Pi depletion induces polymyxin resistance in diverse bacterial species.

(A) Time-kill curves of Escherichia coli MG1655 (left panel) and Salmonella. Typhimurium ATCC14028 (right panel) exposed to polymyxin B (PMXB) (4 μg/ml for E. coli strains, 20 μg/ml for S. Typhimurium) following acute Pi-starvation treatment (Pi-, 0 mM Pi) compared to the group without treatment (Pi+, 1.32 mM Pi). (B) Survival rates for various Enterobacteriaceae strains (E. coli MG1655, O157, UTI-1, UTI-3, UTI-4, UTI-5, S. Typhimurium ATCC14028) and Pseudomonas aeruginosa PAO1 exposed 1-hour to polymyxin B (4 μg/ml for E. coli strains, 20 μg/ml for S. Typhimurium and 8 μg/ml for P. aeruginosa). For all tested strains, phosphate depletion (Pi-, purple bars) significantly increases polymyxin B resistance compared to phosphate-replete (Pi+, teal bars) conditions. Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t-test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Pi depletion induces the expression of ugd-arn system to modify lipid A with L-Ara-4N, conferring stress-induced polymyxin resistance.

(A) Volcano plot depicting differentially expressed proteins in cells following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Red (right) and blue (left) dots represent significantly up-regulated and down-regulated proteins, respectively. The horizontal dashed line depicts a p-value cutoff (0.05), and vertical dashed lines depict 1/2 and 2-fold ratio cutoffs. Selected proteins are labeled. (B) Genetic organization of the arn operon and ugd gene in MG1655. Ugd and ArnABCDEFT catalyze the biosynthesis of 4-amino-4-deoxy-L-arabinose (L-Ara4N) and its modification of lipid A. (C) mRNA levels of arnB and ugd in cells following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). (D) Production of ArnB-3×FLAG in cells following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). The protein band corresponding to ArnB-3×FLAG is marked. (E) β-galactosidase activities of Parn-lacZ and Pugd-lacZ in MG1655 ΔlacZ cells following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). (F) ESI-MS spectra of lipid A molecules extracted from E. coli MG1655 cells following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Peaks corresponding to native lipid A and L-Ara-4N modified lipid A were shown. (G) Survival rates for E. coli MG1655 parent and isogenic ΔarnT, ΔarnB and Δugd mutants exposed 1-hour to polymyxin B (4 μg/ml) following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-tailed Student’s t-test for C and E, and one-way ANOVA followed by Dunnett’s multiple comparisons test for G. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

The PmrAB two-component system is activated during Pi depletion and is responsible for the up-regulation of arn genes under this condition.

(A) Survival rates for E. coli MG1655 parent and isogenic ΔpmrB, ΔpmrA, ΔphoB, and ΔphoR mutants exposed 1-hour to polymyxin B (4 μg/ml) following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). (B) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ parent and isogenic ΔpmrB, ΔpmrA, ΔphoB, and ΔphoR mutants following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). (C) Phosphorylation of PmrA-3×FLAG detected by Phos-Tag SDS–PAGE following immunoblotting with anti-FLAG antibody in cells following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Protein bands representing PmrA-3×FLAG and PmrA-3×FLAG-P are indicated. (D) Schematic diagram of the whole-cell FRET measurement of PmrA-promoter binding assay. A bright fluorescent unnatural amino acid donor CouA (red spheres) is incorporated into G137 of PmrA in DNA-binding domain. SYTO9 (green spheres) is employed to stain chromosome DNA in living E. coli MG1655. (E) Quantification of the FRET effect between the CouA-SYTO9 pair in E. coli cells following acute Pi-starvation treatment (Pi-, 0 mM Pi, 10 μM Fe) or iron excess treatment (Pi+, 1.32 mM Pi, 500 μM FeSO4) compared to the group without treatment (Pi+, 1.32 mM Pi, 10 μM FeSO4). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test for A, B, and E. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Activation of PmrAB during Pi depletion is mediated by the secondary stress signal iron (Fe3+) induced under this condition

