Comparative transcriptome analysis of Brucella melitensis 16M and its hfq mutant under acidified minimum medium (GEM4.0) conditions
Ontology highlight
ABSTRACT:
ORGANISM(S): Brucella melitensis
SUBMITTER: tai 2006
PROVIDER: E-GEOD-46418 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Quorum sensing system (QS) is a global regulator that regulates gene expression in response to cell density and other environmental factors. Two QS genes have been identified in Brucella, one of which (vjbR) is involved in Brucella intracellular survival and virulence. But the regulation mechanism of Brucella intracellular survival by the vjbR remains unknown.To characterize the regulation roles of vjbR, the in vitro vjbR induction conditions simulating intracellular environments were firstly defined, and then, transcriptome of the vjbR mutant is compared to that of the wild type strain. The vjbR gene was highly activated under acidified nutrition limitation condition. Comparative transcriptome analysis showed that a total of 126 genes were greatly differentially expressed in the vjbR mutant. 2 wild type samples and 2 vjbR mutant samples were analyzed.
2010-10-27 | E-GEOD-24942 | biostudies-arrayexpress
Project description:For a pathogen such as Streptococcus suis serotype 2, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analysis of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we used analysis of the global expression profile in response to acidic pH in vitro and identified a set of regulated genes involved in diverse cellular processes. 196 (11%) genes were differentially regulated by the acid stress: 92 (47%) were down-regulated at low acid (pH 5.8) relative to the neutral condition (pH 7.2), whereas 104 (53%) were up-regulated at pH 5.8 versus pH 7.2. To confirm the microarray data, 16 genes were measured by quantitative RT-PCR. There was a strong positive correlation (r = 0.96) between the results obtained by microarray and quantitative RT-PCR. The data showed that S. suis S2 is equally capable of inducing an acid tolerance response with maximal protection provided after adaptation at pH 5.8 for survival. A cDNA microarray imprinted with 2156 genes representing about 98% of the Streptococcus suis serotype 2 genome was used for transcriptome analysis. For two-sample (reference vs. test) microarray hybridization, four independent bacterial cultures from each condition (pH 5.8, pH 7.2) were prepared as biological replicates for RNA isolation. Accordingly, four dual-fluorescence-labeled cDNA probes were prepared to hybridize with four slides. Pairwise comparisons were made using dye-swaps to avoid labeling bias. A ratio of mRNA levels (test/reference) was calculated for each gene. Significant changes of gene expression were identified with the SAM software. After the SAM analysis, only genes with at least 2-fold changes in expression were collected for further analysis.
2011-09-12 | E-GEOD-21295 | biostudies-arrayexpress
Project description:The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis under zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2. The wild-type (WT) Y. pestis strain 201 belongs to a newly established Y. pestis biovar, Microtus, which was thought to be avirulent to humans, but highly virulent to mice. An in-frame deletion of the zur gene was constructed by using one step inactivation method based on the lambda phage recombination in which PCR primers provide the homology to the target gene, as described previously by Datsenko and Wanner. The entire coding region of zur was replaced by a kanamycin resistance (KnR) cassette, which was verified by PCR and DNA sequencing. The resulting mutant strain was referred to as Îzur. Both the WT strain and the Zur mutant were pre-cultivated at 26 ºC to the middle exponential growth phase (OD620 about 1.0) in TMH medium. The cell cultures were then diluted 1:20 in fresh TMH medium and grown at 26°C until an OD620 of about 1.0. Finally, 5mM ZnCl2 was added into each cell culture to ensure zinc rich conditions. Growth was continued for 30 min at 26°C before harvested for total RNA isolation. Gene expression profiles were compared between WT and Îzur. RNA samples were isolated from four individual bacterial cultures, as biological replicates, for each strain. The dual-fluorescently (Cy3 or Cy5 dye) labeled cDNA probes, for which the incorporated dye was reversed, were synthesized from the RNA samples, and then hybridized to four separated microarray slides, respectively.
