ArcA- mutant vs. WT
Ontology highlight
ABSTRACT:
ORGANISM(S): Salmonella Salmonella enterica subsp. enterica serovar Typhimurium
PROVIDER: GSE24564 | GEO | 2011/02/10
SECONDARY ACCESSION(S): PRJNA132633
REPOSITORIES: GEO
Similar Datasets
Project description:The fumarate and nitrate reductase regulator protein, FNR, is a global transcription factor that regulates major biochemical changes as Escherichia coli adapts from aerobic to anaerobic growth. The ability of an fnr mutant to grow anaerobically in the presence of trimethylamine-N-oxide (TMAO) as the terminal electron acceptor was exploited in microarray experiments designed to determine a minimum number of Escherichia coli K-12 MG1655 operons that are regulated directly by FNR. In an anaerobic glycerol-TMAO-fumarate medium, the fnr mutant grew as well as the parental strain, enabling us to reveal the response of the E. coli transcriptome to oxygen, nitrate and nitrite in the absence of glucose repression or artefacts due to variations in growth rate. Many of the discrepancies between previous microarray studies of the E. coli FNR regulon were resolved in this study. First data for 43 previously characterised FNR-dependent operons were analysed. The current microarray data confirmed 32 of these 43 assignments, but alone did not confirm FNR-activation of 5 operons (adhE, glpTQ, cydDC, hlyE and arcA), or FNR repression of 6 operons (hemA, narXL, tpx, yeiL, norVW or ubiCA). Thirty-six operons not previously known to be included in the FNR regulon were activated by FNR and a further 26 operons appeared to be repressed. For each of these operons, an excellent match to the consensus FNR-binding site sequence was identified. The FNR regulon therefore minimally includes at least 94, and possibly as many as 105, operons. Many FNR-activated promoters are also regulated by one or both of two nitrate- and nitrite-responsive two-component regulatory systems, NarX-NarL and NarQ-NarP. Comparison of transcripts in the parental strain and a narXL deletion mutant revealed that transcription of 51 operons is activated, directly or indirectly, by NarL in response to nitrate, and a further 41 are repressed. As phosphorylated NarL can bind to the NarP DNA target sequence, the narP gene was also deleted from the narXL mutant to reveal the extent of regulation by phosphorylated NarP. Fourteen promoters were more active in the narP+ strain than in the mutant, and a further 37 were strongly repressed. This is the first report that NarP might function as a global repressor as well as a transcription activator. The data also revealed possible new biochemical defence mechanisms against reactive nitrogen species. Keywords: genetic modification, growth conditions An fnr mutant is either unable to grow anaerobically in the presence of most terminal electron acceptors and a non-fermentable carbon source such as glycerol or lactate, or grows far more slowly than the parental strain. Under such conditions, any differences in the transcriptomes of an fnr mutant and its parental strain would be due to both direct effects of FNR, and to differences in growth rate. As glucose represses expression from some FNR-activated promoters replacement of glucose by a less repressing fermentable carbohydrate would decrease effects due to glucose repression, but to an unknown extent. We therefore exploited the fact that fnr mutants can be grown anaerobically in the presence of the non-fermentable and non-repressing carbon source, glycerol, in the presence of trimethylamine-N-oxide (TMAO) in addition to fumarate as the terminal electron acceptor. Furthermore, the presence of TMAO has a minimal effect on NarX-NarL or NarQ-NarP-dependent induction or repression. Under these conditions, the fnr mutant grows as well as the parental strain and the use of the glycerol-TMAO-fumarate medium enables us to reveal the response of the E. coli transcriptome to nitrate, nitrite and the two-component regulator system, NarX-NarL. In each large set of experiments a common pool of reference RNA isolated from bacteria that had been grown anaerobically, and in which FNR-activated genes were expressed at a significant level. A potential disadvantage of this approach was the risk that some promoters repressed by FNR would be expressed at such a low level that the microarray signals would be too low to yield reliable data. To check for this artefact, further experiments were completed in which the reference RNA was a pool of samples isolated from bacteria in the early exponential phase of aerobic growth. “Reference” RNA was isolated from at least four independent cultures grown to OD 0.5 to 0.6. “Test” RNA was isolated from three independent cultures grown to OD 0.5 to 0.6.