(A) Cellular metal contents determined by ICP-MS in E. coli MG1655 cells following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). (B) Iron contents in the spent medium of E. coli MG1655 at the indicated time points during Pi depletion treatment (Pi-, 0 mM). (C) Schematic diagram of the sensor kinase PmrB. Two important residues for sensing Fe3+, E36A and E39A, are shown. (D) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ parent and isogenic pmrBE36A and pmrBE36A mutants following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). (E) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ cells following Pi depletion treatment (Pi-, 0 mM Pi, 10 μM Fe) or Fe, Pi depletion treatment (Pi- Fe-, 0 mM Pi, 0 μM Fe) compared to the group without treatment (Pi+, 1.32 mM Pi, 10 μM FeSO4). (F) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ grown under Pi sufficient MOPS medium in the presence of different concentrations of FeSO4 and that subjected to Pi depletion conditions in the presence of different concentrations of FeSO4. Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test for B, and two-tailed Student’s t-test for C, D, and E. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Response to Pi depletion induced iron mobilization to cell surfaces

(A) Iron (Fe) and magnesium (Mg) contents in E. coli MG1655 cells following Pi depletion treatment (Pi-, 0 mM Pi) at the indicated time points. (B) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ cells and Fe contents in E. coli MG1655 cells following Pi depletion treatment at the indicated time points. (C) Fe contents in E. coli MG1655 parent and isogenic ΔpmrA mutant cells following Pi depletion treatment (Pi-, 0 mM Pi) at the indicated time points. (D) Fe contents in E. coli MG1655 cells with Pi depletion treatment (Pi-, 0 mM) and without Pi depletion treatment (Pi+, 1.32 mM) following washing by HEPES or DFOM-supplemented (50μM) HEPES buffer. (E) Reduction of cellular Fe content following washing with DFOM-supplemented (50μM) HEPES buffer. Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-tailed Student’s t-test for C and E, and two-way ANOVA followed by Sidak’s multiple comparisons test for D. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Iron mobilization is driven by Mg2+ dissociation during Pi depletion

(A) Fe contents in E. coli MG1655 cells following acute Pi-starvation treatment (Pi-, 0 mM Pi, 0.53 mM Mg) or Mg excess Pi depletion treatment (Pi-, 0 mM Pi, 10 mM Mg) compared to the group without treatment (Pi+, 1.32 mM, 0.53 mM Mg). (B) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ cells following acute Pi-starvation treatment (Pi-, 0 mM Pi, 0.53 mM Mg) or Mg excess Pi depletion treatment (Pi-, 0 mM Pi, 10 mM Mg) compared to the group without treatment (Pi+, 1.32 mM, 0.53-10 mM Mg). (C) Mg content in the spent medium during Pi depletion treatment at the indicated time points. (D) 2D graph plotted from output signals of two channels of the PI-BactD sensor: fluorescence increase (ΔS/S0) and fluorescence radiometric changes (I482/I375) in each of the treatments indicated. Data are presented as mean ± s.d. of three biological replicates for A, B, and C, and six biological replicates for D. Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-tailed Student’s t-test for a, and one-way ANOVA followed by Dunnett’s multiple comparisons test for B and C. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

A Mg-Fe-PmrAB regulatory circuit underpins the Pi-depletion induced polymyxin resistance through activating the lipid A L-Ara4N modification system

In cell grown under Pi sufficient condition, ribosome and ATP serve as the major intracellular Mg2+ and Pi reservoir. Mg2+ neutralizes negative charge of lipid A and stabilizes outer membrane (OM). The PmrAB two-component system (TCS) is inactive, and the cell is sensitive to polymyxin. During Pi stress, ATP biosynthesis and ribosome biogenesis are halted due to Pi depletion, disrupting the balance between Pi and Mg2+. Mg2+ disassociates from the cell which destabilizes the OM and derives compensatory Fe3+ mobilization to cell surface. Mobilized Fe3+ activates the PmrAB TCS which in turn upregulates the arn operon. Arn and Ugd proteins catalyze the biosynthesis of L-Arn4N and modify lipid A with L-Arn4N to stabilize OM under stressed condition and prevent further Fe mobilization. E. coli cells with L-Arn4N modified lipid A become resistant to polymyxin.

