Carbon and phosphate starvations induce the transcription of E. coli aphA_Ec

The expression of E. coli aphA_Ec is stimulated by carbon starvation 7. To confirm this observation and to study how aphA_Ec transcription is regulated, we constructed four transcriptional reporter plasmids by cloning the serially truncated promoter regions (P1 to P4 from -433 to +30, Figure 1A-C) of aphA_Ec upstream of the lacZYA operon of the low-copy-number vector pGH254 previously used to analyze gene expression in E. coli 10. We first grew the E. coli strains with these reporters in MOPS synthetic rich medium and then shifted the cells to MOPS medium lacking either carbon, amino acid, or phosphate, to quantitate the transcription level of aphA_Ec. Consistent with previous report 7, carbon starvation led to an abrupt increase of aphA_Ec transcription from the promoter region P1 and P2, but moderately from P3, and not from P4 (Figure 1D), suggesting the -200 to -123 region is critical for aphA_Ec transcription during carbon starvation. A downshift of the reporter strains to low phosphate (from 1.32 mM to 0.067 mM) MOPS medium elicited a slow and moderate increase of aphA_Ec transcription from P2 and P3, but not from P4 or P1 (Figure 1E), suggesting that phosphate downshift stimulates the aphA_Ec transcription as well. At last, a downshift to MOPS medium without amino acid revealed no change of aphA_Ec transcription (Figure 1F), suggesting that amino acid starvation, and thus the stringent response, has no effect in aphA_Ec transcription. Of note, both P2 and P3 produced higher basal levels of aphA expression than P1 and P4 before the nutrient downshifts (Figure 1D-1F). Altogether, these data suggest that carbon and phosphate starvations induce the expression of aphA_Ec via overlapping but also distinct promoter regions.

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FIGURE 1: Carbon and phosphate starvations stimulate the expression of E. coli aphA. (A) Schematic overview showing the promoter region of E. coli aphA gene, with the start and stop codons and potential four promoter sequences marked (in bars). (B) Sequence annotation of the potential promoter region of E. coli aphA. Text highlighted in blue is the CytR box; SD, Shine-Dalgarno sequence. (C) Schematic overview showing the promoter regions of aphA, which were transcriptionally fused to a lacZ reporter gene in the plasmid pGH254 [10]. (D, E, F) Beta-galactosidase assays of the various transcriptional reporters during starvations of carbon, phosphate, and amino acid. Time zero indicates the time point right before the downshift of cells. Three biological replicates were performed, and the average and standard deviations are shown. The significance of unpaired t-test is indicated by the symbols ** (p-value < 0.01), *** (p-value < 0.001), and **** (p-value < 0.0001).

CyaA and CRP are essential for the aphA_Ec expression

Carbon starvation in E. coli activates the cAMP-dependent CRP regulon. To confirm the link between carbon starvation and aphA_Ec transcription, we transformed the reporter plasmid pGH254-aphA-P2 into the KEIO deletion strains 11 of CRP or CyaA and performed a beta-galactosidase assay (see method). Besides, we included several other KEIO strains each deleted with a well-studied transcriptional regulator, i.e., Sxy, CytR, RpoS, PhoB, and PurR, given their (plausible) connections to bacterial nucleotide metabolism and AphA production in different organisms 12. Transcription of aphA_Ec dropped to basal levels in both ∆cyaA and ∆crp strains (Figure 2), demonstrating their essential roles in aphA_Ec expression upon carbon starvation. Additionally, in the ∆cytR mutant strain, transcription of aphA increased as compared to the wild type strain, suggesting that CytR suppresses the aphA_Ec transcription (see Discussion below). Indeed, inspection of the promoter region revealed a potential CytR box (Figure 1B, TGCAA, TTGC/tA) 13 within the P4 and N-terminal end of the aphA_Ec gene. The other mutant strains tested showed marginal, if at all, change of the aphA_Ec transcription. These data indicate that the production of AphA_Ec is controlled by both carbon and nucleotide metabolisms (see discussion below).

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FIGURE 2: Beta-galactosidase assays of aphA expression from the promoter P2 during carbon starvation in various mutant strains of E. coli. Time zero indicates the time right before the downshift of cells from the MOPS complete medium to the same medium without glucose. The Miller units were normalized to that of wild type strain at time zero, i.e., before downshift. Three biological replicates were performed, and the average and standard deviations are shown. The significance of unpaired t-test is indicated by the symbol * (p-value < 0.05).

cUMP and cCMP stimulate, while cGMP inhibits, the acid phosphatase activity of AphA

Our previous data 3 suggested that cGMP affects AphA acid phosphatase activity. Given the chemical similarities of cGMP, and cUMP and cCMP with cAMP, we tested their effects on AphA_Hi from Rd KW20. Figure 3B shows that cGMP appears to competitively inhibit AphA_Hi, like cAMP (Figure 3A), with the respective K_i values of 34.5 ± 5.2 and 6.4 ± 1.2 μM. Conversely, both cCMP and cUMP exhibit a concentration dependent stimulation of AphA_Hi phosphatase activity, with cUMP having an apparent higher efficacy than cCMP (Figure 3C, 3D). This intriguing discovery underscores the opposing effects of purine and pyrimidine cyclic nucleotides on AphA_Hi activity.