2005-11-21 | E-GEOD-3591 | biostudies-arrayexpress
Project description:The fumarate and nitrate reductase regulator protein, FNR, is a global transcription factor that regulates major biochemical changes as Escherichia coli adapts from aerobic to anaerobic growth. The ability of an fnr mutant to grow anaerobically in the presence of trimethylamine-N-oxide (TMAO) as the terminal electron acceptor was exploited in microarray experiments designed to determine a minimum number of Escherichia coli K-12 MG1655 operons that are regulated directly by FNR. In an anaerobic glycerol-TMAO-fumarate medium, the fnr mutant grew as well as the parental strain, enabling us to reveal the response of the E. coli transcriptome to oxygen, nitrate and nitrite in the absence of glucose repression or artefacts due to variations in growth rate. Many of the discrepancies between previous microarray studies of the E. coli FNR regulon were resolved in this study. First data for 43 previously characterised FNR-dependent operons were analysed. The current microarray data confirmed 32 of these 43 assignments, but alone did not confirm FNR-activation of 5 operons (adhE, glpTQ, cydDC, hlyE and arcA), or FNR repression of 6 operons (hemA, narXL, tpx, yeiL, norVW or ubiCA). Thirty-six operons not previously known to be included in the FNR regulon were activated by FNR and a further 26 operons appeared to be repressed. For each of these operons, an excellent match to the consensus FNR-binding site sequence was identified. The FNR regulon therefore minimally includes at least 94, and possibly as many as 105, operons. Many FNR-activated promoters are also regulated by one or both of two nitrate- and nitrite-responsive two-component regulatory systems, NarX-NarL and NarQ-NarP. Comparison of transcripts in the parental strain and a narXL deletion mutant revealed that transcription of 51 operons is activated, directly or indirectly, by NarL in response to nitrate, and a further 41 are repressed. As phosphorylated NarL can bind to the NarP DNA target sequence, the narP gene was also deleted from the narXL mutant to reveal the extent of regulation by phosphorylated NarP. Fourteen promoters were more active in the narP+ strain than in the mutant, and a further 37 were strongly repressed. This is the first report that NarP might function as a global repressor as well as a transcription activator. The data also revealed possible new biochemical defence mechanisms against reactive nitrogen species. Keywords: genetic modification, growth conditions
2005-11-21 | GSE3591 | GEO
Project description:Determining how facultative anaerobic organisms sense and direct cellular responses to electron acceptor availability has been a subject of intense study. However, even in the model organism Escherichia coli, established mechanisms only explain a small fraction of the hundreds of genes that are regulated during shifts in electron acceptor availability. Here we propose a qualitative model that accounts for the full breadth of regulated genes by detailing how two global transcription factors (TFs), ArcA and Fnr of E. coli, sense key metabolic redox ratios and act on a genome-wide basis to regulate anabolic, catabolic, and energy generation pathways. We first fill gaps in our knowledge of this transcriptional regulatory network by carrying out ChIP-chip and gene expression experiments to identify 463 regulatory events. We then interfaced this reconstructed regulatory network with a highly curated genome-scale metabolic model to show that ArcA and Fnr regulate > 80% of total metabolic flux and 96% of differential gene expression across fermentative and nitrate respiratory conditions. Finally, based on the data we propose a feedforward with feedback trim regulatory scheme by showing extensive repression of catabolic genes by ArcA and extensive activation of chemiosmotic genes by Fnr. We further corroborated this regulatory scheme by showing a 0.71 r2 (p < 1e-6) correlation between changes in metabolic flux and changes in regulatory activity across fermentative and nitrate respiratory conditions. We also are able to relate the proposed model to a wealth of previously generated data by contextualizing the existing transcriptional regulatory network. We integrated transcription factor binding regions and mRNA transcript abundance to elucidate the ArcA and Fnr regulons experimentally. To measure transcription factor binding at a genome scale, we employed a ChIP-chip method to derivative strains of E. coli K-12 MG1655 harboring ArcA-8myc or Fnr-8myc under various conditions. The E. coli strains harboring Fnr-8myc and ArcA-8myc were generated as described previously [PMID 16454042]. A 12 chip study with two different strains under two different culture conditions.