Disrupting the Mg-Fe-PmrAB signaling circuit abolished Pi-depletion induced polymyxin resistance in Enterobacteriaceae

(A) Time-kill curves of Escherichia coli MG1655 (left panel) and Salmonella. Typhimurium ATCC14028 (right panel) exposed to polymyxin B (PMXB) (4 μg/ml for E. coli strains, 20 μg/ml for S. Typhimurium) following acute Pi-starvation treatment (Pi-, 0 mM Pi), Pi depletion treatment with excess Mg (Pi-, 0 mM Pi, 10mM MgCl2) or Pi depletion treatment with iron chelator (Pi-, 0 mM Pi, 10 μM DFOM) compared to the group without treatment (Pi+, 1.32 mM Pi). (B) Distinct stress adaptation strategies in E. coli and P. aeruginosa under Pi depletion conditions. Genes and regulatory systems activated in the two species are illustrated. Inactive regulatory systems are depicted in faded effects. The regulatory mechanisms for P. aeruginosa are based on findings from Jones et. al.11 (C) A phylogenetic tree of 250 PmrB homologous. A predominant clade is indicated by a blue circle, comprising sequences from bacteria such as Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella, Yersinia, and Enterobacter species. A green circle marks several clades distinct from the predominant one. The domain architecture and AlphaFold3-predicted structure of EcoPmrB and PaePmrB are shown. Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Workflow of acute Pi depletion treatment and following proteomic sample preparation and polymyxin B challenging assay.

Bacteria were cultured to an OD600 of 0.4 in standard MOPS synthetic medium containing 1.32 mM inorganic phosphate (Pi), representing Pi-sufficient conditions. For Pi-depletion conditions, bacteria were first grown to OD600 = 0.4 in the same medium, then transferred to Pi-depleted MOPS medium (0 mM Pi). For comparative proteomic analysis, the collected bacterial cells were subjected to sample preparation and proteomic analysis. For the polymyxin challenging assay, the collected bacterial cells were resuspended in standard MOPS medium and treated with the designated concentration of polymyxin B. Cell viability was assessed at the beginning and end of treatment by serial dilution and plating.

Killing of E. coli MG1655 with different concentration of polymyxin B.

Survival rates for E. coli MG1655 cells exposed 1-hour to polymyxin B (1-16 μg/ml) following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t-test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Quantitative proteomic analysis of E. coli MG1655 in response to acute Pi depletion.

Pie chart of total and differentially expressed proteins detected by LC-MS/MS analysis in E. coli MG1655 cells subjected to Pi depletion for 3 hours, relative to cells under Pi sufficient conditions. Red and blue colors represent significantly upregulated and downregulated proteins, respectively. Clusters of Orthologous Groups (COG) functional categories of differentially expressed proteins in E. coli MG1655 in response to Pi depletion. Red and blue colors represent significantly upregulated and downregulated proteins, respectively.

The ratio of L-Ara-4N modified Lipid A molecule increases in E. coli O157, E. coli UTI-3, S. Typhimurium ATCC14028 and P. aeruginosa PAO1 cells subjected to Pi depletion.

