Effect of curcumin treatment on the <i>B</i>. <i>subtilis</i> AH75 growth and cell morphology.

<p><b>(A)</b><i>B</i>. <i>subtilis</i> AH75 strain was grown in LB media having spectinomycin antibiotic (100 μg/mL) till the OD₆₀₀ reached to 0.1. Then the cultures were treated with DMSO (control), 20 μM (IC₅₀ concentration) and 100 μM (MIC concentration) curcumin. Growth curve was plotted by measuring the OD₆₀₀ for all the samples at every 20 min interval till 360 min (mid-exponential phase). The three time points of curcumin treatment (20, 60 and 120 min) used in proteomic analysis are indicated by arrows. IC₅₀ concentration was used for subsequent proteomic analysis. (<b>B)</b><i>B</i>. <i>subtilis</i> AH75 strain was grown in the presence of 20 μM (IC₅₀ concentration) curcumin and the samples was collected after 20, 60 and 120 min of the drug treatment. Cultures treated with only DMSO was used as control. The nuclear materials were stained using 1 μg/μL DAPI for 20 min at room temperature in dark for all the samples. The fluorescence microscopic images were captured with both DAPI and DIC filters. The control <i>B</i>. <i>subtilis</i> cells showed normal cell length with one or two nucleoids per cell whereas after 20, 60 and 120 min of incubation with 20 μM (IC₅₀ concentration) curcumin, most of the cells turned into filamentous structure with multiple nucleoids. I- DIC image, II- DAPI image and III- overlay image.</p

(A) CTC staining and flow cytometric analysis for respiratory activity.

<p>Graphical representation of CTC mean intensity (PE-Texas Red-A) <i>vs</i>. FSC-A obtained in the FACS analysis of control, 20, 60 and 120 min curcumin treated samples and negative control. Both dot plot and histogram representations are displayed for each sample. <b>(B) & (C)</b> Potassium and phosphorus leakage assay; curcumin (20 and 40 μM) was added to the <i>B</i>. <i>subtilis</i> in HEPES-glucose medium and K⁺ and P levels were measured at 20, 60, 90 and 120 min time intervals, and also in the untreated control and positive control (heated at 70° C for 30 min) samples using ICP-AES and data was normalized with baseline HEPES-glucose medium. <b>(D)</b> Metabolic activity assay using resazurin. 20 min curcumin treatment has showed lower metabolic activity whereas the metabolic activity increased as time of exposure increased to 60 min and 120 min as compared to control. * indicates <i>p</i> < 0.05. <b>(E)</b> Gene expression analysis using RT-PCR for <i>murAA</i>, <i>spoVG</i> and <i>ftsH</i> genes and the relative expression was calculated by taking mean C_t values from triplicate runs. * indicates <i>p</i> < 0.05 and ** indicates <i>p</i> < 0.001. <b>(F)</b> Physical interaction analysis of curumin with <i>B</i>. <i>subtilis</i> FtsZ immobilized on CM-5 sensor chip. The interaction was monitored by measuring the response unit and the response unit was increased as the concentration of curcumin increased. Both sensorgram and the bar diagram showing the binding to FtsZ was displayed.</p

Schematic representation of experimental strategy for temporal proteome analysis of <i>B</i>. <i>subtilis</i> under curcumin treatment by iTRAQ-based quantitative proteomics.

<p><b>(A)</b> Samples processed in triplicate were pooled from control, 20, 60 and 120 min curcumin treated cultures and labelled with iTRAQ reagent 114, 115, 116 and 117, respectively. The labelled peptides were fractionated in OFFGEL fractionators using high resolution (24 cm; 3–10 pH) IPG strips and each fraction was desalted using C18 tips. Desalted fractions were subjected to LTQ-Orbitrap Velos mass spectrometer for protein identification and quantitation. <b>(B)</b> Representative MS/MS spectrum of a few selected differentially expressed proteins identified after curcumin treatment. UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 (MurAA), ATP-dependent zinc metalloprotease FtsH, Septum site-determining protein (DivIVA), and 3-oxoacyl-[acyl-carrier-protein] synthase 3 protein 1 (FabHB). Inset showing the iTRAQ reporter ion intensities for representative peptides in control and curcumin treated samples. <b>(C)</b> S-curve analysis exhibiting distribution of the differentially expressed proteins in <i>B</i>. <i>subtilis</i> after 20, 60 and 120 min of curcumin treatment identified using Q-TOF (average of three triplicate runs). <b>(D)</b> S-curve analysis exhibiting distribution of the differentially expressed proteins in 20, 60 and 120 min curcumin treated <i>B</i>. <i>subtilis</i> identified using LTQ-orbitrap.</p

Quantitative profiles of the differentially expressed proteins involved in diverse biological processes identified in iTRAQ-based quantitative proteomics analysis using LTQ-Orbitrap.

<p><b>(A)</b> Peptidoglycan biosynthesis, <b>(B)</b> Fatty acid synthesis, <b>(C)</b> Cell division and sporulation, <b>(D)</b> TCA cycle, <b>(E)</b> Stress response and <b>(F)</b> Nucleotide biosynthesis. Data from QTOF is provided in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120620#pone.0120620.s002" target="_blank">S2 Fig</a>.</p

Modulation of essential physiological pathways in <i>B</i>. <i>subtilis</i> due to curcumin treatment.

<p>The pathway was built based on DAVID, KEGG and KOBAS analysis and the combined pathway was built manually. Red bar or name indicates down-regulation and blue bar and name indicates up-regulation. The three bars indicate the protein expression at 20, 60 and 120 min of curcumin treatment (expression levels obtained from LTQ-Orbitrap analysis). PRPP-5-Phospho-alpha-D-ribose 1-diphosphate; GAR-5'-Phosphoribosylglycinamide; FGAM-5'-Phosphoribosyl-N-formylglycinamidine; CAIR-5'-Phosphoribosyl-5-amino-4-imidazolecarboxylate; SAICAR-5'-Phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole; AICAR-5-Phosphoribosyl-4-carbamoyl-5-aminoimidazole; FAICAR-5'-Phosphoribosyl-5-formamido-4-imidazolecarboxamide; IMP-Inosine monophosphate.</p

List of differentially expressed proteins in <i>B</i>. <i>subtilis</i> due to curcumin treatment obtained from DIGE analysis and its comparison with iTRAQ analysis^$.

<p> This is a partial list having selected candidates with >1.5 fold change and complete list is provided in S2 Table

#: Proteins unique in DIGE, Bold: Same trend in both DIGE and iTRAQ (Orbitrap data);

* or NS: No significant change in iTRAQ in Orbitrap data (less than 1.2 fold up and down);

NI- Not identified in iTRAQ analysis.

List of differentially expressed proteins in B. subtilis due to curcumin treatment obtained from DIGE analysis and its comparison with iTRAQ analysis</a>.</p</sup>

List of the primers used for quantitative RT-PCR analysis for selected genes.

<p>List of the primers used for quantitative RT-PCR analysis for selected genes.</p