Comment[ArrayExpressAccession] E-GEOD-23588 MAGE-TAB Version 1.1 Public Release Date 2015-07-31 Investigation Title Influence of Autoinducer-2 (AI-2) in Salmonella Typhimurium Gene Expression Comment[Submitted Name] Influence of Autoinducer-2 (AI-2) in Salmonella Typhimurium Gene Expression Experiment Description Salmonella enterica serovar Typhimurium is known to synthesize and respond to the cell signaling molecule, autoinducer-2 (AI-2). The luxS gene is involved in the synthesis of AI-2. We have previously shown that luxS controls a variety of bacterial processes in S. Typhimurium. In this study we identified the genes regulated by AI-2 using mRNA samples from isogenic luxS gene mutant of S. Typhimurium strain 87-26254 grown in LB media in the presence/absence of in vitro synthesized AI-2. In the presence of in vitro synthesized AI-2, 536 genes were significantly (p<0.05) regulated. Interestingly, in vitro synthesized AI-2 caused the down-regulation of 39 genes in Salmonella Pathogenicity Island-1 (SPI-1) including the transcriptional regulators hilA, hilD, and invF. Purified cDNAs from the mutant supplemented with AI-2 and mutant without AI-2 (PBS) were each labeled with Cy-3 mono-Reactive Dye and Cy-5 mono-Reactive Dye (GE Health Care, Piscataway, NJ) and were processed using a dye-swapping design. A total of eight microarray arrays, each with duplicate spots for each gene and used for mutant supplemented with AI-2. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Jesudhasan Jesudhasan Pillai Person First Name Palmy Palmy Suresh Person Mid Initials R R D Person Email palmy.jesudhasan@utsouthwestern.edu Person Affiliation University of Texas Southwestern Medical Center Person Phone 214-648-5627 Person Address Microbiology, University of Texas Southwestern Medical Center, NL4.110M, Dallas, TX, USA Person Roles submitter Protocol Name P-GSE23588-5 P-GSE23588-3 P-GSE23588-2 P-GSE23588-1 P-GSE23588-4 P-GSE23588-13 P-GSE23588-9 P-GSE23588-14 P-GSE23588-25 P-GSE23588-20 P-GSE23588-10 P-GSE23588-16 P-GSE23588-6 P-GSE23588-18 P-GSE23588-23 P-GSE23588-19 P-GSE23588-7 P-GSE23588-21 P-GSE23588-11 P-GSE23588-17 P-GSE23588-24 P-GSE23588-8 P-GSE23588-12 P-GSE23588-22 P-GSE23588-15 Protocol Description The signal intensity of each gene was globally normalized using LOWESS within the R statistics package. The normalized data was analyzed using commercial SAS 9.1.3 program (SAS Institute Inc. Cary, NC). ID_REF = VALUE = MA Lowess normalized of value cy5 / MA Lowess normalized value of cy3 F635 Median = CH1_ SIG_median B635 Median = CH1_BKD_median F532 Median = CH2_ SIG_median B532 Median = CH2_BKD_median The signal intensity of each gene was globally normalized using LOWESS within the R statistics package. The normalized data was analyzed using commercial SAS 9.1.3 program (SAS Institute Inc. Cary, NC). ID_REF = VALUE = Lowess M log ratio test/reference F635 Median = CH1_ SIG_median B635 Median = CH1_BKD_median F532 Median = CH2_ SIG_median B532 Median = CH2_BKD_median The signal intensity of each gene was globally normalized using LOWESS within the R statistics package. The normalized data was analyzed using commercial SAS 9.1.3 program (SAS Institute Inc. Cary, NC). ID_REF = F635 Median = CH1_ SIG_median B635 Median = CH1_BKD_median F532 Median = CH2_ SIG_median B532 Median = CH2_BKD_median VALUE = Lowess M log ratio test/reference The signal intensity of each gene was globally normalized using LOWESS within the R statistics package. The normalized data was analyzed using commercial SAS 9.1.3 program (SAS Institute Inc. Cary, NC). ID_REF = VALUE = Lowess M log ratio test/reference F635 Median = CH2_ SIG_median B635 Median = CH2_BKD_median F532 Median = CH1_ SIG_median B532 Median = CH1_BKD_median The signal intensity of each gene was globally normalized using LOWESS within the R statistics package. The normalized data was analyzed using commercial SAS 9.1.3 program (SAS Institute Inc. Cary, NC). ID_REF = VALUE = MA Lowess normalized of value cy3 / MA Lowess normalized value of cy5 F635 Median = CH1_ SIG_median B635 Median = CH1_BKD_median F532 Median = CH2_ SIG_median B532 Median = CH2_BKD_median Total RNA (5 µg) was used to synthesize cDNA using a random primer for reverse transcription (Invitrogen Life Technologies, Carlsbad, CA). The primer was annealed at 70°C for 10 min, followed by snap-freezing in a dry ice-ethanol bath for 30 s and centrifugation for 1 min. The reaction mixture (Superscript III buffer, 0.1 M dithiothreitol, and aminoallyl-deoxyuridine triphosphate mix) was then incubated overnight with Superscript III reverse transcriptase (Invitrogen) at 42°C. Residual RNA was removed by alkaline treatment followed by neutralization, and the cDNA was purified with a QIAquick PCR purification kit (Qiagen). Purified cDNA was labeled with Cy-3 mono-Reactive Dye (GE Health Care). Total RNA (5 µg) was used to synthesize cDNA using a random primer for reverse transcription (Invitrogen Life Technologies, Carlsbad, CA). The primer was annealed at 70°C for 10 min, followed by snap-freezing in a dry ice-ethanol bath for 30 s and centrifugation for 1 min. The reaction mixture (Superscript III buffer, 0.1 M dithiothreitol, and aminoallyl-deoxyuridine triphosphate mix) was then incubated overnight with Superscript III reverse transcriptase (Invitrogen) at 42°C. Residual RNA was removed by