DOI: 10.6084/m9.figshare.20375965
title: Additional file 1 of Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication

description: Additional file 1: Fig. S1. Key amino-acids of the Cat and PEPut domains of PykA. A. Cat domain analysis. Clustalw and Chimera analysis of the pyruvate kinase of B. subtilis (PykA), human cells (PKM2) and Mycobacterium tuberculosis (PYK) identified key amino acids of the catalytic site of the B. subtilis protein. B. PEPut domain analysis. Alignment of the PEPut domain of PykA to related domains of various metabolic enzymes. The red arrow highlights the conserved LTSH motif (coordinates 536-539). Fig. S2. Effect of Cat and PEPut mutations on growth in MC. Wild-type and pykA mutants were first grown over-night in MC supplemented with antibiotic when appropriate. Upon saturation, cultures were diluted 1000-fold in the same medium without antibiotic and growth was monitored spectrophometrically. Left panel: Analysis of catalytic mutants (pykAΔcat, pykAR32A, pykAR73A, pykAK220A, pykAGD245/6AA, pykAT278A, pykAJP). Right panel: Analysis of PEPut and Cat-PEPut interaction mutants (pykAΔPEP, pykAT&gt;A, pykAS&gt;A, pykAH&gt;A, pykATSH&gt;AAA, pykAT&gt;D, pykAS&gt;D, pykAH&gt;D, pykATSH&gt;DDD, pykAE209A, pykAL536A). Controls: TF8A (wild-type) and ΔpykA. Fig. S3. Analysis of NTP in the metabolome of wild-type and pykAT&gt;D cells. ATP, GTP and CTP were detected in the positive ionization mode. UTP was detected in the negative ionization mode. Note that TTP signals were too low for quantifications. Data correspond to 3 independent extractions (solid cultures).*, p &gt; 0.05 ; **, p &lt; 0.05 (Welch's T-test). Values in bold indicate the fold change for each metabolite (WT vs pykAT&gt;D). Fig. S4. LC/MS analysis of legionaminic acid in the metabolome. A. Extracted ion chromatogram (EIC) corresponds to the deprotonated molecule [M-H]- at m/z 333.1303 (5 ppm accuracy). B. Zoom on the mass spectrum of legionaminic acid in the negative mode. C. Collision Induced dissociation (CID) spectrum of legionaminic acid in the negative mode at 22% Normalized Collision Energy (NCE). D. CID spectrum of legionaminic acid in the positive mode at 22% NCE. E and F. Zoom on the mass spectrum of the deuterated forms of legionaminic acid in the negative and positive ionization mode, respectively. G and H. Comparison of legionaminic acid (G) and CMP-legionaminic (H) acid contents in wild-type (WT), ΔspsE and ΔspsF cells, respectively. Data correspond to 3 independent extractions (liquid cultures). ***, p &lt; 0.001; **, p &lt; 0.01 (Welch's T-test). Fig. S5. Representative cell cycle results in Cat and PEPut mutants. Top raw: Microscopy of exponentially growing cells stained with FM4-64 (membrane staining, red) and DAPI (nucleoid staining, blue). Middle raw: Representative runout DNA histograms (experiments were reiterated 3-12 times). Bottom raw: Representative marker frequency analysis along the right arm of the chromosome (experiments were reiterated at least three times). Fig. S6. Cell cycle parameters of wild-type and pykAT&gt;D cells grown in proline and malate, respectively. Left panel: Growth in malate of wild-type (WT), ΔpykA (Δ), pykAT&gt;D (T&gt;D) and other pykA mutants (pykAT&gt;A, pykAGD245/6AA and pykAK220A, blue lines). Right panel: Runout DNA histograms and cell cycle parameters. Fig. S7. PykA-mCherryBSU localization. Strains deleted for the natural pykA gene and encoding the PykA-mCherryBSU fusion from an inducible promoter (Physpank) were grown in MC and microscopy analysis was carried out at OD600nm = 0.1 to 0.2. Top raw: analysis of the mCherryBSU signal produced at different IPTG concentrations. Bottom raw: analysis