Transcriptional profiling of nitrogen fixation and the role of NifA in the diazotrophic endophyte Azoarcus sp. strain BH72
Ontology highlight
ABSTRACT:
ORGANISM(S): Azoarcus sp.
SUBMITTER: 
Barbara Reinhold-Hurek
PROVIDER: E-GEOD-49913 | ArrayExpress | 2013-08-15
SECONDARY ACCESSION(S): GSE49913PRJNA215319
REPOSITORIES: GEO, ArrayExpress
Similar Datasets
Project description:Azoarcus sp. BH72 is known to express nitrogenase genes endophytically in rice seedlings in gnotobiotic culture. Availability of fixed nitrogen is one of the important signals regulating the transcription of nitrogenase genes and hence nitrogen fixing activity. NifA is the essential transcription activator of nif genes. RNA isolated from the nifA knockout mutant of strain BH72 was compared with the transcriptome of wild type under nitrogen fixing condition using a global genome wide microarray approach and the differences in the gene expression profile were monitered. RNA isolated from wild type strain BH72 and nifLA mutant strain BHLAO grown respectively under microaerobic nitrogen fixing condition with glutamate as poor nitrogen source was used for two color whole genome microarray approach
2013-08-15 | E-GEOD-49912 | ArrayExpress
Project description:Model endophyte Azoarcus sp. BH72 is known to contribute fixed nitrogen to its host Kallar grass by nitrogen fixation and also expresses nitrogenase genes endophytically in rice seedlings in gnotobiotic culture. Availability of fixed nitrogen is one of the important signals regulating the transcription of nitrogenase genes and hence nitrogen fixing activity. Therefore, we analysed global transcription in response to differences in the nitrogen source. Transcription profiles of cells grown microaerobically (0.6% oxygen) on minimal medium with nitrogen (N2-fixing) versus ammonium (combined nitrogen) were compared using a genome-wide microarray approach and differences in the gene expression profile were monitored. RNA from cells grown on nitrogen-free synthetic medium under nitrogen fixation (experiment) and combined nitrogen source as ammonium chloride (control) was used for two-color whole-genome microarray approach.
2013-08-01 | E-GEOD-49394 | ArrayExpress
Project description:Low oxygen tensions are often encountered in flooded soils of rice fields by root-associated, strictly respiratory, beta proteobacterium, Azoarcus sp. BH72 which fixes nitrogen only under microaerobic condition. In this study, genome wide oligonucleotide microarrays were used compare the global transcription profile of Azoarcus sp. BH72 under microaerobic condition with cells grown under aerobic condition, both with ammonia as sole nitrogen source. The outcome of this study will provide a better insight about the establishment of this endophyte in the microaerobic environment, probably prevailing inside of the rice root niche . RNA from cells grown under microaerobic condition with 0.3% oxygen (experiment) and aerobic condition with 21% oxygen (control), respectively was used for two color whole genome microarray approach.
2014-09-13 | E-GEOD-61387 | ArrayExpress
Project description:Azoarcus sp. BH72 is able to communicate via cell density-dependent gene regulation. Here, the impact of cell-free conditioned culture supernatants, obtained from stationary phase Azoarcus wild type cultures, on gene expression was investigated determining changes in transcript profiles when early exponential aerobic cultures were incubated with cell-free culture supernatants for one and four hours. Bacterial communication via quorum sensing (QS) is involved in the regulation of several cellular mechanisms such as metabolic processes, microbe-host interactions or biofilm formation. The nitrogen-fixing model endophyte of grasses Azoarcus sp. strain BH72 shows density-dependent gene regulation in the absence of common hydrophobic autoinducers for pilA encoding the structural protein of type IV pili that are essential for plant colonization. Here, we used a transcriptomic approach to identify target genes differentially regulated under QS conditions in conditioned supernatants in comparison to standard growth conditions. Analysis used RNA from the early exponential growth phase as control samples for comparison to the quorum-sensing condition samples taken at one hour and four hours after incubation with cell-free culture supernatants.
2011-12-01 | E-GEOD-25939 | ArrayExpress
Project description:Endophytic colonization is a very complex process which is not yet completely understood. Molecules exuded by the plants may act as signals which influence the ability of the microbe to colonize the host or survive in the rhizosphere. Here we investigated whether root exudates of the host might play a role in initiating the endophyte-rice interaction. The whole genome microarray approach was used to investigate the response of the diazotrophic model endophyte, Azoarcus sp. strain BH72, to exudates of O. sativa cv. Nipponbare in order to identify differentially regulated genes. Azoarcus sp. strain BH72 was grown in the presence or absence of root exudates of Oryza sativa cv. Nipponbare for two different time points, and differences in the gene expression profile were monitored. RNA from cells grown on synthetic medium for 1 and 4 hours respectively in presence (experiment) and absence (control) of exudates was used for two color whole genome microarray approach.
