ABSTRACT: The aim of this study is to investigate the transcriptional response of S. Typhimurium to heat, osmotic, oxidative and acid stress under anoxic and oxic conditions and to non-stressed anoxic conditions.
Project description:The purpose of this study was to examine how Mtb integrates acidic pH and available carbon sources as environmental cues to regulate its metabolism and growth rate. RNA-seq transcriptional profiling of M. tuberculosis growing at acidic or neutral pH, in pyruvate or glycerol, was examined. These studies identified carbon source-dependent and -independent pH-dependent adaptations. Mtb strain CDC1551 was grown in standing T-75 flasks in 40 mL of medium seeded an initial OD of 0.1. We examined medium in four conditions pH 7.0 10 mM glycerol, pH 5.7 10 mM glycerol, pH 7.0 10 mM pyruvate, pH 5.7 10 mM pyruvate. Following 3 days of incubation at 37C, RNA was isolated from the bacterial cultures and used for RNA-seq.
Project description:We sought to determine the effect of TbPARN-1 overexpression on global mRNA steady-state levels in T. brucei. The mRNA expression profile of tet-induced TbPARN-1 OvEx procyclic cells was compared to the mRNA profile of control cells (expressing tet-induced TAP tag alone) by microarray analysis. Since overexpression of TbPARN-1 showed increased deadenylation activity in cytoplasmic extracts, overexpressing PARN-1 in vivo should result in more rapid deadenylation and decay of mRNAs targeted by PARN-1. Thus, mRNAs with lower steady state levels in PARN-1 OvEx cells can be considered likely targets for PARN-1-dependent deadenylation. Three different clones of the PARN-1 OvEx and Control cell lines were used for analysis. Each experiment was run in duplicate, with the dye-labeling reversed between duplicates. The T. brucei version 3 microarray chip (PFGRC) was used for this experiment. Each gene on the microarray was represented in duplicate, so for each experiment, mRNA levels were obtained from both points and averaged. As a control between arrays, we used a control probe set obtained from PFGRC. "The probe set consists of Cy3 and Cy5 end labeled 40-mer probes that are complementary to a set of 500 Arabidopsis thaliana 70-mer targets on PFGRC DNA microarrays. The UMSS is supplied as a ready to use mix of the Cy3 and Cy5 labeled oligonucleotides. A small aliquot is spiked into Cy-dye labeled nucleic acid samples just prior to hybridization on PFGRC microarrays. The UMSS produces a set of reference fluorescent signal intensities and ratios that are generated independent of the user-specific experimental samples. Since PFGRC intentionally targeted a heavy representation of Cy3 and Cy5 signals in the mid-range of detection, the Cy3 and Cy5 signal intensities and ratios generated by the UMSS should be highly reproducible. These signals serve as a reference point to judge the success of microarray experiments, and troubleshoot potential problems."
Project description:Transcriptional profiling of Myxococcus xanthus comparing wild-type and mutant gene expression at different developmental time points The mutants in our study, nla4, nla18, nla6 and nla28 are strains that carry a mutation in the corresponding genes nla4 (MXAN2516), nla18 (MXAN3692), nla6(MXAN4042), and nla28(MXAN1167). (MXAN numbers are the conventional gene names; nlas are common names). All four of these genes are response regulators of the ntrC family of transcriptional activators. The mutations were made by insertion of a plasmid (pCR2.1TOPO, invitrogen)into the particular activator gene. Plasmids used carry a region of homology (usually 350-500 bp in length) to the sequence of the activator gene coresponding to the highly conserved central domain that is characteristic of these ntrC family of activators. Plasmids were inserted into the chromosome via homologous recombination at the site of homology to the activator gene. These for genes were originally identified because their mutants have phenotypes with strong defects during development. In all four cases, a mutation in the particular gene gives a phenotype with incomplete or abolished development. In this study, the effects of these mutants were examined on developmental gene expression. Two condition experiment, normal wild-type vs mutant
Project description:Salmonella Enteritidis is the major food-borne pathogen primarily causing human infection through contaminated chicken meat and eggs. We recently demonstrated that S. Enteritidis strains from poultry differ in their ability to invade human intestinal cells and cause disease in orally challenged mice. Here we hypothesized that the differential pathogenicity of S. Enteritidis strains is due to the differential fitness in the adverse environments that may be encountered during infection in the host. The response of a panel of six S. Enteritidis strains to acid stress, oxidative stress, survival in egg albumen and the ability to cause infection in chickens were analyzed. This analysis allowed classification of strains into two categories: stress- sensitive and stress- resistant, with the former showing significantly (P<0.05) reduced survival in acidic (gastric phase of infection) and oxidative (intestinal and systemic phase of infection) stress. Stress-sensitive strains also showed impaired intestinal colonization and systemic dissemination in orally inoculated chickens and failed to survive/grow in egg albumen. Comparative genomic hybridization microarray analysis revealed no differences at the discriminatory level of the whole gene content between stress-sensitive and stress-resistant strains. However, sequencing of rpoS, a stress-regulatory gene, revealed that one of the three stress-sensitive strains carried an insertion mutation in the rpoS resulting in truncation of σS. Finding that one of the stress-sensitive strains carried an easily identifiable small polymorphism within a stress-response gene suggests that the other strains may also have small polymorphisms elsewhere in the genome, which likely impact regulation of stress or virulence associated genes in some manner. To determine if the differential stress response and virulence observed in this study could be due to the differences in the gene content between different strains, we compared the genomes of all the strains using CGH microarray designed based on the genomic information available for the sequenced strain. All strains (n=6) were hybridized in triplicate.
