ABSTRACT: We performed Hi-C analysis of Escherichia coli MG1655 cells in exponential and stationary growth phase. We could detect long-range interactions predominantly in the Ori domain of the chromosome. Interestingly, the use of a new type of control revealed that these interactions are mostly crosslinking independent. 6 samples from exponential phase growth in M9 medium; 6 samples from stationary phase growth in M9 medium; for each growth condition, 3 samples were with crosslinking and 3 samples were without.
ORGANISM(S): Escherichia coli str. K-12 substr. MG1655
Project description:Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of glutamate decarboxylase (GAD) system, the most efficient acid resistance mechanism in E. coli. The full contribution of GadE to the acid resistance and virulence of pathogenic E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles with that of wild type in exponential and stationary phases of growth using microarrays containing 6088 ORFs from three E. coli genomes. gadE inactivation significantly altered the expression of 60 genes independent of growth phase and 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly down-regulated the expression of gadA, gadB, gadC and many acid fitness island genes in a growth phase-dependent manner. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Altogether, our data indicate that GadE is critical for acid resistance of E. coli O157:H7 and plays an important role in virulence by down-regulating expression of LEE. The results are based on O157:H7 Sakai wild type and gadE mutant exponential and stationary phase cultures grown in MOPS minimal medium. Differences in transcript levels were determined using a mixed model ANOVA in R/MAANOVA which tested for significant differences due to growth phase (exponential or stationary), strain (wild type or mutant) and the interaction of these two factors using the following linear model: array+dye+sample (biological replicate)+ phase+strain+phase*strain. We incorporated the dye-swaps among the biological replicates.
Project description:Escherichia coli O157:H7 has caused serious outbreaks of foodborne illness via transmission in a variety of food vehicles, including unpasteurized apple juice, dried salami, and spinach. To understand how this pathogen responds to the multiple stresses of the food environment, we compared global transcription patterns after exposure to apple juice. Transcriptomes of mid-exponential and stationary phase cells were evaluated after 10 minutes in model apple juice (pH3.5) using microarrays probing 4,886 ORFs. Significant changes in gene expression were determined using R/MAANOVA and the Fs test. A total of 331 ORFs were significantly induced upon exposure of cells to model apple juice and included genes involved in the acid and osmotic stress responses as well as the oxidative stress response and envelope stress. Genes involved in the acid and osmotic stress responses, including asr, osmC, osmB, and osmY were significantly induced in response to model apple juice. Genes involved in the envelope stress response, known to be controlled by CpxR (cpxP, degP, and htpX), were significantly induced 2 to 15 fold upon exposure to apple juice, independent of growth phase. Inactivation of CpxRA resulted in a significant decrease in survival of O157:H7 in model apple juice compared to the isogenic parent strain. Of the 331 ORFs induced in model apple juice, 104 are O157-specific ORFs, including those encoding type three secretion effectors espJ, espB, espM2, espL3 and espZ. By elucidating the response of O157:H7 to acidic foods, we hope to gain insights into how this pathogen is able to survive in food matrices and how exposure to foods affects subsequent transmission and virulence. Keywords: stress The results are based on O157:H7 Sakai exponential and stationary phase cultures grown in MOPS minimal medium and then exposed to model apple juice (pH 3.5, 37C) for 10 minutes. Differences in transcript levels were determined using a mixed model ANOVA in R/MAANOVA which tested for significant differences due to growth phase (exponential or stationary), treatment (MOPS or MAJ) and the interaction of these two factors using the following linear model: array+dye+sample (biological replicate)+ phase+treatment+phase*treatment. We incorporated the dye-swaps among the biological replicates.
Project description:Growth phase dependent transcriptional and translational changes of Hb. salinarum were examined. In the genome-wide transcriptome analysis, gene expression in exponential versus stationary growth phase was monitored on DNA microarrays. In the global study of translational regulation, the relative amounts of free versus polysome-bound mRNA (separated by gradient centrifugation) were quantified in exponential as well as stationary growth phase using DNA microarrays.
Project description:Growth phase dependent translational regulation of Hf. volcanii was analyzed on a genome-wide scale. The relative amounts of free versus polysome-bound mRNA (separated by gradient centrifugation) were quantified in exponential as well as stationary growth phase by the use of shotgun DNA microarrays.
Project description:Lactobacillus casei Zhang is a probiotic bacterium isolated from koumiss in Inner Mongolia of China. Gene expression dynamics of L. casei Zhang during growth in soymilk was investigated in attempt to reveal the mechanisms involved in growth stimulation for growing probiotics in. Comparison of different transcripts next to each other revealed 162 and 63 significantly induced genes in late logarithmic phase and stationary phase, of which the expression was at least 3 fold up-regulated and down-regulated, respectively. Approximately, 38.4% of the up-regulated genes were associated with amino acid transport and metabolism notably for histidine and lysine biosynthesis, followed by genes/gene clusters involved in carbohydrate transport and metabolism, lipid transport and metabolism, and inorganic ion transport and metabolism. The analysis results suggest that the stimulatory effect of soymilk-based ecosystem on the L. casei Zhang growth is more complex than amino acids or oligopeptides supply. To study gene expression dynamics of L. casei Zhang during growth in soymilk, a whole genome microarray was used to screen for differentially expressed genes when grown to lag phase, late logarithmic phase, and stationary phase.