2012-11-30 | E-GEOD-35187 | ArrayExpress
Project description:Analysis of the role of the Candida dubliniensis Telomeric (TLO) genes and MED3, encoding subunits of Mediator, in regulating transcription The transcriptional response of a Candida dubliniensis TLO1/TLO2 double (null) mutant was analysed. Reintegrant strains harboring TLO1 or TLO2 were compared to this null mutant to elucidate the individual role of each ORF. The role of MED3 was compared also. 9 experiemental parameters were analysed, each carried out in triplicate or quadruplicate
2014-07-11 | E-GEOD-59113 | ArrayExpress
Project description:Aspergillus fumigatus was cultured in a chemostat for 12.5 hours, and switched to hypoxia (0.2% oxygen). Samples were collected at the beginning of the experiment, before the switch to hypoxia, and 2, 6, 12 and 24 hours after the switch. RNA was extracted and microarrays performed to compare each time point to the time the experiment was switched. There are 3 biological replicates and 2 technical replicates.
2012-03-01 | E-MEXP-3251 | ArrayExpress
Project description:Transcriptional comparisons between wild-type Streptococcus mutans UA159 and two mutant strains (∆ackA and ∆ackA pta). On growth condition in defined FMC medium, WT vs. either ∆ackA or ∆ackA pta mutants. Biological replicates: 10 control, 5 each mutant, independently grown and harvested. One replicate per array.
2013-05-11 | E-GEOD-46835 | ArrayExpress
Project description:To determine the transcriptional effects of a novel plant-based compound, dehydrobrachylaenolide, on P. falciparum, parasite cultures were treated with the compound over time. Samples were taken for analysis 2, 6, and 12 hours post-invasion of human red blood cells. Control cultures were treated simultaneously with DMSO, and samples isolated at 2, 6, and 12 hours for transcriptional analysis. Background Antimalarial drug resistance threatens to undermine efforts to eliminate this deadly disease. The resulting omnipresent requirement for drugs with novel modes of action prompted a national consortium initiative to discover new antiplasmodial agents from South African medicinal plants. One of the plants selected for investigation was Dicoma anomala subsp. gerrardii, based on its ethnomedicinal profile. Methods Standard phytochemical analysis techniques including solvent-solvent extraction, thin-layer and column chromatography, were used to isolate the main active constituent of Dicoma anomala subsp. gerrardii. The crystallised pure compound was identified using nuclear magnetic resonance spectroscopy, mass spectrometry and X-ray crystallography. The compound was tested in vitro on Plasmodium falciparum cultures using the parasite lactate dehydrogenase assay. The effects of treatment on the P. falciparum transcriptome were subsequently investigated by treating ring-stage parasites (alongside untreated controls) with the pure compound, followed by oligonucleotide microarray and data analysis. Results The main active constituent was identified as dehydrobrachylaenolide, a eudesmanolide-type sesquiterpene lactone. The compound demonstrated an in vitro IC50 of 245.6 nM, which was comparable to the IC50 of chloroquine, against a chloroquine-resistant strain (K1) of P. falciparum. Microarray data analysis identified a cluster of unique genes that were differentially expressed as a result of the treatment and gene ontology analysis identified various biological processes that were significantly affected. Comparison of the dehydrobrachylaenolide treatment transcriptional dataset with a published artesunate (also a sesquiterpene lactone) dataset revealed little overlap. This suggests differentiated modes of action between the two compounds. Conclusions Dehydrobrachylaenolide could play a valuable role as a drug candidate to generate new antimalarial compounds with novel modes of action and favourable ADMET properties. Reference design. Transcriptional analysis was performed as described in GSE18075 with the following modification. Profiles from parasite cultures isolated 2hrs after drug treatment during early ring-stage development were contrasted against untreated control cultures isolated at this timepoint. Profiles from parasites isolated 6 and 12 hours following drug treatment were contrasted to against untreated control cultures isolated at 6 hours. A detailed description of the statistical methodology used for this dataset is outlined in the accompanying manuscript. A reference design was employed for array hybridisation, utilising the URR pool described previously in the NCBI GEO Series GSE18075 dataset. All solvent-control and drug-treated samples were hybridised to Operon slides, along with the URR. In contrast to the hybridisation protocol described in GSE18075, 40 pmoles of each dye was hybridised to each array, utilising Cy3 (sample) and Cy5 (reference) dyes from GE Healthcare (#RPN5661), specifically optimised for nucleic acid labelling. A total of nineteen slides were processed in the study. Two independent cDNA samples (biological replicates) were prepared for each untreated and drug-treated sample at each time point. One of the biological replicate cDNA samples were additionally hybridised to a third slide (representing the technical replicate). In the case of the 2 hour DMSO treated control culture, an additional technical microarray replicate was included for