2014-02-27 | E-GEOD-55366 | biostudies-arrayexpress
Project description: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. Keywords: FNR, Salmonella, anaerobic, virulence
2007-01-05 | GSE3657 | GEO
Project description: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.
2010-07-01 | E-GEOD-3657 | biostudies-arrayexpress
Project description:Determining how facultative anaerobic organisms sense and direct cellular responses to electron acceptor availability has been a subject of intense study. However, even in the model organism Escherichia coli, established mechanisms only explain a small fraction of the hundreds of genes that are regulated during shifts in electron acceptor availability. Here we propose a qualitative model that accounts for the full breadth of regulated genes by detailing how two global transcription factors (TFs), ArcA and Fnr of E. coli, sense key metabolic redox ratios and act on a genome-wide basis to regulate anabolic, catabolic, and energy generation pathways. We first fill gaps in our knowledge of this transcriptional regulatory network by carrying out ChIP-chip and gene expression experiments to identify 463 regulatory events. We then interfaced this reconstructed regulatory network with a highly curated genome-scale metabolic model to show that ArcA and Fnr regulate > 80% of total metabolic flux and 96% of differential gene expression across fermentative and nitrate respiratory conditions. Finally, based on the data we propose a feedforward with feedback trim regulatory scheme by showing extensive repression of catabolic genes by ArcA and extensive activation of chemiosmotic genes by Fnr. We further corroborated this regulatory scheme by showing a 0.71 r2 (p < 1e-6) correlation between changes in metabolic flux and changes in regulatory activity across fermentative and nitrate respiratory conditions. We also are able to relate the proposed model to a wealth of previously generated data by contextualizing the existing transcriptional regulatory network. Biological replicates were performed in triplicate for M-NM-^Tfnr, M-NM-^TarcA, and wild type Escherichia coli K12 MG1655 strains under both fully fermentative and nitrate respiratory conditions. An 18 chip study with three different strains under two different culture conditions.
2014-02-27 | E-GEOD-55365 | biostudies-arrayexpress
Project description:Determining how facultative anaerobic organisms sense and direct cellular responses to electron acceptor availability has been a subject of intense study. However, even in the model organism Escherichia coli, established mechanisms only explain a small fraction of the hundreds of genes that are regulated during shifts in electron acceptor availability. Here we propose a qualitative model that accounts for the full breadth of regulated genes by detailing how two global transcription factors (TFs), ArcA and Fnr of E. coli, sense key metabolic redox ratios and act on a genome-wide basis to regulate anabolic, catabolic, and energy generation pathways. We first fill gaps in our knowledge of this transcriptional regulatory network by carrying out ChIP-chip and gene expression experiments to identify 463 regulatory events. We then interfaced this reconstructed regulatory network with a highly curated genome-scale metabolic model to show that ArcA and Fnr regulate > 80% of total metabolic flux and 96% of differential gene expression across fermentative and nitrate respiratory conditions. Finally, based on the data we propose a feedforward with feedback trim regulatory scheme by showing extensive repression of catabolic genes by ArcA and extensive activation of chemiosmotic genes by Fnr. We further corroborated this regulatory scheme by showing a 0.71 r2 (p < 1e-6) correlation between changes in metabolic flux and changes in regulatory activity across fermentative and nitrate respiratory conditions. We also are able to relate the proposed model to a wealth of previously generated data by contextualizing the existing transcriptional regulatory network.