(A) ESI-MS spectra of lipid A molecules extracted from E. coli O157 cells following acute Pi-starvation treatment (Pi-, 0 mM) (right) compared to the group without treatment (Pi+, 1.32 mM) (left). Peaks correspond to native lipid A lost a HPO3 (1718), native lipid A (1798), L-Ara-4N modified lipid A lost a HPO3 (1848) and L-Ara-4N modified lipid A (1928) are shown. (B) ESI-MS spectra of lipid A molecules extracted from E. coli UTI-3 cells following acute Pi-starvation treatment (Pi-, 0 mM) (right) compared to the group without treatment (Pi+, 1.32 mM) (left). Peaks correspond to native lipid A lost a HPO3 (1718), native lipid A (1798), L-Ara-4N modified lipid A lost a HPO3 (1848) and L-Ara-4N modified lipid A (1928) are shown. (C) ESI-MS spectra of lipid A molecules extracted from S. Typhimurium ATCC14028 cells following acute Pi-starvation treatment (Pi-, 0 mM) (right) compared to the group without treatment (Pi+, 1.32 mM) (left). Peaks correspond to native lipid A lost a HPO3 (1718), native lipid A (1798), L-Ara-4N modified lipid A lost a HPO3 (1848), L-Ara-4N and -OH modified lipid A lost a HPO3 (1863) and L-Ara-4N modified lipid A (1928) are shown. (D) ESI-MS spectra of lipid A molecules extracted from PAO1 cells following acute Pi-starvation treatment (Pi-, 0 mM) (right) compared to the group without treatment (Pi+, 1.32 mM) (left). Peaks correspond to hydroxylated lipid A lost a HPO3 (1366), di-hydroxylated lipid A lost a HPO3 (1382) are shown.

Parn and Pugd promoter activities in E. coli MG1655 during Pi depletion supplemented with different concentration of Pi.

(A) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ cells following Pi-starvation treatment with different concentration of Pi (Pi-, 0-0.4 mM) compared to the group without treatment (Pi+, 1.32 mM). (B) β-galactosidase activities of Pugd-lacZ in MG1655 ΔlacZ cells following Pi-starvation treatment with different concentration of Pi (Pi-, 0-0.4 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Up-regulation of arn genes during Pi depletion is independent of PhoBR and PhoPQ TCS in E. coli MG1655.

β-galactosidase activities of Pugd-lacZ in MG1655 ΔlacZ parent and isogenic ΔpmrB, ΔpmrA, ΔphoB and ΔphoR mutants following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Up-regulation of ugd gene during Pi depletion is independent of PmrAB and PhoPQ TCS, but partially dependent on PhoBR TCS in E. coli MG1655.

β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ parent and isogenic ΔphoR, ΔphoB, ΔphoP and ΔphoQ mutants following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Pi depletion induced polymyxin tolerance is not affected in eptA deletion strain.

Survival of E. coli MG1655 parent and isogenic ΔeptA mutant exposed 1-hour to polymyxin B (4 μg/ml) following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-tailed Student’s t-test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

pH of spent medium of E. coli MG1655 after 5hrs Pi sufficient growth and after 3hrs Pi depletion treatment.

pH of the medium before bacteria inoculating and pH of the spent medium after 5hrs Pi sufficient growth or 3hrs Pi depletion treatment were measured. Error bars represent the standard deviation of at least three independent experiments.

E36A and E39A mutation abolish the Fe3+ signaling capacity of PmrA.

β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ parent and isogenic pmrBE36A, pmrBE39A mutants growth under MOPS minimal medium supplemented with different concentration of FeSO4. Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Deletion of canonical Fe3+ up-take genes does not affect the activation of PmrAB system and increase of Fe mobilization to E. coli cell during Pi depletion.

(A) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ parent and isogenic ΔfecA, ΔfepA, ΔentE, Δfiu, ΔcirA, and Δ5Fe (ΔfecAΔfepAΔentEΔfiuΔcirA) mutants following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). (B) Fe contents in E. coli MG1655 parent and isogenic ΔfecA ΔfepA ΔentE Δfiu ΔcirA mutant following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test for A and two-way ANOVA followed by Sidak’s multiple comparisons test for B. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Decrease of cellular magnesium during Pi depletion is independent of Fe availability in medium.

