Fig 1.
Genomic organization and biochemical characterization of PrpN.
(A) Schematic description of PrpN phosphatase domain. PrpN primary sequence analysis depicted the presence of metallophosphatase (MPP) phosphoprotein phosphatase (PPP) conserved domain family. The conserved active site and metal binding site residues are illustrated in the schematic (B) Pictorial representation of prpN gene organization in B. anthracis genome. The reference organization was fetched from NCBI GenBank database. (C) Operon prediction by RT-PCR analysis of BAS0538-40 region. BAS0538, 39 and 40 internal gene specific primers as depicted in Fig 1B were designed for PCR to amplify gene products of around 1200-bp. Agarose gel showing the amplified products by the primer pairs (OC Fp1 and OC Rp1) and (OC Fp2 and OC Rp2). Left panel- Lane 1: 1Kb DNA Ladder RTU (GeneDirex, Cat. No. DM010-R500), Lane 2 and 4: gDNA as template, Lane 3 and 5: cDNA as template, Lane 6 and 7 (No-RT cDNA as template). Right panel- Agarose gel showing successful cDNA preparation using rpoB and prpN intergenic primers amplifying a gene product of 104-bp and 120-bp, respectively. Lane 1: 100bp DNA Ladder H3 RTU (GeneDirex, Cat. No. SD003-R600), Lane 2: cDNA as template with rpoB primers, Lane 3: No-RT cDNA as template with rpoB primers, Lane 4: cDNA as template with prpN primers and Lane 5: No-RT cDNA as template with prpN primers. (D) Enzymatic and biochemical characterization of PrpN. (i) PrpN phosphatase activity was evaluated by pNPP hydrolysis assay in the presence of different ions to assess cofactor requirement. (ii) Kinetic plot of PrpN using serine/threonine phosphopeptides. The data were fitted to a Michaelis-Menten curve to determine enzyme kinetics parameters (Km and Vmax) using GraphPad Prism. Error bars reflect the variation of triplicate measurements.
Fig 2.
PrpN is critical for optimum vegetative growth and is maximally expressed during stationary growth phase.
Growth patterns of indicated strains at 37°C monitored by measuring A600nm at intervals of 2 hours till 14 hours. Average values and standard deviation calculated from biological triplicates are shown in the graph. Triplicate cultures were grown in LB medium to 0.1 A600nm and then stresses imposed by adding (A) no addition, (B) 1 M NaCl, and (C) 2.5 mM H2O2. (D) Representative phase contrast microscopy images of BAS WT and BAS ΔprpN strains at different time points. Scale bars are depicted in the images. (E) Expression of PrpN at different growth phases. Representative immunoblot showing growth dependent differential expression of PrpN in BAS WT strain. Equal amounts of protein lysates prepared from different growth phases (as indicated in Fig 2A and 2D) were loaded and probed using anti-PrpN and anti-GroEL. Ladder- PageRuler Prestained Protein Ladder, Thermo-Scientific (Cat. No. 26616). (F) Densitometer analyses were done using Amersham Imager600 software and the corresponding PrpN/GroEL ratios were plotted using GraphPad Prism. Densitometer readings calculated from three experiments executed independently are shown in the bar graph.
Fig 3.
Effect of prpN deletion on vegetative cell morphology.
(A) Representative phase contrast images of bacterial cells at exponential and stationary growth phase. Bacterial cells were visualized under 100x/1.4 oil DIC objective of Zeiss Axio Imager Z2 upright microscope. Scale bars represents 10 μm. (B) Representative scanning electron microscope images of indicated vegetative bacterial cells. Cells were visualized under Zeiss Evo LS15. Scale bars represent 2 μm, magnification-5000X. (C) Cell septation properties of BAS WT, BAS ΔprpN and BAS ΔprpN::prpN. Bacterial strains grown in an LB agarose pad setup for 6 hours containing 1 μg/mL FM4-64 membrane staining dye. Live vegetative bacterial cells were visualized using Leica SP8 confocal laser scanning microscope. Arrow heads indicate multi-septation in the BAS ΔprpN strain (middle panel). Scale bars represents 10 μm. (D) Graph indicating % distribution of multi-septa in 1500 BAS WT and BAS ΔprpN cells.
