Dataset Information

Proteomic response of Staphylococcus sp. NIOSBK35 to varying arsenic (III) concentrations

ABSTRACT: Protein expression in Staphylococcus sp. NIOSBK35 isolated from marine environment (mangrove sediments) to different concentrations of arsenic (III)

INSTRUMENT(S): 6538 Q-TOF LC/MS

ORGANISM(S): Staphylococcus Cohnii

SUBMITTER: Samir Damare

LAB HEAD: Dr Samir R. Damare

PROVIDER: PXD013569 | Pride | 2019-12-02

REPOSITORIES: Pride

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Differential protein expression in a marine-derived <i>Staphylococcus</i> sp. NIOSBK35 in response to arsenic(III).

Shah Shruti S Damare Samir R SR

3 Biotech 20180605 6

Peptide mass fingerprinting of Gram-positive marine-derived <i>Staphylococcus cohnii</i> #NIOSBK35 gave us an insight into the proteins involved in conferring arsenic resistance as well as the probable metabolic pathways affected under metal stress. Analysis of the protein profiles obtained from LC/MS QToF (Liquid Chromatography-Mass Spectrometry-Quadrupole Time of Flight) resulted in the identification of 689 proteins. Further grouping of these proteins based on the arsenic concentration (0, 25 ...[more]

PMID: 29881665

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Project description:YEAST STRAIN: A genetically modified Saccharomyces cerevisiae yeast strain with PMI40 knockout (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2 PMI40::HIS3). The parent strain was CEN.PK113-7A (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2) obtained from Euroscarf (9). CULTIVATION MEDIUM: The mineral medium used for the bioreactor cultivations was based on the composition described by Verduyn and co-workers (11). The medium consisted of 5 g/l (NH4)2SO4 (Sigma-Aldrich, USA), 3 g/l KH2PO4 (Fluka, Swizerland) and 0.5 g/l MgSO4 × 7 H2O (Sigma-Aldrich, USA), as well as 1 ml/l trace element and 1 ml/l vitamin solution. The composition of the trace element solution was 15 g/l EDTA, 4.5 g/l ZnSO4 × 7 H2O, 0.84 g/l MnCl2 × H2O, 0.3 g/l CuSO4 × 5 H2O, 3 g/l FeSO4 × 7 H2O (Riedel-de-Haën AG, Germany), 0.1 g/l KI, 0.3 g/l CoCl3 × 6 H2O, 1 g/l H3BO3, 4.5 g/l CaCl2 × 2 H2O (Merck, Germany), and 0.4 g/l Na2MoO4 × 2 H2O (BDH Laboratory Supplies, UK). The composition of the vitamin solution was 0.05 g/l D-Biotin, 1 g/l Ca-pantothenate, 1 g/l Nicotinic acid, 25 g/l myo-inositol, 1 g/l Thiamine-HCl, 1 g/l Pyridoxine HCl, and 0.2 g/l p-aminobenzoic acid (Sigma-Aldrich, USA). The media in each cultivation contained initially 15 g/L D-glucose and a varying amount of D-mannose (0.75 g/L, 1.0 g/L, 1.5 g/L, 3.0 g/L, or 5.0 g/L). CULTIVATIONS AND SAMPLING: Bioreactor cultivations were performed in 3.2 l Braun Biostat CT2-DCU 3 instruments (B. Braun Biotech International GmbH, Germany). The cultivation temperature was +30 °C, agitation speed 1000 rpm, initial cultivation volume 2.0 l, airflow 1.0 l/min, and pH 5.0. In each cultivation, samples for the measurement of gene expression levels were taken 6 hours after inoculation. MEASUREMENT OF GENE EXPRESSION LEVELS: 0.9 mg CDW of cells were quenched in cold (-40 °C) methanol (45%) and centrifuged (1 min, +4 °C, 16100 G). The pellets were suspended in 400 µl of breaking buffer (20 mM Tris-HCl pH 7.4, 100 mM KCl, 2 mM MgCl2, 2mM DTT), 400 µl 1:1 phenol-chloroform, and 5 µl 20% SDS. Equal volume of glass beads (diameter 0.40-0.60 mm, B.Braun Biotech International, Germany) were added and samples were shaken in a bead beater (Mini-BeadBeater-8, BioSpec Products, USA) three times 1 minute at +4 °C. The mixtures were centrifuged (15 min, +4 °C, 16100 G in 5415 R, Eppendorf AG, Germany) and supernatants were recovered. The total RNA was purified from the supernatants using the QIAGEN RNeasy Mini kit (Qiagen, USA) according to manufacturers instructions. After this step the double-stranded cDNA was synthesized from 15 µg of total RNA with SuperScript Double-Stranded cDNA synthesis kit (Invitrogen, USA) using a T7-oligo(dT) primer. Biotin-labeled cRNA was then synthesized employing an Enzo High Yield RNA Transcript Labeling Kit (Enzo Life Science, USA). The biotin-labeled cRNA was heat-fragmented and biotinylated cRNAs were hybridized to Affymetrix YG-S98 microarrays in Affymetrix Hybridization Oven 640. Washing and staining of arrays were performed using the GeneChip Fluidics Station 450 and scanning with the Affymetrix GeneChip Scanner 3000. Acquisition and quantification of array images were performed using the Affymetrix GeneChip Operating System 1.0. The signals were scaled into target intensity of 100. All samples were assayed in triplicates. Keywords: dose response

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