Project description:Chromosomes are the physical realization of genetic information and thus form the basis for its reading, hindering and propagation. Here we present a high-resolution chromosomal contact map derived from a new genome-wide chromosome conformation capture approach (a simplified version of the Hi-C method) applied to Drosophila embryonic nuclei. The data show that the entire genome is linearly partitioned into well-demarcated physical domains that overlap extensively with active and repressive epigenetic marks. Chromosomal contacts are hierarchically organized between domains. Global modeling of compaction and clustering of domains show that inactive domains are condensed and confined to their chromosomal territories, while active domains reach out of the territory to form remote intra- and inter-chromosomal contacts. One pilot sample, comprising seven paired-end Illumina GA-II lanes; one deep-sequenced sample, comprising seven paired-end Illumina HiSeq lanes
Project description:In this experiment, we've examined chromatin conformation of OG2 (B6; CBA-Tg(Pou5f1-EGFP)2Mnn/J; stock number 004654) mouse stem cells cultured as described in (Shi et al., 2008), using different amounts of starting cells. We performed a modified in situ Hi-C protocol for 6 samples digested with MboI restriction enzyme having as starting material 1 million (M), 100 thousand (k), 50k, 25k, 10k or 1k cells. As well as, to 2 samples digested with HindIII restriction enzyme that had as starting material 5M or 100k cells. Traditional in situ Hi-C protocols recommend 5-10 million starting cells. The aim of the experiment was to assess the impact of decreasing the cell number on reproducibility, library complexity, chromatin structure visualization in order to adapt the method to the study of rare cell populations. Furthermore, we have characterised the 3D structure of peripheral blood mononuclear cells (PBMCs) obtained from a blood extraction from a healthy donor and from a lymph node biopsy from a DLBCL patient as a proof of concept for the suitability of Low-C for rare cell population analysis.
Project description:In this experiment we examined higher order chromatin structure during early Drosophila melanogaster development. We performed in situ Hi-C for hand-sorted non-mitotic embryos at nuclear cycle number 12, 13 and 14, and for embryos at 3-4 hours post fertilisation. During this time in development, the zygotic genome is activated and zygotic transcription is taking place for the first time. To assess the impact of transcription on chromatin structure we injected the transcription inhibitors alpha-amanitin or triptolide before zygotic genome activation and performed Hi-C and ChIP-seq for RNA Pol II. Furthermore, we used Hi-C to study genome architecture in embryos lacking the transcription factor Zelda.
Project description:Capture Hi-C (CHi-C) is a state-of-the art method for profiling chromosomal interactions involving targeted regions of interest (such as gene promoters) globally and at high resolution. Signal detection in CHi-C data involves a number of statistical challenges that are not observed when using other Hi-C-like techniques. We present a background model, and algorithms for normalisation and multiple testing that are specifically adapted to CHi-C experiments, in which many spatially dispersed regions are captured, such as in Promoter CHi-C. We implement these procedures in CHiCAGO (http://regulatorygenomicsgroup.org/chicago), an open-source package for robust interaction detection in CHi-C. We validate CHiCAGO by showing that promoter-interacting regions detected with this method are enriched for regulatory features and disease-associated SNPs. Three human CHi-C biological replicates were generated (comprising 1, 2and 3 technical replicates). Two mouse CHi-C biological replicates were generated (both comprising three technical replicates) and a mouse Hi-C dataset. The publicly available HiCUP pipeline (doi: 10.12688/f1000research.7334.1) was used to process the raw sequencing reads. This pipeline was used to map the read pairs against the mouse (mm9) and human (hg19) genomes, to filter experimental artefacts (such as circularized reads and re-ligations), and to remove duplicate reads. For the CHi-C data, the resulting BAM files were processed into CHiCAGO input files, retaining only those read pairs that mapped, at least on one end, to a captured bait. CHiCAGO then identified Hi-C restriction fragments interacting, with statistical significant, to captured baits.
Project description:Lactobacillus casei Zhang is a probiotic bacterium isolated from koumiss in Inner Mongolia of China that has been commercially used as a starter in the manufacture of dairy products. To study the gene expression profiles of L. casei Zhang during growth in milk, a whole-genome microarray was used. Compared to L. casei Zhang grown to late logarithmic phase in milk, 61 genes were significantly up-regulated (>5 fold) in stationary phase, whereas 26 genes were down-regulated. Collectively, these data showed that the majority of the identified genes was involved in carbohydrate metabolism and energy production, followed by genes involved in nucleotide metabolism, inorganic ion transport, amino acid transport and metabolism, chaperone, etc. This study demonstrates the fundamental effects of cultural conditions on the transcriptome of L. casei Zhang. Moreover, it improves the understanding of the growth and survival mechanism of the bacterium during the late stage of milk fermentation. L. casei Zhang was grown in milk for 14h (late logarithmic phase) or 18h (stationary phase). 2 biological replicates each.