Fig 4.
Effect of prpN deletion on sporulation.
(A) Representative phase contrast images of BAS WT and BAS ΔprpN strains grown in agarose pad prepared in sporulation medium. Images were captured at the indicated time points as depicted in the Fig panel using Zeiss Axio Imager Z2 upright microscope. Scale bars represents 20 μm. (B) and (C) Bar graphs depicting sporulation efficiency and total spores in indicated strains. Average values and standard deviations calculated from three experiments executed independently are shown in the bar graphs. Asterisks indicate statistical significance of the data set calculated using two-tailed Student’s t test. **** denotes p<0.0001. (D) Representative transmission electron micrographs of indicated strains spores. The strains were grown in sporulation medium for 72 hours and washed with water to remove vegetative cell debris. The spore pellet was processed for TEM imaging and visualized using FEI Tecnai G2 Spirit at 200 KV. Spore layers are depicted in the images with arrows- ES: exosporium, OSM: outer spore membrane, ISM: inner spore membrane, C: cortex. Scale bars are depicted in the images. Magnification is 550X (upper panel) and 15000X (lower panel). (E) Representative transmission electron micrographs of BAS ΔprpN sporulating cells. FS denotes forespore and MC denotes mother cell. White arrow indicates asymmetric septation. Images were captured using FEI Tecnai G2 Spirit at 200 KV. Scale bars represents 0.5 μm. (F) Bar graph depicting viable cell count in BAS WT and BAS ΔprpN post complete sporulation. Data is represented as mean CFU log10/mL. Error bars denote standard deviations of three independent experiments.
Fig 5.
Defective PA, LF and AtxA expression in BAS ΔprpN strain.
Representative immunoblots and bar graphs showing PA, LF, and AtxA synthesis in indicated strains. The strains were grown in NBY medium containing 1% NaHCO3. Whole cell lysates (A,B,E) or supernates (C,D) were loaded in equal amounts and probed using anti-PA (A,C), anti-LF (B,D), or anti-AtxA (E). The blots were stripped and probed using anti-GroEL. Densitometer analyses were done using Amersham Imager600 or ImageLab software and the corresponding PA, LF, and AtxA ratios to GroEL were plotted using GraphPad Prism. Average values and standard deviations calculated from minimum three independent experiments are shown in the bar graphs. Ladder- BlueRAY Prestained Protein Ladder, GeneDirex (Cat. No. PM006-0500). Statistical Analysis: Asterisks indicate statistical significance of the data set calculated using two-tailed Student’s t test. * corresponds to p<0.05; ** corresponds to p<0.01; *** corresponds to p<0.001 and **** corresponds to p<0.0001.
Fig 6.
CodY—A substrate of PrkC and PrpN.
(A) Coomassie stained gel image of CodY elution’s from BAS WT and BAS ΔprpN. (B) and (C) Representative immunoblots and bar graph showing phosphorylation status of CodY protein purified from indicated strains. Equal amounts (1 μg) of purified CodY proteins were loaded and probed using anti-phosphoserine. The same blot was stripped and probed again using anti-CodY. Indicated MWs were derived from adjacent lanes containing PageRuler Prestained Protein Ladder, Thermo Scientific (Cat. No. 26616). Densitometer analysis were done using Amersham Imager600 software and the corresponding phosphoserine/CodY ratio with respect to BAS WT were plotted in a bar graph using GraphPad Prism. Average values and standard deviations calculated from five independent experiments are shown in the bar graph. Asterisks indicate statistical significance of the data set calculated using two-tailed Student’s t test. *** corresponds to p<0.001. (D) Phosphate standard curve and results for amounts of inorganic phosphate released from phosphorylated CodY proteins purified from BAS ΔprpN strain by PrpN treatment. (E and F) PrkC mediated phosphorylation of CodY. (E) Representative immunoblots and Ponceau-S stained image of in-vitro kinase assay performed using autophosphorylated PrkCcat and CodY. (F) Co-expression of CodY-His6 with and without PrkCcat. Proteins were resolved on SDS-PAGE and visualized using Pro-Q diamond phospho specific gel stain to examine the phosphorylation status (upper image) and Coomassie stain to confirm loading pattern (lower image). Lane 1: CodY-His6 produced in E. coli using pETDuet without PrkCcat; Lane 2: CodY-His6 produced using pETDuet with PrkCcat; Lane 3 and 4: CodY-His6 produced using pProEXHTc. Indicated MWs were derived from adjacent lanes containing PageRuler Prestained Protein Ladder, Thermo Scientific (Cat. No. 26616).
