Project description:Nitrogen fixation is a highly energy-demanding process and highly regulated at multiple levels. The two major signals that regulate nitrogen fixation in most diazotrophs are oxygen and ammonia. In order to study the complex regulated mechanism and to highlight the complete nitrogen fixing system in genome level, here we present the transcriptional profiles of the nitrogen fixation genes of P.stutzeri A1501 in different growth conditions with a genome-wide DNA microarray. In this study, the three samples of "P.stutzeri A1501 treated with 0.1mM ammonia and 0.5% Oxygen tension","P.stutzeri A1501 treated with 0.1mM ammonia and 0.5% Oxygen tension-2" and "P.stutzeri A1501 treated with 0.1mM ammonia and 0.5% Oxygen tension-3" were three repeat experiments, while, the other three samples of "P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-1", "P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-2" and "P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-3" were three repeat experiments. The gene expressions under these two growth phases were compared to investigate which genes' expression were effected by different ammonia concentrations. Keywords: nitrogen fixation, nitrogen repression P.stutzeri whole genome microarray, which was based on our sequenced strain A1501. The microarray used in this study featured 3988 of the 4145 ORFs identified in P.stutzeri strain A1501, because 157 ORFs were not on the microarray of the inability to amplify these products. ORFs were amplified with specific primer pairs (Invitrogen). To ensure that the elements of our array would detect specifically their corresponding genes and no others, the ORF coordinates fed into the primer program were circumscribed such that they would exclude regions of any ORF that contained significant similarity to any other ORF. Agarose gel electrophoresis was used to perform quality control on all PCR products. Oligonucleotides were removed from the PCR mix by PCR 96 cleanup plate (Millipore). DNA was resuspended in 12 Î¼l of spotting solution containing 50% dimethyl sulfoxide. PCR products were spotted onto gamma amino propylsilan coated GAPII slides (Corning) with a OmniGrid â¢ microarrayer (GeneMachines).The negative control genes were from the type III secretion system genes of shigella flexneri 2a and the commercial Arabidopsis genes (SpotReportâ¢ cDNA Array Validation System, Stratagene). The positive control genes were from the 16S rRNA of P.stutzeri A1501. We used Cy3-labeled genomic DNA and Cy5 dye-labeled cDNA, for hybridization. The cDNA was synthesized by reverse transcription of 5 Î¼g of total RNA. The labeled cDNA and genomic DNA samples were mixed and hybridized at 65â for 16 hours. Genomic DNA was used as a universal internal control for the quality of the microarray and also allowed for the comparison of results across multiple experiments. Each experiment was repeated three times from the beginning of inoculation.

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.

Project description:Background: Glucocorticoids are important pharmaceutical agents in the treatment of acute lymphoblastic leukemia in children. Resistance or sensitivity to glucocorticoids is considered to be of crucial importance for disease prognosis. Prednisolone is a first-line chemotherapeutic agent in the treatment of acute lymphoblastic leukemia. Here, the effects of prednisolone on the resistant CCRF-CEM leukemic cell line were studied. Methods: Prednisolone’s cytotoxic and cell cycle effects were studied with flow cytometry. NF-κB translocation was studied with Western Blotting and differential gene expression was studied with cDNA microarrays. Results: Prednisolone exerted a delayed biphasic effect, necrotic at low doses and apoptotic at higher doses. At low doses, prednisolone exerted a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolone’s mitogenic and cell death effects were counterbalanced. NF-κB was constitutively present in the nucleus. Early gene microarray analysis revealed 40 differentially expressed genes upon 4 hours of prednisolone’s exposure. Notable differences in gene regulation were observed between the lowest and the highest glucocorticoid doses. Prednisolone activated genes related to apoptosis/tumor suppression, cell cycle progression, metabolism and intra-/ extra-cellular signaling pathways. Conclusions: The mitogenic/biphasic effects of prednisolone are of clinical importance in the case of resistant leukemic cells. This approach might lead to the identification of gene candidates for future molecular drug targets in combination therapy with glucocorticoids, along with early markers for glucocorticoid resistance. Elucidation of the mechanisms of GC action may lead to identification of gene targets responsible for GC resistance. Key tools in this process are high-throughput technologies such as microarray-based gene expression analysis. For this purpose, the parental CCRF-CEM cell line was chosen as the system of study for the effects of prednisolone treatment. This is a T-cell leukemia cell line characterized by a mutation (L753F) on one GR gene allele that impairs ligand binding (Thompson and Johnson 2003). It is known that both the DNA and ligand binding domains of the GR are required in order to repress NF-κB transactivation (Wissink, van Heerde et al. 1997). Interestingly, concerning the question whether this mutation would affect GC resistance, it has been reported previously that both the GC-resistant as well as the GC-sensitive CCRF-CEM subclones express heterogeneous populations of the GR (GRwt/GRL753F) (Palmer and Harmon 1991; Powers, Hillmann et al. 1993). The CCRF-CEM cell line has been reported to be resistant to GCs, presumably due to the accumulation of more resistant variants after long periods of prolonged culture (Norman and Thompson 1977). It is possible that these cells are clonally inhomogeneous, as possibly the cells obtained in vivo by patients. Moreover, the large number of the CCRF-CEM subclone studies in the literature makes it difficult to choose an appropriate resistant cell model. In addition, the utilization of an in vitro system for this study offered reproducibility, an opportunity to closely examine intracellular signals and avoid interference from other in vivo-participating systems. Thus, the cell line used for this study was considered to be useful in studying GC action and resistance in leukemic cells. The aim of this work was to determine the cytotoxic, cell cycle phase distribution and early cancer-specific gene expression effects of prednisolone in CCRF-CEM cells, as an in vitro model of ALL resistance to glucocorticoids. The early gene expression profile allowed identification of genes initiating pivotal, early onset regulatory mechanisms activated by GC and excluded ensuing feedback responses and further downstream signals. Samples tested were in the form of a loop-design i.e. control vs. 10nM prednisolone (designated 0vs1) , 10nM prednisolone vs.700uM prednisolone (designated 1vs3) and control vs. 700uM prednisolone (designated 0vs3), the sum of the logarithms of the first two should equal the third. In other words if Rj,i is the ratio of the ith gene in the jth experiment then R0vs1,i+R1vs3,i=R0vs3,i. We tested this using a statistical test. We have applied an intensity-dependent z-score where the sum of the ratios was compared to the ratio of the third experiment. If the difference was significant genes, in a standard deviation threshold of ±1.5 in relative units, were rejected from further analysis (Kerr and Churchill 2001; Kerr and Churchill 2001; Altman and Hua 2006; Kerr and Churchill 2007).

