biostudies-arrayexpressStreptococcus pneumoniaeGenePixhttps://www.ebi.ac.uk/biostudies/studies/E-BUGS-89Data is also available from <ahref=http://bugs.sgul.ac.uk/E-BUGS-89 target=_blank>BuG@Sbase</a>biostudies-arrayexpressGrowth Protocol - Title: S. pneumoniae growth without MnSO4. Description: Cultures from frozen stocks were added to C+Y. Cultures were incubated at 37°C and growth measured at regular intervals until A600 = 0.25 was reachedHybridization - Microarray slides were prehybridized for 20 min at 65°C in a solution of 3.5× SSC, 0.1% SDS and 10 mg ml−1 BSA. Prehybridization buffer was removed by washing in water for 1 min, propan-2-ol for 1 min and drying by centrifugation at 1500 r.p.m. for 5 min. The purified cDNA mixture was adjusted to 16 µl in 4× SSC and 0.3% SDS, heated to 95°C for 2 min and applied to the microarray. Hybridization was carried out for 16–20 h at 65°C in the dark. Before scanning using an Affymetrix 428 scanner the slides were washed for 2 min at 65°C in 1× SSC/0.05% SDS and 2 min at room temperature in 0.06× SSC.Nucleic Acid Extraction - Total RNA was extracted from in vitro- and in vivo-harvested bacteria by a modification of the method of Mortier-Barrière et al. (1998)Down . The bacterial pellet was resuspended completely in 300 µl prewarmed (65 °C) acid-phenol and incubated for 5 min at 65 °C. To this, 300 µl prewarmed NAES buffer (50 mM sodium acetate pH 5·1, 10 mM EDTA, 1% SDS) was added, and the mixture was incubated for another 5 min at 65 °C, with intermittent mixing. The mixture was cooled on ice for 1 min and the phases were separated by centrifugation at 15500 g for 1 min. The aqueous phase was re-extracted twice with acid-phenol followed by two further extractions with chloroform. Sodium acetate was added to the resulting supernatant to a final concentration of 300 mM and RNA was then Labeling - 3 micrograms RNA was mixed with 3 micrograms random hexamers in 11 microlitres H2O, heated to 95C and snap-cooled. In a total volume of 25 µl, the labelling reaction was initiated by the addition of 5 µl First Strand Buffer, 25 mM DTT, 1 mM each dATP, dGTP and dTTP, 0·4 mM dCTP, 2 nmol Cy3- or Cy5-dCTP and 500 U Superscript II reverse transcriptase (Life Technologies). The reaction was incubated in the dark at 25 °C for 10 min and then at 42 °C for 90 min. The relevant pairs of Cy3- (wild-type H37Rv) and Cy5- (mutant strain or heat-shocked cells) labelled cDNA were mixed and purified using a Qiagen MinElute kit, eluting in water.MIAME ScoreRaw DataAssays and DataMAGE-TAB FilesArray DesignsImage Adquisition - Title: BuG@S Axon Scanning (4000B). Description: Microarray slides were scanned with a GenePix 4000B Microarray Scanner (Axon Instruments) using PMT voltages usually of 800 for cDNA experiments. Basic analysis was performed using the software provided with the scanner, GenePix Pro. Care was taken to ensure that control spots and spots with high local background were removed from the results so that they were unable to interfere with later analysis.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsGenePix 4000B Microarray ScannerThe importance of Mn(2+) for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn(2+) is required are yet to be fully elucidated. Here, we analyzed the effect of Mn(2+) limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn(2+) transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn(2+)-dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn(2+)-SodA and virulence-associated genes pcpA and prtA. We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-responstranscription profiling by arrayStreptococcus pneumoniaeCentral Role of Manganese in Regulation of Stress Responses, Physiology, and Metabolism in Streptococcus pneumoniaeOgunniyi, Abiodun D.; Mahdi, Layla K.; Jennings, Michael P.; McEwan, Alastair G.; McDevitt, Christopher A.; Van der Hoek, Mark B.; Bagley, Christopher J.; Hoffmann, Peter; Gould, Katherine A.; Paton, James C.falseTranscription profiling of by array of Streptococcus pneumoniae D39 to investigate the effect of manganese limitationData is also available from <ahref=http://bugs.sgul.ac.uk/E-BUGS-89 target=_blank>BuG@Sbase</a>2010-07-22T00:00:00Z2022-03-07T00:51:07.244Z2022-03-07T00:51:07.244ZE-BUGS-8920601473EFO_000276820601473