Mg contents in E. coli MG1655 cell following Pi depletion treatment (0 Pi, 10 μM FeSO4) or Fe-free Pi depletion treatment (0 Pi, 0 FeSO4) at the indicated time points. Error bars represent the standard deviation of at least three independent experiments.

Magnesium disassociates from E. coli cells when subjected to Mg-free Pi-depleted medium.

Mg content in the spent medium during Mg-free Pi depletion treatment (0 Pi, 0 MgCl2) at the indicated time points. Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Cellular metal content in P. aeruginosa PAO1 cells with Pi depletion treatment and without Pi depletion treatment.

Cellular metal contents determined by ICP-MS in P. aeruginosa PAO1 cells following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-tailed Student’s t-test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Disruption of Mg and Fe signaling showed no effect on Pi depletion induced polymyxin resistance in P. aeruginosa.

(A) Time-kill curves of P. aeruginosa PAO1 exposed to PMXB (8 μg/ml) following acute Pi-starvation treatment (Pi-, 0 mM Pi), Pi depletion treatment with excess Mg (Pi-, 0 mM Pi, 10mM MgCl2) or Pi depletion treatment with iron chelator (Pi-, 0 mM Pi, 10 μM DFOM) compared to the group without treatment (Pi+, 1.32 mM Pi). (B) Time-kill curves of P. aeruginosa PAO1 exposed to PMXB (8 μg/ml) following acute Pi-starvation treatment (Pi-, 0 mM Pi), Pi depletion treatment with Fe2+ chelator (Pi-, 0 mM Pi, 30 μM DIP) or Pi depletion treatment with both Fe2+ and Fe3+chelator (Pi-, 0 mM Pi, 30 μM DIP, 10 μM DFOM) compared to the group without treatment (Pi+, 1.32 mM Pi). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Cellular ATP level in E. coli MG1655 cells continuously decrease during Pi depletion.

Cellular ATP levels in E. coli MG1655 cells following Pi depletion treatment at the specified time points. Error bars represent the standard deviation of at least three independent experiments.

Lack of upregulation of PphoQ and PmgtA promoter activities indicates that the PhoPQ TCS is not activated during Pi depletion in E. coli MG1655.

β-galactosidase activities of PphoQ-lacZ and PmgtA-lacZ in MG1655 ΔlacZ parent following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-tailed Student’s t-test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Up-regulation of both Parn and Pugd promoter activities during Pi depletion are not affected in ppk deletion strain.

(A) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ parent and isogenic Δppk mutant following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). (B) β-galactosidase activities of Pugd-lacZ in MG1655 ΔlacZ parent and isogenic Δppk mutant following acute Pi-starvation treatment (Pi-, 0 mM) compared to the group without treatment (Pi+, 1.32 mM). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Pi depletion, amino acid depletion, and combined Pi–amino acid stress upregulate Parn promoter activity, while ppk deletion only attenuates upregulation under combined stress.

(A) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ parent and isogenic Δppk mutant following acute nutrients downshift (MOPS 0.1 mM Pi) compared to the group without treatment (LB). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. (B) β-galactosidase activities of Parn-lacZ in MG1655 ΔlacZ parent and isogenic Δppk mutant following Pi depletion treatment (MOPS 0 Pi, 0.1% CAA), amino acid depletion treatment (MOPS 1.32 mM Pi, 0 CAA) and combined Pi–amino acid stress treatment (MOPS 0 Pi, 0 CAA) compared to the group without treatment (MOPS 1.32 mM Pi, 0.1% CAA). Error bars represent the standard deviation of at least three independent experiments. Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. Significance levels are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Minimum inhibitory concentrations (MICs) of strains in MOPS minimal medium

Proteins identified to be different expressed in E. coli MG1655 growing under Pi sufficient and depletion conditions

Bacteria strains used in this study.

Plasmids used in this study.