2010-05-18 | E-GEOD-15183 | biostudies-arrayexpress
Project description:Glucose-lysine based Maillard reaction products (MRPs) are introduced to the human body via food ingestion or are synthesized in vivo. Depending on reaction conditions, they represent a mixture of compounds with diverse chemical structures and physiological properties. MRPs may also be a potential nutrient source for Salmonella, a major foodborne gastrointestinal pathogen. This study was designed to determine the growth and transcriptional responses of S. Typhimurium LT2 to MRPs generated at low water activity to simulate the reaction occurring during food processing and storage. Maillard reactions between N-α-acetyl-lysine and glucose were varied in time (4, 23, and 143 h) to generate sub-samples with prevailing Amadori compound, advanced glycation end products (AGEs), or melanoidines respectively. The addition of MRP up to 5 mg mL-1 to defined media with 0.4% glucose had no effect on S. Typhimurium LT2 growth. In media, where the MRP sub-samples (4 and 23 h) served as the only carbon source, MRP assimilation by S. Typhimurium LT2 was observed as secondary logarithmic growth phases (0.063 h-1 and 0.072 h-1) after the growth on glucose available in the MRP reaction mixtures (0.479 h-1). The MRPs sub-sample with the highest concentration of melanoidines (143 h) also supported S. Typhimurium LT2 growth (0.368 h-1). Of the three MRPs sub-samples, the Amadori compound was the preferred carbon source for S. Typhimurium LT2 as evidenced by its almost complete disappearance (98%). Decreases in AGEs (37%) and melanoidines (15%), when incubated with S. Typhimurium LT2, also occurred. Transcription profiles of the cells grown on the MRPs fractions revealed predominant up-regulation of genes associated with the functional groups of energy metabolism, fatty and phospholipid metabolism, cellular process, and regulatory functions, and general down-regulation of the genes in the groups of amino acid biosynthesis, protein synthesis and transcription, transport and binding proteins, and DNA metabolism. The carbon flow in tricarboxylic acid (TCA) cycle partitioned by the glyoxylate cycle appeared to play an essential role in the assimilation of glucose-lysine-derived MRPs. The high expression level of numerous genes encoding hypothetical proteins or proteins with unknown function suggested the presence of genes whose role in glucose-lysine MRPs catabolism in Salmonella remains to be determined. This study was designed to determine the growth and transcriptional responses of S. Typhimurium LT2 to MRPs generated at low water activity to simulate the reaction occurring during food processing and storage. Maillard reactions between N-α-acetyl-lysine and glucose were varied in time (4, 23, and 143 h) to generate sub-samples with prevailing Amadori compound, advanced glycation end products (AGEs), or melanoidines respectively. Total bacterial RNA was isolated as previously described and the RNA samples were then converted to fluorescently-labeled cDNA and hybridized to S. Typhimurium microarrays version 5 (NIAID's Pathogen Functional Genomics Resource Center). Real-time PCR and physiological experiments were performed to validate the microarray data. In all supplementary files channel A = Cy3, channel B = Cy5.
2010-05-16 | E-GEOD-18950 | biostudies-arrayexpress
Project description:In the present study we have determined the global gene expression and biomolecular composition in an Escherichia coli model strain exposed to ten adverse conditions (sodium chloride, ethanol, glycerol, two acids (hydrochloric acid and acetic acid), sodium hydroxide, heat (46°C) and cold (15°C) as well as ethidium bromide and the disinfectant benzalkonium chloride). The large variation in responses and few common genes illustrates the adaptation potential of E. coli and its ability to survive and colonize a wide range of environments. Keywords: gene expression study, stress response The strain used in this study was the genome-sequenced strain E. coli K-12 MG1655 (1) and the following concentrations/conditions were used; CH3COOH (pH 5.9 ± 0.05); HCl (pH 4.75 ± 0.05); NaOH (pH 9.6 ± 0.05); EtOH (5%); NaCl (4.5%); Glycerol (15%); BC (7 and 9 mg/ml); EtBr (150 mg/ml); 46°C; 15°C. The cultures were prepared by inoculating one colony from tryptone soya agar (TSA) (Oxoid) (overnight growth at 37°C) to 5 ml TSB and incubating overnight at 37°C, with shaking (200 rpm). This culture was initially diluted 1:10 in medium and used to inoculate room-temperature TSB for each of the different stress factors (40 ml total volume) to a final concentration of approx. 1X10^7 CFU/ml (1:100 dilution of overnight culture). For cold stress experiment the medium was chilled to 15°C prior to inoculation. The cultures were incubated at 37°C unless otherwise stated (Table 1), shaking at 200 rpm and samples for global gene expression and biomolecular analyses were collected during exponential growth at a cell density of approx. 