2014-02-27 | GSE55366 | GEO
Project description:Determining how facultative anaerobic organisms sense and direct cellular responses to electron acceptor availability has been a subject of intense study. However, even in the model organism Escherichia coli, established mechanisms only explain a small fraction of the hundreds of genes that are regulated during shifts in electron acceptor availability. Here we propose a qualitative model that accounts for the full breadth of regulated genes by detailing how two global transcription factors (TFs), ArcA and Fnr of E. coli, sense key metabolic redox ratios and act on a genome-wide basis to regulate anabolic, catabolic, and energy generation pathways. We first fill gaps in our knowledge of this transcriptional regulatory network by carrying out ChIP-chip and gene expression experiments to identify 463 regulatory events. We then interfaced this reconstructed regulatory network with a highly curated genome-scale metabolic model to show that ArcA and Fnr regulate > 80% of total metabolic flux and 96% of differential gene expression across fermentative and nitrate respiratory conditions. Finally, based on the data we propose a feedforward with feedback trim regulatory scheme by showing extensive repression of catabolic genes by ArcA and extensive activation of chemiosmotic genes by Fnr. We further corroborated this regulatory scheme by showing a 0.71 r2 (p < 1e-6) correlation between changes in metabolic flux and changes in regulatory activity across fermentative and nitrate respiratory conditions. We also are able to relate the proposed model to a wealth of previously generated data by contextualizing the existing transcriptional regulatory network.
2014-02-27 | GSE55365 | GEO
Project description:Flagella-driven motility of Salmonella enterica serovar Typhimurium facilitates host colonization. However, the large extracellular flagellum is also a prime target for the immune system. As consequence, expression of flagella is bistable within a population of Salmonella, resulting in flagellated and non-flagellated subpopulations. This allows the bacteria to maximize fitness in hostile environments. The degenerate EAL-domain protein RflP (formerly YdiV) is responsible for the bistable expression of flagella by directing the flagellar master regulatory complex FlhD4C2 to proteolytic degradation. The environmental cues controlling expression of rflP and thus the bistable flagellar biosynthesis remain ambiguous. Here, we demonstrate that RflP responds to cell envelope stress and alterations of outer membrane integrity. Lipopolysaccharide (LPS) truncation mutants of Salmonella Typhimurium exhibited increasing motility defects due to downregulation of flagellar gene expression. Transposon mutagenesis and genetic profiling revealed that σ24 (RpoE) and Rcs phosphorelay-dependent cell envelope stress response systems sense modifications of lipopolysaccharide, low pH activity of the complement system. This subsequently results in activation of RflP expression and degradation of FlhD4C2 via ClpXP. We speculate that diverse hostile environments inside the host might result in cell envelope damage and would thus trigger the repression of resource-costly and immunogenic flagella biosynthesis via activation of the cell envelope stress response.
2018-04-13 | GSE87447 | GEO
Project description:Salmonella enterica serotype Typhimurium produces a variety of fimbrial appendages, among which the type 1 fimbriae is the most common type. In vitro static broth culture favors S. Typhimurium to produce type 1 fimbriae, while solid agar inhibits its expression. A transposon inserted in the stbC gene, which would encode an usher protein for Stb fimbriae of a non-flagellar S. Typhimurium LB5010 strain, conferred it to agglutinate yeast cells on both cultures, and was mannose-sensitive. Reverse transcription polymerase chain reaction (RT-PCR) revealed that the expression of the fimbrial major subunit gene fimA, and fimZ, a positive regulator gene of fimA, were both increased in the stbC mutant strain when grown on LB agar; fimW, a repressor gene of fimA, exhibited lower expression. Flagella were observed in the stbC mutant and this phenotype was correlated with the motile phenotype detected by MSRV agar medium and reaction with flagella antiserum. Microarray data and RT-PCR also indicated that the expression of three genes, motA, motB, and cheM, was enhanced in the stbC mutant. The S. Typhimurium stbC mutant was resistant to a variety of antibiotics, consistent with the finding that expression of yhcQ and ramA, two genes involved in multidrug resistance, was enhanced. A complementation test revealed that transforming a recombinant plasmid possessing the coding sequence of the stbC gene restored the mannose-sensitive agglutination phenotype to the stbC mutant much as that in the parental S. Typhimurium LB5010 strain, indicating the possibility of an interplay of different fimbrial systems in coordinating their expression. Key Words: Salmonella enterica serotype Typhimurium, fimbriae, type 1 fimbriae, stbC, transposon, multidrug resistant, flagella
2011-12-25 | GSE34685 | GEO