of cells grown in the absence of IPTG, fixed in a 1x PBS solution supplemented with 1% paraformaldehyde and stained with DAPI. Scale bar: 4 μm. Similar results were obtained with fusions mutated in the Cat or PEPut domain of PykA. Fig. S8. PykA purification and characterization of its function and oligomeric state. A. SDS-PAGE (15% polyacrylamide gel) showing over-expression of the 6His-MBP-PykA in Rosetta (DE3) E. coli. The soluble expressed tagged PykA protein is shown in a red rectangular in lane 4, whereas lanes M, 1, 2 and 3 show molecular weight standards, the control uninduced insoluble fraction, the control uninduced soluble fraction and the IPTG-induced insoluble fraction, respectively. B.1. SDS-PAGE (15% polyacrylamide gel) showing fractions from the first IMAC purification step of PykA. From left to right, lanes represent molecular weight standards (M), the flow-through (1), the eluted tagged PykA (2), the overnight TEV treated tagged PykA (3), the flow-through fractions containing untagged PykA from the second IMAC step after TEV proteolysis (4-10). B.2. SDS-PAGE (15% polyacrylamide gel) showing the final gel filtration column (HiLoad 26/60 Superdex 200 Prep Grade Gel Filtration Column). From left to right, lanes represent molecular weight standards (M) and fractions of the size exclusion chromatography (1-9). C. The graph shows a Hill plot for the activity of PykA at 25°C. The Rate/(Vmax-Rate) (Y-axis) was plotted against the PEP substrate concentration (X-axis) using GraphPad Prism 4 software and the Vmax (19.3 μmol/min), Km (2.7 mM) and the Hill coefficient n (0.8111) values are shown below the graph. The n value is &lt;1 indicating negative cooperative binding of PykA to its PEP substrate. D. The graph shows a Michaelis-Menten plot for the activity of PykA at 25°C. The initial rate of the reaction (Y-axis) was plotted against the PEP substrate concentration (X-axis) using GraphPad Prism 4 software and the Vmax (16.3 μmol/min) and Km (1.7 mM) values are shown below the graph. E. A native mass spectrum showing the PykA tetramer and miniscule amounts of the dimer and monomer. The theoretical mass of the PykA monomer (62,314.9 Da), dimer (124,629.8 Da) and tetramer (249,259.6 Da). Native mass spectrometry showed that PykA was found to be predominantly tetrameric (250,092 ± 72 Da), with very low abundance dimer (124,811 ± 38 Da) and monomer (62,437 ± 8 Da) peaks. F. Collision induced dissociation of the 33+ charge state of the tetramer shows it to be very stable in the gas-phase, with no apparent dissociation to lower-order oligomers. G. Comparative analytical gel filtration of the PykA tetramer against molecular weight standards (Thyroglobulin 670 kDa, g-globulin 158 kDa, ovalbumin 44 kDa, myoglobin 17 kDa and vitamin B12 1.3 kDa) through a Superdex 200 10/300 GL prepacked Tricorn gel filtration column (GE Healthcare). H. Selectivity trendline constructed from the molecular weight standards (shown in graph G) for the estimation of the PykA MW. The x axis is in logarithmic scale. Graphpad was used for plotting the data points. The theoretical value of our PykA (249,259.6 Da) is close to the estimated (285,000 Da) which along with the MS data verifies the tetramer in solution. Kd is the equilibrium distribution coefficient. The numbers (1-5) on the data points correspond to the proteins shown in graph G. Fig. S9. Stimulation of DnaE activity by PykA but not by BSA. A. Primer extension assays monitoring the extension of a 5′-32P-radioactively labelled 60mer DNA primer annealed onto M13 ssDNA over time by the B. subtilis DnaE. The activity of DnaE polymerase (10 nM) was monitored in the presence and absence of PykA (10 