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FIGURE 3: AphAHi activity is stimulated by cCMP and cUMP and inhibited by cAMP and cGMP. (A, B) Lineweaver-Burk plot of H. influenzae AphAHi kinetics in the presence of varied concentration of cAMP (A) and cGMP (B). (C, D) Michaelis-Menten plots of H. influenzae AphAHi in the absence and presence of cCMP (C), and cUMP (D). pNPP was used as the substrate and formation of the yellow p-nitrophenol was quantified spectrophotometrically at 405 nm. Data represent two biological replicates, each with two technical replicates.

Construction of transcriptional reporters of aphA-lacZ

Strains used in this study are listed in Table 1 and primers are listed in Table 2.

To make the APHA-1 transcriptional reporter, the primers pYZ262 and pYZ266 were used to amplify the P1 promoter region (Figure 1) of aphA_Ec and cloned into the plasmid vector pGH254 10 via the EcoRI and BamHI restriction sites. This places the P1 promoter region of aphA_Ec in front of the lacZYA genes on pGH254 resulting in the transcriptional reporter pGH254-APHA-1. Likewise, P2, P3 and P4 promoter regions of aphA_Ec were amplified using primers pYZ263 and pYZ266, primers pYZ264 and pYZ266, and primers pYZ265 and pYZ266, respectively, and similarly cloned into pGH254 to construct the pGH254-APHA-2, pGH254-APHA-3, pGH254-APHA-4, respectively.

Beta-galactosidase assay

Overnight cultures of E. coli strains grown in MOPS medium (18 h, 37°C, 160 rpm) were back-diluted into fresh MOPS medium and grown to OD₆₀₀=0.3-0.5 before they were shifted to a MOPS medium lacking either phosphate, glucose, or amino acids. During growth, pelleted cell samples were collected and immediately frozen until use. For beta-galactosidase assays, the collected sample cells were thawed and resuspended in a resuspension buffer composed of 100 mM Tris (pH 7.8), 32 mM Na₃PO₄ (pH 7.8), 1 mM PMSF and 19 mM β-mercaptoethanol and were sonicated (10% amplitude) until complete lysis was observed. Protein concentration was measured using the Bradford assay, and equal amounts of total protein from each lysed sample were resuspended in a reaction buffer composed of 76 mM Tris (pH 7.8), 24 mM Na₃PO₄ (pH 7.8), 0.03% SDS, 1.3 mM MgSO₄, 0.4 mg/ml DNase I, 1 mM PMSF and 19 mM β-mercaptoethanol to a final volume of 75 μl. The β-galactosidase reaction was started by adding 60 μl 4 mg/ml ONPG to the reaction mixture and was terminated by the subsequent addition of 150 μl 1 M Na₂CO₃. Finally, the samples were spun down for 2 min at 14600 rpm and 200 μl supernatant was transferred to a clear flat-bottom 96-well plate (Greiner) and the absorbance was read at 420 nm in a plate reader. Three biological replicates each with two technical replicates were performed, and the average and standard deviations are shown.

AphA purification and enzymatic assays

Both the purification and enzymatic assay of AphA were performed in the same method as in 3. Briefly, E. coli BL21 DE3 strains containing the vector pET28a-his.aphAhi were grown up in LB medium to OD₆₀₀=0.6-0.8, when 0.5-1 mM IPTG was added to induce the expression of His-AphA_Hi for 4 hours at 37°C. Harvested Cells were lysed via sonication (Branson) in cold lysis buffer (5% glycerol 50 mM Tris pH=7.6, 150 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol) containing cOmplete^TM Protease Inhibitor Cocktail. The cleared supernatant was then loaded on Ni-NTA resins on a Poly-Prep column. The resins were washed with cold wash buffer (5% glycerol 50 mM Tris pH=7.6, 150 mM NaCl, 20 mM imidazole), eluted with elution buffer (5% glycerol 50 mM Tris pH=7.6, 150 mM NaCl, 500 mM imidazole) and further purified via the ÄKTA system on a Superdex 200 Increase 10/300 GL column. The purity of purified protein was verified using SDS-PAGE gel and its concentration was quantified using Bradford assay. We then used the pNPP as the substrate to assess the enzymatic reaction of His-AphA_Hi. For this, 15 nM of His-AphA_Hi was used in reaction buffer containing 50 mM sodium acetate (pH 5.6), 0.1 M NaCl, 1 mM MgCl₂, 0.01% Triton X-100 and varying concentrations of cAMP, cGMP, cCMP and cUMP. The enzymatic reaction was started by adding varied pNPP and stopped by transferring 60 μl of the reaction mixture into 100 μl 3 M NaOH in a 96 well plate (Greiner, flat bottom) at defined time points. The amount of reaction product was quantitated by the absorption at 405 nm. Two biological replicates each with two technical replicates were performed, and the average and standard deviations are shown.

Preparation of chemicals

cAMP (SIGMA, A9501) was dissolved in Milli Q (MQ) H₂O. cUMP (BioLog, 56632-58-7) and cCMP (BioLog, 54925-33-6) were each dissolved in a buffer of 25 mM Tris-HCl (pH 7.4) and 100 mM NaCl to a concentration of 250 mM and were then diluted to the desired concentrations in MQ H₂O. p-nitrophenyl phosphate (pNPP, SIGMA, S0942) was dissolved in 50 mM NaOAc to a concentration of 50 mM and was then serially diluted in the same buffer to the desired concentrations.