Fig 7.
In-vivo confirmation of CodY serine215 phosphosite in B. anthracis and impact of CodY phosphorylation on its DNA binding activity.
(A) Representative immunoblots to confirm in-vivo phosphorylation of CodY at serine215 residue. 2 μg CodYS215A-His6 produced in BAS WT (lane 2) and BAS ΔprpN (lane 3) strains was resolved and probed using anti-phosphoserine. The same blot was stripped and probed using anti-CodY. 2 μg PrkCcat (lane 4) and native CodY (lane 5) produced in E. coli BL21 were used as positive and negative controls, respectively. Indicated MWs were derived from adjacent lanes containing PageRuler Prestained Protein Ladder (Lane 1), Thermo Scientific (Cat. No. 26616). (B) Representative immunoblots of native CodY and CodYS215A purified from bacillus strains resolved on Phos-tag precast gel (upper image) and SDS-PAGE (lower image). Lane 1: CodYS215A-His6 produced in BAS WT, Lane 2: CodY-His6 produced in BAS WT, Lane 3: CodY-His6 produced in BAS ΔprpN, Lane 4: CodYS215A-His6 produced in BAS ΔprpN. pCodY and CodY denotes phosphorylated and unphosphorylated form of CodY. Indicated MWs were derived from adjacent lanes containing PageRuler Prestained Protein Ladder, Thermo Scientific (Cat. No. 26616). (C) Electrophoretic Mobility-Shift Assay using SYBR Green and SYPRO Ruby stains. Increasing amounts (1, 1.5, 3, 4, 5, 6, 8, 10 and 12μM- lane 2 to 10) of native CodY, CodY S215E and CodY S215A proteins were added in a binding reaction containing 4 nM atxA promoter region as probe. Lane 1 represents only DNA control. Upper gel image represents DNA bands stained using SYBR Green and lower image represents protein bands stained using SYPRO Ruby. (D) Comparative expression of atxA mRNA in BAS ΔprpN with respect to BAS WT strain. The RT-PCR data were normalized to the expression of rpoB from each strain. Error bars represents an average of three independent biological triplicates, each performed in three technical replicates.
Fig 8.
Unphosphorylated CodY is an activator of toxin synthesis.
Representative immunoblots and bar graphs showing expression of PA (A), LF (B) and AtxA (C) in BAS WT, BAS ΔprpN, and BAS ΔprpN expressing CodY S215A (BAS ΔprpN::codYS215A). Immunoblots were analyzed as in Fig 5. Indicated MWs were derived from adjacent lanes containing PageRuler Prestained Protein Ladder, Thermo Scientific (Cat. No. 26616). Mean and standard deviation from minimum three independent experiments are shown in the bar graph. Asterisks indicate statistical significance of the data set calculated using one-way ANOVA followed by a post hoc test (Tukey test). P values < 0.05 were considered as statistically significant.
Fig 9.
Schematic illustration of PrpN mediated toxin synthesis regulation via CodY. Binding of unphosphorylated CodY protein to atxA promoter region activates AtxA expression, which thereby promotes toxin proteins (PA, LF and EF) synthesis. PrpN positively regulates anthrax toxin synthesis by dephosphorylation of CodY protein (Green arrow), while CodY phosphorylation abrogates its DNA binding ability to atxA promoter region (Red arrow) ultimately leading to downregulation of AtxA and anthrax toxin synthesis. The schematic was prepared by AG using BioRender.