Project description:The study aimed to identify role of OxyR during growth on different electron acceptors when E. coli are growing anaerobically. Wt and OxyR null cells were grown on nitrate and fumarate medium anaerobically. Each condition was done in duplicate. RNA was extracted using Qiagen RNeasy kit after stabilization of RNA with RNA protect bacteria reagent. Hybridization and further processing was done based on Affymetrix protocols on E. coli Genome 2.0 arrays.
Project description:Oxidative stress caused by exposure to reactive oxygen species (ROS), is a major challenge for aerobic and especially anaerobic organisms. Bacteria coordinate the response to oxidative stress through the LysR-type transcriptional regulator (LTTR) OxyR. Extensive studies have focused on the classical Escherichia coli system to shed light on the mode of action of defensive weapons against oxidative stress. The underlying mechanism is mediated via the formation of redox-dependent disulfide bond between two conserved cysteines of OxyR, thus activating transcription of members of the OxyR regulon. However, only fragmentary information on the regulation and function of OxyR has been gleaned through genetic and biochemical analyses in the important opportunistic pathogen P. aeruginosa. In this report, we used a comprehensive transcriptional profiling analysis to delineate the OxyR regulon under three conditions (King’s A medium [Pseudomonas medium or PM], Luria Broth (LB), and LB when oxyR is overexpressed), to investigate its roles in different cellular aspects that are independent of the classical oxidative stress response. Interestingly, when grown in LB, OxyR was found to regulating many genes involved in the process of inter-cellular communication known as quorum sensing (QS). In contrast, when grown in PM, OxyR regulate the expression of a newly identified CSS (cell-surface signaling) system in an OxyR-dependent fashion. In addition, the results from oxyR overexpression further confirmed that OxyR was linked to regulation of QS and Type 3 Secretion (T3SS) in addition to the regulation of antioxidative genes. Taken together, our results show that, apart from its dominant role in defense against oxidative stress in P. aeruginosa, OxyR acts as a global regulator that provides a link between the regulation of oxidative stress response, QS and virulence.
Project description:Oxidative stress caused by exposure to reactive oxygen species (ROS), is a major challenge for aerobic and especially anaerobic organisms. Bacteria coordinate the response to oxidative stress through the LysR-type transcriptional regulator (LTTR) OxyR. Extensive studies have focused on the classical Escherichia coli system to shed light on the mode of action of defensive weapons against oxidative stress. The underlying mechanism is mediated via the formation of redox-dependent disulfide bond between two conserved cysteines of OxyR, thus activating transcription of members of the OxyR regulon. However, only fragmentary information on the regulation and function of OxyR has been gleaned through genetic and biochemical analyses in the important opportunistic pathogen P. aeruginosa. In this report, we used a comprehensive transcriptional profiling analysis to delineate the OxyR regulon under three conditions (KingM-bM-^@M-^Ys A medium [Pseudomonas medium or PM], Luria Broth (LB), and LB when oxyR is overexpressed), to investigate its roles in different cellular aspects that are independent of the classical oxidative stress response. Interestingly, when grown in LB, OxyR was found to regulating many genes involved in the process of inter-cellular communication known as quorum sensing (QS). In contrast, when grown in PM, OxyR regulate the expression of a newly identified CSS (cell-surface signaling) system in an OxyR-dependent fashion. In addition, the results from oxyR overexpression further confirmed that OxyR was linked to regulation of QS and Type 3 Secretion (T3SS) in addition to the regulation of antioxidative genes. Taken together, our results show that, apart from its dominant role in defense against oxidative stress in P. aeruginosa, OxyR acts as a global regulator that provides a link between the regulation of oxidative stress response, QS and virulence. 15 samples, representing 5 different biological conditions, including 3 biological replicates for each condition
Project description:Oxidative stress that originates from reactive oxygen species (ROS) is an inevitable consequence of aerobic respiration in bacteria. Three transcription factors (TFs), OxyR, SoxR, and SoxS play a critical role in transcriptional regulation of the defense system. However, the full genome-wide regulatory potential of them remains elusive. Here, we comprehensively reconstruct genome-wide OxyR, SoxR, and SoxS transcriptional regulatory networks in Escherichia coli under oxidative stress. Integrative data analysis reveals that OxyR, SoxR, and SoxS regulons are comprised of 38 genes in 28 transcription units (TUs), 11 genes in 10 TUs, and 34 genes in 25 TUs, respectively, significantly expanding the current knowledge of their regulatory networks. Comparison of them to other stress-response regulatory networks highlights minimal overlap between their regulons, indicating that E. coli has a series of relatively distinct stress responses covering the range of different stresses. We also demonstrate that these intricate networks coordinate detoxification process with DNA and protein damage repair, cell wall synthesis, divalent metal ion homeostasis, as well as metabolic robustness to produce overall response of E. coli to oxidative stress.
Project description:Oxidative stress that originates from reactive oxygen species (ROS) is an inevitable consequence of aerobic respiration in bacteria. Three transcription factors (TFs), OxyR, SoxR, and SoxS play a critical role in transcriptional regulation of the defense system. However, the full genome-wide regulatory potential of them remains elusive. Here, we comprehensively reconstruct genome-wide OxyR, SoxR, and SoxS transcriptional regulatory networks in Escherichia coli under oxidative stress. Integrative data analysis reveals that OxyR, SoxR, and SoxS regulons are comprised of 38 genes in 28 transcription units (TUs), 11 genes in 10 TUs, and 34 genes in 25 TUs, respectively, significantly expanding the current knowledge of their regulatory networks. Comparison of them to other stress-response regulatory networks highlights minimal overlap between their regulons, indicating that E. coli has a series of relatively distinct stress responses covering the range of different stresses. We also demonstrate that these intricate networks coordinate detoxification process with DNA and protein damage repair, cell wall synthesis, divalent metal ion homeostasis, as well as metabolic robustness to produce overall response of E. coli to oxidative stress.
Project description:Oxidative stress that originates from reactive oxygen species (ROS) is an inevitable consequence of aerobic respiration in bacteria. Three transcription factors (TFs), OxyR, SoxR, and SoxS play a critical role in transcriptional regulation of the defense system. However, the full genome-wide regulatory potential of them remains elusive. Here, we comprehensively reconstruct genome-wide OxyR, SoxR, and SoxS transcriptional regulatory networks in Escherichia coli under oxidative stress. Integrative data analysis reveals that OxyR, SoxR, and SoxS regulons are comprised of 38 genes in 28 transcription units (TUs), 11 genes in 10 TUs, and 34 genes in 25 TUs, respectively, significantly expanding the current knowledge of their regulatory networks. Comparison of them to other stress-response regulatory networks highlights minimal overlap between their regulons, indicating that E. coli has a series of relatively distinct stress responses covering the range of different stresses. We also demonstrate that these intricate networks coordinate detoxification process with DNA and protein damage repair, cell wall synthesis, divalent metal ion homeostasis, as well as metabolic robustness to produce overall response of E. coli to oxidative stress. A total of six samples were analyzed. oxyR-8myc, soxR-8myc, and soxS-8myc tagged cells were cultured in M9 minimal media with 0.2% glucose. Then cells were treated with 250 uM of paraquat at mid-log pahse for 20 min with agitation.
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.