Project description:Escherichia coli O157 presents a number of specific problems in terms of food safety and public health. It has been found that E. coli O157 is more resistant to a number of the stresses encountered during food production such as heat, pH and osmotic shock. This greater resistance is thought to contribute to the low infectious dose of E. coli O157 (<100 organisms). Moreover, E. coli O157 is associated with debilitating conditions such as haemorrhagic colitis and haemoytic uraemic syndrome, particularly in children and the elderly. We have been studying the stress responses of E. coli O157:H7 (Sakai) and comparing with a commensal strain of E. coli K-12, MG1655. We found that E. coli O157 (Sakai) is more sensitive to oxidative stress than MG1655. A microarray study of these strains treated with sub-lethal concentrations (0.5mg/ml) of menadione revealed big differences in their responses. In E. coli O157 (Sakai), 540 genes responded significantly to the treatment compared to 121 genes in MG1655. One surprising finding from the microarray data was the observation that many iron-transport genes were up-regulated in E. coli O157 (Sakai) whereas relatively few were induced in MG1655 despite the fact that the bacteria were grown in a medium containing ample iron. We speculated that the induction of iron transport genes in an iron-rich medium might have contributed to the enhanced killing of E. coli O157 (Sakai) through triggering of a Fenton reaction. We speculated that the difference in sensitivity to oxidative stress might be due to differences in the intracellular iron content of E. coli O157 and MG1655. We found that E. coli O157 contains ~50% more iron than MG1655 and believe that during oxidative stress, this iron is released by damaged proteins. The greater levels of free iron in E. coli O157 will trigger a greater Fenton reaction that can damage the ferric uptake regulator (Fur), resulting in unregulated iron transport. In MG1655, the lower iron content results in a smaller Fenton reaction, enabling the cellular protection systems to limit damage and protect Fur. Overnight cultures of E. coli O157 (Sakai) and E. coli K-12 MG1655 were grown in Neidhardt's EZ Rich Defined Medium and diluted 1:100 in 50 ml fresh medium in 125 ml Ehrlenmeyer flasks. The cultures were shaken at 37C until the optical density (OD600) reached 0.4. Each culture was divided into 2 equal parts in identical flasks. One flask contained menadione bisulphite to a final concentration of 0.5 mg/ml; the other flask contained an equivalent volume of distilled water. The flasks were shaken for a further 10 mins and then treated with RNAprotect™ to stabilise the mRNA. The experiment was performed 3 times on different days. Six custom-made microarray slides were used in this study; each slide was hybridised with labelled cDNA made from mRNA taken from untreated and treated E. coli O157 (Sakai) or MG1655.

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Transcriptional profile of EHEC O157 TUV93-0 overexpressing PsrA or PsrB

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