Epigenetic control of the master regulator fumarate nitrate reductase (FNR) contributes to survival of Haemophilus influenzae during lung infection
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PROVIDER: PRJNA1158767 | ENA |
REPOSITORIES: ENA
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Project description:Regulatory elements that dictate how Haemophilus influenzae adapts to the airways of COPD patients are poorly understood. In this work, global fitness profiling of H. influenzae mutants by transposon mutagenesis/sequencing (Tn-seq) within the mouse lung identified Dam methyltransferase as an in vivo requirement, even though dam mutants grow normally during in vitro culture. In turn, methylation sequencing found that genome-wide Dam methylation patterns were undermethylated at GATC motifs with putative regulatory elements. This was confirmed for the htpG heat-shock chaperone gene, making it the first case of phenotypic variation controlled by variable Dam methylation for H. influenzae. Moreover, RNA sequencing revealed a novel multifactorial regulatory network, in which Dam methyltransferase activity is coordinated with the FNR and Fur transcriptional regulators to control the expression anaerobic defences against host-produced reactive species. Our results indicate key bacterial pathways for survival in diseased environments with high nitrosative stress, where damage to the airways reduces the amount of oxygen in the lungs as is encountered in COPD.
2025-08-20 | GSE276728 | GEO
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: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: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: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:Here, we report the overexpression, purification, and characterization of the transcriptional activator fumarate and nitrate reductase regulator from the pathogenic bacterium Neisseria meningitidis (NmFNR). Like its homologue from Escherichia coli (EcFNR), NmFNR binds a 4Fe-4S cluster, which breaks down in the presence of oxygen to a 2Fe-2S cluster and subsequently to apo-FNR. The kinetics of NmFNR cluster disassembly in the presence of oxygen are 2-3x slower than those previously reported for wild-type EcFNR, but similar to constitutively active EcFNR* mutants, consistent with earlier work in which we reported that the activity of FNR-dependent promoters in N. meningitidis is only weakly inhibited by the presence of oxygen (Rock, J. D., Thomson, M. J., Read, R. C., and Moir, J. W. (2007) J. Bacteriol. 189, 1138-1144). NmFNR binds to DNA containing a consensus FNR box sequence, and this binding stabilizes the iron-sulfur cluster in the presence of oxygen. Partial degradation of the 4Fe-4S cluster to a 3Fe-4S occurs, and this form remains bound to the DNA. The 3Fe-4S cluster is converted spontaneously back to a 4Fe-4S cluster under subsequent anaerobic reducing conditions in the presence of ferrous iron. The finding that binding to DNA stabilizes FNR in the presence of oxygen such that it has a half-life of approximately 30 min on the DNA has implications for our appreciation of how oxygen switches off FNR activatable genes in vivo.
| S-EPMC2801238 | biostudies-literature
Project description:Haemophilus influenzae responds to the steroid beclomethasone used in asthma therapy by modulation of biofilm formation and antibiotic tolerance
2019-06-30 | GSE125415 | GEO
Project description:The microbiome is an underappreciated contributor to intestinal drug metabolism with broad implications for drug efficacy and toxicity. While considerable progress has been made towards identifying the gut bacterial genes and enzymes involved, the role of environmental factors in shaping their activity remains poorly understood. Here, we focus on the gut bacterial reduction of azo bonds (R-N=N-R’), found in diverse chemicals in both food and drugs. Surprisingly, the canonical azoR gene in Escherichia coli was dispensable for azo bond reduction. Instead, azo reductase activity was controlled by the fumarate and nitrate reduction (fnr) regulator, consistent with a requirement for the anoxic conditions found within the gastrointestinal tract. Paired transcriptomic and proteomic analysis of the fnr regulon revealed that in addition to altering the expression of multiple reductases, FNR is necessary for the metabolism of L-Cysteine to hydrogen sulfide, enabling the degradation of azo bonds. Furthermore, we found that FNR indirectly regulates this process though the small non-coding regulatory RNA fnrS. Taken together, these results show how gut bacteria sense and respond to their intestinal environment to enable the metabolism of chemical groups found in both dietary and pharmaceutical compounds.
2023-08-09 | GSE235465 | GEO