1X10^7 CFU/ml. All the stress conditions, including the control, were inoculated with the same overnight culture and started at the same time point. The experiment was performed 3 times at different days and with freshly prepared solutions, resulting in 3 biological replicates. Total RNA was extracted from 1 ml cells using the RNeasy Protect Bacteria Mini Prep kit (Qiagen) as recommended by the manufacturer including the "on-column" DNase treatment. The concentration and purity of the total RNA was analyzed using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.) and the RNA 600 Nano LabChip system (Agilent Technologies). Total RNA (5 ug) and 0.5 ul test/reference Lucidea spike controls RNA (Amersham Biosciences) were reverse transcribed by using random hexamers in the presence of aminoallyl-dUTP (2 aa-dUTP (Ambion): 3 dTTP (Invitrogen)) as described in standard operating procedure no. M007 (The Institute for Genomic Research [TIGR] protocol). The removal of unincorporated aa-dUTP and free amines was performed according to the TIGR protocol using the Qiagen QIAquick PCR purification kit. After drying in a Speed vac, the samples were stored at -20°C. Coupling aminoallyl-labeled cDNA to Cy3/Cy5 mono reactive dyes (Amersham Biosciences) was done according to the TIGR protocol. The E. coli microarrays consisted of E. coli oligos (Operon) printed on Corning Ultra GAPS slides as described previously (2, 6, 7). Microarray slides were treated with a pre-soak solution (Promega/Corning) before incubation in a pre-hybridization solution (5XSSC [1XSSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% dodecyl sulfate [SDS], 1 mg/ml bovine serum albumin) at 42°C for 30-60 minutes. After pre-hybridization, the slides were washed briefly in distilled water, followed by a brief wash in isopropanol. The dried Cy3- and Cy5-labeled cDNA was resuspended in 55 ul hybridization solution (30% formamide, 5XSSC, 0.1%SDS and 0.1 mg/ml Salmon Sperm DNA), denatured and applied to the prehybridized microarray underneath a 25- by 60-mm mSeries LifterSlip coverslip (Erie Scientific Company). The slides were hybridized at 42°C overnight (~16 h). After hybridization the slides were washed twice in 2XSSC buffer with 0.1% SDS at 42°C for 5 minutes, followed by two washes in 0.1XSSC buffer with 0.1% SDS for 10 min at room temperature and four washes in 0.1X SSC buffer for 1 minute at room temperature. The slides were dried by centrifugation (90xg for 12 min). The reference used in the microarray experiments was pooled RNA from all the growth conditions including the control. Slides were scanned with the ScanArray Express 1.0 scanner (Packard BioScience) following the manufacturers guidelines. The fluorescent spot intensities were quantified using ImaGene 5.6.1 (BioDiscovery Inc.) software. Background subtraction and normalization (LOWESS (3, 5)) was performed in GeneSpring 7 (Silicon Genetics). (A lowess curve was fit to the log-intensity versus log-ratio plot. 20% of the data was used to calculate the lowess fit at each point. The curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then the value was set to 10). Only data from spots representing E. coli K-12 MG1655 genes were analyzed in our studies. All analyses were based on 3 biological replicates with the exception of the second biological replicate from conditions HCl and NaOH. These two observations were removed due to bad hybridization giving a total of 34 array hybridizations (observations). Genes not present in any of the 34 observations were filtered out, resulting in the analysis of 4279 out of 4289 MG1655 genes on the array. Missing values (data points) were replaced by using the KNNimpute procedure (4) (k=10) on log2 transformed data. 1. Blattner, F. R., G. Plunkett, 3rd, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453-74. 2. Constantinidou, C., J. L. Hobman, L. Griffiths, M. D. Patel, C. W. Penn, J. A. Cole, and T. W. Overton. 2006. A reassessment of the FNR regulon and transcriptomic analysis of the effects of nitrate, nitrite, NarXL, and NarQP as Escherichia coli K12 adapts from aerobic to anaerobic growth. J Biol Chem 281:4802-15. 3. Quackenbush, J. 2002. Microarray data normalization and transformation. Nat Genet 32 Suppl:496-501. 4. Troyanskaya, O., M. Cantor, G. Sherlock, P. Brown, T. Hastie, R. Tibshirani, D. Botstein, and R. B. Altman. 2001. Missing value estimation methods for DNA microarrays. Bioinformatics 17:520-5. 5. Yang, Y. H., S. Dudoit, P. Luu, D. M. Lin, V. Peng, J. Ngai, and T. P. Speed. 2002. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30:e15. 6. Zhang, L., R. R. Chaudhuri, C. Constantinidou, J. L. Hobman, M. D. Patel, A. C. Jones, D. Sarti, A. J. Roe, I. Vlisidou, R. K. Shaw, F. Falciani, M. P. Stevens, D. L. Gally, S. Knutton, G. Frankel, C. W. Penn, and M. J. Pallen. 2004. Regulators encoded in the Escherichia coli type III secretion system 2 gene cluster influence expression of genes within the locus for enterocyte effacement in enterohemorrhagic E. coli O157:H7. Infect Immun 72:7282-93. 7. Zheng, D., C. Constantinidou, J. L. Hobman, and S. D. Minchin. 2004. Identification of the CRP regulon using in vitro and in vivo transcriptional profiling. Nucleic Acids Res 32:5874-93.