quality control purposes. GenePix results (gpr) files were generated using GenePix 6.0 (Molecular Devices) software, without normalization. For clustering analyses, results files were normalized with DNMAD (Diagnosis and Normalization for MicroArray Data) using print-tip loess. The normalized values were subsequently downloaded and analyzed with the Multiexperiment Viewer (MeV) in the TM4 software suite. Hierarchical Clustering (HCL, average linkage) was performed to estimate technical and biological variation between samples and at which point cytostasis most likely occurred for comparative purposes in downstream analyses. Intensity data for individual slides were imported into LIMMA (linear models for microarray data) in the R computing environment. Pre- and post-normalization diagnostic plots were performed using MARRAY. Data from each microarray slide was normalized using print-tip loess. Data between microarrays was normalized using R-Quantile normalisation. Pearson correlations were computed in MS Excel to estimate variation between technical and biological replicates. Spots excluded from slide correlations and normalisation were those weighted by the limma script or flagged in the genepix results file (gpr). Additionally, spots termed Alien, Empty, Null and Operon Use Only were excluded from the correlation analyses. These spots were similarly excluded for correlations between untreated and treated samples at each time point following normalisation. Results from biological and slide replicates within each of the time points were collated, and linear models were computed to contrast gene expression between time points. A two-fold change in gene expression was used as cut-off, in conjunction with correction for false discovery (false discovery rate (FDR) = 5%). Analysis of differentially expressed genes was performed in MADIBA (Micro Array Data Interface for Biological Annotation).
2011-11-01 | E-GEOD-29874 | ArrayExpress
Project description:Transcriptional profiling of P. falciparum cultures treated with cyclohexylamine over time (18, 25 and 30 hours post invasion) A control experiment was also set up in which P. falciparum was not treated with cyclohexylamine, and samples were taken at 18, 25 and 30 h post invasion). Background Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level. Results Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels. Conclusions This study details the malaria parasite’s response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies. Keywords: Time course experiment in response to a drug treatment Reference design. All timepoints compared with t = 18 hours (untreated) post invasion. Two biological replicates and one technical sample per treatment and per time point. A reference design was employed for array hybridisation, utilising the URR pool described previously. All solvent-control and drug-treated sampled were hybridised to Operon slides, along with the URR. For each time point and each untreated/treated sample, three microarray slides were processed, such that a total of eighteen slides were processed in the study. Two independent cDNA samples (biological replicates) were prepared for each untreated and drug-treated sample at each time point. One of the biological replicate cDNA samples were additionally hybridised to a third slide (representing the technical replicate). GenePix results (gpr) files were generated using GenePix 6.0 (Molecular Devices) software, without normalization. For clustering analyses, results files were normalized with DNMAD (Diagnosis and Normalization for MicroArray Data) using print-tip loess. The normalized values were subsequently downloaded and analyzed with the Multiexperiment Viewer (MeV) in the TM4 software suite. Hierarchical Clustering (HCL, average linkage) was performed to estimate technical and biological variation between samples and at which point cytostasis most likely occurred for comparative purposes in downstream analyses. Intensity data for individual slides were imported into LIMMA (linear models for microarray data) in the R computing environment. Pre- and post-normalization diagnostic plots were performed using MARRAY. Data from each microarray slide was normalized using print-tip loess. Data between microarrays was normalized using rquantile normalisation. Pearson correlations were computed in ExCel to estimate variation between technical and biological replicates. Spots excluded from slide correlations and normalisation were those weighted by the limma script or flagged in the genepix results file (gpr). Additionally, spots termed Alien, Empty, Null and Operon Use Only were excluded from the correlation analyses. These spots were similarly excluded for correlations between untreated and treated samples at each time point following normalisation. Results from biological and slide replicates within each of the time points were collated, and linear models were computed to contrast gene expression between time points. A two-fold change in gene expression was used as cut-off, in conjunction with correction for false discovery (false discovery rate (FDR) = 5%). Normalised data was deposited in the Gene Expression Omnibus (GEO) database, number GSE18075. Analysis of differentially expressed genes was performed in MADIBA (Micro Array Data Interface for Biological Annotation).
2010-04-19 | E-GEOD-18075 | ArrayExpress