bacterial counts were determined at 1 h (T1) and 21 h (T21) post-infection. Fold replication was calculated as [CFU at T21 / CFU at T1]. Data represent means ± SD from three independent infections. Statistical significance between strains was assessed using a paired two-tailed t-test; ns = not significant, p* < 0.05. (B) The phoUA147E mutation also decreases virulence in mice. Survival of C3H/HeN mice (n = 5 per group) inoculated intraperitoneally with ~103 CFU of the strains listed above was monitored over 21 days. According to the log-rank test, there were significant differences among three groups (P < 0.0001).

Fig. 5. The phoUA147E substitution does not impair bacterial growth under either high- or low-phosphate conditions. (A, B) Growth curves of Salmonella strains 14028s (wild-type), SM323 (phoUA147E), and SM101 (ΔphoU). Bacteria were cultured in N-minimal medium containing 10 mM Pi (A) or 0.01 mM Pi (B) at 37°C for 8 h. Optical density at 600 nm (OD600) was measured hourly.

Fig. 6. The phoUA147E substitution does not affect in vivo polyphosphate accumulation. Confocal laser scanning microscopy (CLSM) images of wild-type Salmonella (14028s), the chromosomal phoUA147E mutant (SM323), and the phoU deletion mutant (SM101). Bacteria were grown for 5 h in N-minimal medium containing 0.01 mM Mg2+. Polyphosphate granules and nucleoids were stained with DAPI, and plasma membranes were stained with FM4-64, as described in ‘Materials and Methods.’ Merged fluorescence images are shown to visualize polyphosphate localization. polyP: polyphosphate; PM: plasma membrane.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

PhoU interaction with the PhoR PAS domain is required for repression of the pho regulon and Salmonella virulence, but not for polyphosphate accumulation