nM, tetramer) through a time course (30-150 sec). Lanes in the gels from left to right indicate: (M): DNA-ladder and then the time course (0, 30, 60, 90, 120 and 150 sec) depicted by the rectangular triangle. B. Primer extension assays as above with or without 10 nM (monomer) BSA instead of PykA. C. DnaE (1 nM) polymerase activity at increasing BSA concentrations (0, 5, 50, 500 nM), as indicated by the rectangular triangle, monitored by alkaline agarose electrophoresis. The DNA substrate is a labelled 20mer (5′-CAGTGCCAAGCTTGCATGCC-3′) primer annealed onto ssM13 ssDNA (2nM). The primer extension reaction was carried out for a longer time than above (5 min instead of 30-150 sec) and the film was over-exposed to compensate for the lower DnaE concentration. The assay was carried out at 37 °C in 50 mM Tris-HCl 7.5, 50 mM NaCl, 10 mM MgCl2 mM DTT, 1 mM dNTPs. No stimulation of the DnaE polymerase activity was observed in the presence of 5 and 50 nM BSA. The marginal stimulation observed at 500 nM BSA excess is likely because at this high concentration, BSA acts as a blocking agent preventing adhesion of DnaE to the plastic reaction tubes. Fig. S10. Stimulation of DnaE activity by PykA does not result from stimulation of DnaE binding to primed templates. EMSA investigation of the effect of PykA on the DNA binding of DnaE polymerase. The DNA substrate was constructed by annealing a 5′-32P-radioactively labelled 15mer (5′-AAGGGGGTGTGTGTG-3′) primer annealed onto a 30mer (5′-ACACACACACACACACACACACACCCCCTT-3′) oligonucleotide. Binding reactions were carried out with 1 nM DNA substrate, DnaE (500nM) and increasing concentrations (0, 12.5, 125 and 1,250 nM tetramer) of PykA, as indicated by the rectangular triangle for 10 min at 37°C in 50 mM NaCl, 10 mM MgCl2, 50 mM Tris-HCl pH 7.5. Lanes C and PykA represent the radioactive substrate in the absence of any proteins and in the presence of PykA (1,250 nM tetramer), respectively, showing that PykA does not bind to the DNA substrate. No stimulation of DnaE binding to DNA was observed in the presence of increasing concentrations of PykA indicating that PykA does not enhance the DNA binding activity of DnaE. Fig. S11. PEPut purification. A. SDS-PAGE showing overexpression of the His-MBP tagged PEPut in Rosetta (DE3) E. coli. From left to right, lanes show protein MW markers (M), the insoluble uninduced (1), soluble uninduced (2), insoluble induced (3) and soluble induced (4) fractions. The expressed soluble His-MBP tagged PEPut is shown by a red rectangular. B. SDS-PAGE showing the final purified untagged PEPut after removal of the His-MBP tag with TEV proteolysis. Lanes from left to right show protein MW markers (M) and fractions from the flow through the HisTrap column containing the pure untagged PEPut (lanes 1,2 and 3). Fig. S12. PEPut does not stimulate DnaE activity. Primer extension time course (30, 60, 90, 120 and 150 sec) assays using a primed DNA substrate (133 pM) constructed by annealing a radioactively labelled 5′-32P 15mer primer (5′-AAGGGGGTGTGTGTG-3′) onto a 110mer oligonucleotide (5′-CACACACACACACACACACACACACACACACACACACACACACACACACACACACACACCCCTTTAAAAAAAAAAAAAAAAGCCAAAAGCAGTGCCAAGCTTGCATGCC-3′), at suboptimal 25 pM DnaE concentration (left), in the presence of 25 pM PykA tetramer (middle) and 25 pM PEPut domain monomer (right). At this suboptimal DnaE concentration, there is no detectable DnaE primer extension activity in the absence of PykA but clear activity is visible in the presence of PykA. By comparison, no DnaE activity is detectable in the presence of the purified PEPut domain. These data show that full length PykA stimulates the DnaE activity while the PEPut domain alone does not.