alkaline treatment followed by neutralization, and the cDNA was purified with a QIAquick PCR purification kit (Qiagen). Purified cDNA was labeled with Cy-5 mono-Reactive Dye (GE Health Care). The labeled mixtures were further purified using a QIAquick PCR purification kit (Qiagen). Equal amounts of labeled cDNA from the treatment and control were used to hybridize on S. Typhimurium LT2 genome microarrays (version 2), obtained from JCVI (formerly, TIGR, Rockville, MD) and provided by the Pathogen Functional Genome Resource Center (PFGRC). These arrays were amplicon arrays with 6780 ORF each, with 2 replicate spots per ORF. The labeled cDNA was applied to the above arrays. Hybridization was carried out overnight at 42°C in a water bath using Corning hybridization chamber. The pre- and post-hybridization was carried out using the the PFGRC SOP #M008. The labeled mixtures were further purified using a QIAquick PCR purification kit (Qiagen). Equal amounts of labeled cDNA from the treatment and control were used to hybridize on S. Typhimurium LT2 genome microarrays (version 2), obtained from JCVI (formerly, TIGR, Rockville, MD) and provided by the Pathogen Functional Genome Resource Center (PFGRC). These arrays were amplicon arrays with 6780 ORF each, with 2 replicate spots per ORF. The labeled cDNA was applied to the arrays. Hybridization was carried out overnight at 42°C in a water bath using Corning hybridization chamber. The pre- and post-hybridization was carried out using the the PFGRC SOP #M008. Salmonella Typhimurium with 10% of luxS mutant cell free supernatant (control) Salmonella Typhimurium with 10% PBS (control) Salmonella Typhimurium treated with 25 mM in vitro synthesized AI-2 (Treatment). Salmonella Typhimurium treated with 25 mM in vitro synthesized AI-2 (Treatment) Salmonella Typhimurium with 10% PBS (control). Salmonella Typhimurium treated with 10% of Salmonella Typhimurium wild type cell free supernatant (wCFS) that has AI-2 for 3 hours (Treatment) Fresh Salmonella Typhimurium was allowed to grow in the presence of 10% PBS for 3 hours. Fresh Salmonella Typhimurium was allowed to grow in the presence of 10% (25 mM) in vitro synthesized AI-2 for 3 hours Fresh Salmonella Typhimurium was allowed to grow in the presence of Salmonella Typhimurium luxS mutant cell free supernatant (PBS) for 3 hours. Fresh Salmonella Typhimurium was allowed to grow in the presence of 10% PBS for 3 hours Fresh Salmonella Typhimurium was allowed to grow in the presence of 10% (25 mM) in vitro synthesized AI-2 for 3 hours. Fresh Salmonella Typhimurium was allowed to grow in the presence of 10% of Salmonella Typhimurium wild type cell free supernatant (wCFS) that has AI-2 for 3 hours. Total RNA was isolated from the cultures using an RNeasy mini kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer's protocol. RNAprotect bacteria reagent (QIAGEN, Inc., Valencia, CA) was added to the cultures to stabilize RNA before isolation. The RNase-free DNase set (QIAGEN, Inc., Valencia, CA) was used for on-column DNase digestion to remove residual genomic DNA. The quantity and quality of RNA was examined using ND-1000 spectrophotometer (NanoDrop® Technologies, Wilmington, DE) and bioanalyser 2100 (Agilent Technologies, Santa Clara, CA) respectively. Total RNA was isolated from the cultures using an RNeasy mini kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer's protocol. RNAprotect bacteria reagent (QIAGEN, Inc., Valencia, CA) was added to the cultures to stabilize RNA before isolation. The RNase-free DNase set (QIAGEN, Inc., Valencia, CA) was used for on-column DNase digestion to remove residual genomic DNA. The quantity and quality of RNA was examined using ND-1000 spectrophotometer (NanoDrop® Technologies, Wilmington, DE) and bioanalyser 2100 (Agilent Technologies, Santa Clara, CA) respectively Total RNA was isolated from the cultures using an RNeasy mini kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer's protocol. RNAprotect bacteria reagent (QIAGEN, Inc., Valencia, CA) was added to the cultures to stabilize RNA before isolation. The RNase-free DNase set (QIAGEN, Inc., Valencia, CA) was used for on-column DNase digestion to remove residual genomic DNA. The quantity and quality of RNA was examined using ND-1000 spectrophotometer (NanoDrop® Technologies, Wilmington, DE) and bioanalyser 2100 (Agilent Technologies, Santa Clara, CA) respectively. The slides were washed and scanned using a GenePix 4100A scanner (Axon Instruments Inc., Union City, CA) at 532 nm (Cy3 channel) and 635 nm (Cy5 channel), and the images were stored for further analysis. Protocol Type normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol labelling protocol labelling protocol hybridization protocol hybridization protocol sample treatment protocol sample treatment protocol sample treatment protocol sample treatment protocol sample treatment protocol sample treatment protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol nucleic acid extraction protocol nucleic acid extraction protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name treatment Experimental Factor Type treatment Comment[SecondaryAccession] GSE23588 Comment[GEOReleaseDate] 2015-07-31 Comment[ArrayExpressSubmissionDate] 2010-08-12 Comment[GEOLastUpdateDate] 2015-07-31 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-23588.sdrf.txt