Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

FNR- vs WT in anoxic conditions

ABSTRACT: Salmonella enterica var. Typhimurium (S. Typhimurium) is a Gram-negative, facultative intracellular pathogen that infects the intestinal tracts of humans and animals. In the host, S. Typhimurium encounters a wide range of oxygen concentrations going from oxic conditions in the stomach to near anoxic conditions in the distal sigmoid colon-rectal junction. In Escherichia coli, FNR (Fumarate Nitrate Reductase) is one of the main regulatory proteins involved in oxygen sensing and in controlling the transcription of the genes required for the aerobic/anaerobic transition.. However, the role of FNR in S. Typhimurium is largely unknown. To assess its role in S. Typhimurium, we constructed an FNR- mutant (NC983) in the pathogenic wild-type (WT) strain, ATCC14028s. The WT and the fnr- mutant strains were grown under anaerobic conditions in a Coy Anaerobic Chamber. Total RNAs were isolated when the cultures reached an OD600 of 0.3 (mid-log). Microarray slides were used to compare the global expression patterns of the two strains (i.e., 14028s vs. NC983). On each slides there were three replicas of the Salmonella genome and we swapped dyes, thus having a total of six replicates for each gene.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium

SUBMITTER: Hosni Hassan

PROVIDER: E-GEOD-3657 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

FNR is a global regulator of virulence and anaerobic metabolism in Salmonella enterica serovar Typhimurium (ATCC 14028s).

Fink Ryan C RC Evans Matthew R MR Porwollik Steffen S Vazquez-Torres Andres A Jones-Carson Jessica J Troxell Bryan B Libby Stephen J SJ McClelland Michael M Hassan Hosni M HM

Journal of bacteriology 20070112 6

Salmonella enterica serovar Typhimurium must successfully transition the broad fluctuations in oxygen concentrations encountered in the host. In Escherichia coli, FNR is one of the main regulatory proteins involved in O2 sensing. To assess the role of FNR in serovar Typhimurium, we constructed an isogenic fnr mutant in the virulent wild-type strain (ATCC 14028s) and compared their transcriptional profiles and pathogenicities in mice. Here, we report that, under anaerobic conditions, 311 genes (6 ...[more]

PMID: 17220229

Similar Datasets

Project description:Ferric uptake regulator (Fur) is the major regulator of iron acquisition in Escherichia coli and other bacteria. In the present study, the role of Fur in anaerobic S. enterica serovar Typhimurium was determined by transcriptome analysis, reporter assays, and enzymatic assays. In anaerobic ?fur, 298 genes were differentially expressed. In general, Fur repressed genes required for iron acquisition/storage, metabolism, electron transport, oxidative/nitrosative stress, and modulators of virulence. There were 73 genes whose expression required Fur, eleven of which contain a putative Fur box 5? of the gene. Transcription of sodA was >9-fold higher in ?fur but there was no corresponding increase in the activity of SodA. Anaerobic cultures of the fur mutant and the WT were used to inoculate two sets of three independent flasks each containing 150 ml of anoxic LB-MOPS-X. The three independent cultures were grown to an optical density at 600 nm (OD600) of 0.25 to 0.35, pooled, and treated with RNAlater (QIAGEN, Valencia, CA) to fix the cells and preserve the quality of the RNA. Total RNA was extracted with the RNeasy RNA extraction kit (QIAGEN), and the samples were treated with RNase-free DNase (Invitrogen, Carlsbad, CA). The absence of contaminating DNA and the quality of the RNA was confirmed by PCR amplification of known genes and by using agarose gel electrophoresis. Aliquots of the RNA samples were kept at –80°C for use in the microarray and quantitative real-time reverse transcription-PCR (qRT-PCR) studies. The SuperScript Indirect cDNA labeling system (Invitrogen) was used to synthesize the cDNA for the hybridizations. Each experiment consisted of two hybridizations, on two slides, and was carried out in Corning Hybridization Chambers at 42°C overnight. Dye swapping was performed to avoid dye-associated effects on cDNA synthesis. The slides were washed at increasing stringencies (2x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% sodium dodecyl sulfate [SDS], 42°C; 0.1% SSC, 0.1% SDS, room temperature; 0.1% SSC, room temperature). Following hybridization, the microarrays were scanned for the Cy3 and Cy5 fluorescent signals with a ScanArray 4000 microarray scanner from GSI Lumonics (Watertown, MA). The intensity of every spot was codified as the sum of the intensities of all the pixels within a circle positioned over the spot itself and the background as the sum of the intensities of an identical number of pixels in the immediate surroundings of the circled spot. Cy3 and Cy5 values for each spot were normalized over the total intensity for each dye to account for differences in total intensity between the two scanned images. The consistency of the data obtained from the microarray analysis was evaluated by a pair-wise comparison, calculated with a two-tailed Student's t test and analyzed by the MEAN and TTEST procedures of SAS-STAT statistical software (SAS Institute, Cary, NC) . The signal intensity at each spot from the fur mutant and the WT were background subtracted, normalized, and used to calculate the ratio of gene expression between the two strains. All replicas were combined, and the median expression ratios and standard deviations were calculated for open reading frames (ORFs) showing 2.5-fold change.

Dataset Information

FNR- vs WT in anoxic conditions

Publications

FNR is a global regulator of virulence and anaerobic metabolism in Salmonella enterica serovar Typhimurium (ATCC 14028s).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets