Investigation Title Transcription profiling of Pseudomonas aeruginosa of the sulfate starvation response Comment[Submitted Name] Transcriptomic analysis of the sulfate starvation response in Pseudomonas aeruginosa Experimental Design unknown_experiment_design_type transcription profiling by array Experimental Design Term Source REF EFO Comment[ArrayExpressReleaseDate] 2008-06-16 Comment[SecondaryAccession] GSE8408 Comment[AEMIAMESCORE] 3 Comment[ArrayExpressAccession] E-GEOD-8408 Comment[MAGETAB TimeStamp_Version] 2010-08-09 10:39:13 Last Changed Rev: 13058 Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Person Last Name Kertesz Person First Name Michael Person Mid Initials A. Person Email michael.kertesz@manchester.ac.uk Person Phone Person Fax Person Address Faculty of Life Sciences, University of Manchester, Oxford Rd, Manchester, M13 9PT, United Kingdom Person Affiliation University of Manchester Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2008-06-16 PubMed ID 17675390 Publication DOI 17675390 Publication Author List Tewes Tralau, Stéphane Vuilleumier, Christelle Thibault, Barry J Campbell, C Anthony Hart, Michael A Kertesz Publication Title Transcriptomic analysis of the sulfate starvation response of Pseudomonas aeruginosa. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Gene expression in response to changes in sulfur supply was studied in P. aeruginosa E601, a cystic fibrosis isolate that displays mucin sulfatase activity, and in P. aeruginosa PAO1. A large family of genes was found to be upregulated by sulfate limitation in both isolates, encoding sulfatases and sulfonatases, transport systems, oxidative stress proteins, and a sulfate-regulated TonB/ExbBD complex. These genes were localized in five distinct islands on the genome, and encoded proteins with a significantly reduced content of cysteine and methionine. Growth of P. aeruginosa E601 with mucin as sulfur source led to a sulfate starvation response, but also to induction of genes involved with type III secretion systems. Experiment Overall Design: Gene expression in exponential-phase cells was analysed using Affymetrix arrays. Sulfur was supplied either as sulfate, or as the non-sulfate S sources cyclamate (sodium cyclohexylsulfamate) and mucin (human colon cell line mucin LS174T). Control experiments were also done with combinations of these sulfur sources. Studies with mucin as sulfur source were carried out with P. aeruginosa E601 (a CF isolate with mucin sulfatase activity) and comparative studies were performed with P. aeruginosa PAO1. Experiments with strain E601 were done in 3-4 fold replication, experiments with strain PAO1 with 2-3 fold replication. Protocol Name P-G8408-11 P-G8408-7 P-G8408-5 P-G8408-14 P-G8408-12 P-G8408-10 P-G8408-8 P-G8408-1 P-G8408-9 P-G8408-13 P-G8408-2 P-G8408-6 P-G8408-4 P-G8408-3 Affymetrix:Protocol:Hybridization-Unknown P-AFFY-6 Protocol Type grow grow grow grow grow grow grow grow grow grow specified_biomaterial_action nucleic_acid_extraction nucleic_acid_extraction labeling hybridization feature_extraction Protocol Description 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. .These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM sulfate as S source. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2. 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. The cells were then grown for at least 12 generations with 500 uM cyclamate (sodium cyclohexylsulfamate) as sulfur source.These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM cyclamate as S source). For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2. 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. These cells were then used to inoculate the starter culture needed for RNA preparation. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2. 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. They wer then grown for at least 12 generations with 500 uM sulfate and 500 uM cyclamate (sodium cyclohexylsulfamate) as S source. These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM cyclamate/500 uM sulfate as S source. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2. 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM sulfate as S source. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2. 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. The cells were then grown for at least 12 generations with a combination of 500 uM cyclamate (sodium cyclohexylsulfamate) and 25 ug/ml human colon cell line mucin LS174T as sulfur source.These cells were then used to inoculate the starter culture needed for RNA preparation, grown with the same mixture of cyclamate and mucin as S source). For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2. 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. The cells were then grown for at least 12 generations with 25 ug/ml human colon cell line mucin LS174T as sulfur source.These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 25 ug/ml LS174T mucin as S source). For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2. 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. These cells were then used to inoculate the starter culture needed for RNA preparation. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. The cells were then grown for at least 12 generations with a combination of 500 uM cyclamate (sodium cyclohexylsulfamate) and 25 ug/ml human colon cell line mucin LS174T as sulfur source.These cells were then used to inoculate the starter culture needed for RNA preparation, grown with the same mixtire of cyclamate and mucin as S source). For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2. 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. They were then grown for at least 12 generations with 500 uM cyclamate (sodium cyclohexylsulfamate) as S source. These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM cyclamate as S source. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2. no treatment Qiagen RNeasy mini kit â€" bacterial protocol. The washing step with 700 ul of buffer RW1 was split up into 2 steps and an on column digest of DNA using Qiagen DNase was performed in between Qiagen RNeasy mini kit â€" bacterial protocol. The washing step with 700ul of buffer RW1 was split up into 2 steps and an on column digest of DNA using Qiagen DNase was performed in between Standard Affymetrix procedures, starting with 5-10 ug RNA Title: Affymetrix Generic Hybridization. Description: Title: Affymetrix CEL analysis. Description: Protocol Parameters Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF mo SDRF File E-GEOD-8408.sdrf.txt Term Source Name The MGED Ontology ArrayExpress EFO The MGED Ontology mo Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version