2010-05-18 | E-GEOD-11041 | biostudies-arrayexpress
Project description:Hfq is a bacterial Sm-like protein which serves as a post-transcriptional regulator of global gene expression. It has been shown to be involved in a wide range of cellular processes and pathways. To characterize the regulation roles of Hfq in Brucella melitensis, the in vitro hfq induction conditions simulating intracellular environments were firstly defined, and then, the transcriptome of the hfq mutant was compared to that of the wild type strain. Comparative transcriptome analysis showed that a total of 359 genes showed a>2.0-fold change in transcript abundance in Δhfq when compared to that in 16M.
2013-04-27 | GSE46418 | GEO
Project description:Transcriptional profiling of K. pneumoniae comparing the wild type strain (S5) with a hfq deletion mutant (H0201). Goal was to determine the effects of Hfq absence on global K. pneumoniae gene expression. WT vs. Hfq mutant. Eight replicates for each ORF presented in the 44K array.
2013-01-11 | E-GEOD-29448 | biostudies-arrayexpress
Project description:To gain novel insights into the molecular mechanisms underlying Pima cotton fiber quality, expression profiling of the Pima fiber transcriptome was performed as a function of development. The profiling was performed at 6 developmental time points: 8, 11, 14, 17, 21, and 24dpa. The expression profiling using long oligonucleotide microarray showed dynamic changes to the fiber transcriptome that correlate to morphogenesis, and biological cellular processes. Differentially expressed genes were identified by being differentially regulated at various stages of fiber development, and they were used to develop stage-specific regulation patterns that correlate the dynamics of gene expression to fiber morphogenesis. A bi-directional double loop hybridization (Kerr and Churchill, 2001) with dye swap experimental design encompassing a total of 28 hybridizations was employed to maximize discovery of significant changes between developmental stages. Self-hybridization controls were conducted to evaluate both within-array correlation and dye bias.
2010-05-26 | E-GEOD-11689 | biostudies-arrayexpress
Project description:The study searched for the correlates of vaccine protection against furunculosis. After pathogen challenge of vaccinated (V) and unvaccinated (N) fish, hepatic gene expression was compared in salmon with high resistance (HR, individual samples) and low resistance (LR, pooled samples) Two-condition experiment - vaccinated and unvaccinated salmon, HR vs. LR livers. Biological replicates (HR): 6 vaccinated, 5 unvaccinated, pooled samples of LR were used as reference. One replicate per array.
2010-05-05 | E-GEOD-18120 | biostudies-arrayexpress
Project description:As production of virulence factors by Pseudomonas aeruginosa is influenced by the host environment, we examined the effect of 10% adult bovine serum (ABS) on the global transcription with P. aeruginoas PAO1 at different phases of growth. At early exponential phase (4 h), serum significantly enhanced expression of 138 genes, most of which are repressed by iron and carry binding sequences for the ferric uptake regulator or the Fur-regulated extracytoplasmic function sigma factor PvdS. The expression of another 40 genes was reduced at 4h. However, the expression of only a few genes was significantly altered (reduced) at the early stationary phase of growth (8 h). Transcriptional fusion analyses confirmed serum enhances the expression of toxA, regA, and their regulatory genes regA and pvdS, yet does not interfere with the repression of these genes by iron. The P. aeruginosa strain PAO1 was grown in the iron-limited medium TSB-DC with or without 10% (v/v) ABS. The presence of 10% ABS reduced PAO1 growth, but despite this reduction, the cultures reached early exponential phase at 4 h and early stationary phase at 8 h in both media. Accordingly, we determined the PAO1 transcriptome in both media at these two specific time points. To standardize the comprison, we adjusted the cultures to the same optical density at 600 nm (biomass) by diluting with fresh TSB-DC PAO1 grown in TSB-DC to the same OD as PAO1 grown in TSB-DC/ABS. We obtained the transcriptome profile of 5,552 genes representing the complete P. aeruginosa PAO1 genome.
2012-07-08 | E-GEOD-30698 | biostudies-arrayexpress