Project description:Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes (Lm) expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500nt) isolated from extracellularly growing bacteria and from Lm-infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here we report on the discovery of 150 regulatory RNAs of which 71 have never been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by Northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50 for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNAs and the absence of sRNA loci in genomes of naturally-occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.
Project description:L. monocytogenes EGD-e (Glaser et al., 2001) was grown in Brain Hearth Infusion Broth (Difco) with shaking (200 rpm, New Brunswick C24KC) using 10 ml culture medium in 100 ml conical flasks. An exponentially growing culture was used to inoculate 100 ml of fresh pre-warmed BHI broth in a 500 ml flask. This culture was incubated at 48°C (GFL Type 1083, 60 % shaking speed) and samples for RNA extraction were taken after 3, 10, 20, and 40 min.
Project description:We infected wild type L. monocytogenes EGD-e (1) and its isogenic deltahlydeltaplcA (2) (lacking the ability to breach the vacuolar compartment of host cells following uptake) mutant strain to human intestinal epithelial cell line (Caco-2) with an MOI of 100 and 500 respectively. Bacterial total RNA was isolated at 1 h (deltahlydeltaplcA) and 4 h (EGD-e) post infection, reverse transcribed, hybridised to whole genome microarray and microarray data was analysed as described previously (3) 1. Glaser et al. 2001. Comparative genomics of Listeria species. Science 294:849-852. 2. Paschen et al. 2000. Human dendritic cells infected by Listeria monocytogenes: induction of maturation, requirements for phagolysosomal escape and antigen presentation capacity. Eur.J.Immunol. 30:3447-3456. 3. Chatterjee et al. 2006. Intracellular gene expression profile of Listeria monocytogenes. Infect.Immun. 74:1323-1338.
Project description:A screening process was used to determine the aciduric capacities of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at pH 5.0 in the presence 4 different organic acids. Food and clinical isolates tended to be more resistant but strain genetic groupings were found to be only weakly linked to sodium diacetate (SDA)-resistance. Representative and comparatively aciduric food isolates FW04/0023 and FW04/0025 were found to accumulate reduced levels of acetate anion and K+ ion during growth in the presence of SDA, compared to more acid sensitive reference strains EGD (ATCC BAA-739) and ATCC 19111. The aciduric nature of FW04/0023 and FW04/0025 was also reflected by their comparatively high tolerance to pH 2.4 acid challenge; a property boosted by the presence of SDA, and growth-phase dependent acid tolerance. Transcriptomic analyses revealed increased levels of SDA did not activate or promulgate the response of any of the known acid protective mechanisms, except for acetoin biosynthesis. Elevated levels of unprotonated SDA (20 mM SDA at pH 5.0) was found to have broad effects on gene expression that could be differentiated from effects caused by mildly acidic conditions (pH 5.0) and substantial strain variation was also found. Collated SDA mainly effect genes associated with virulence, cell wall processes, DNA repair, and cofactor and lipid biosynthesis. Strain FW04/0025 was more responsive to elevated unprotonated SDA increasing the expression of 222 genes (>2-fold change, p<0.05), compared to 112 genes for strain EGD. Key differences between the strains in relation to SDA-enhanced transcript abundance in FW04/0023 were primarily associated with the cell wall, oxidative stress management, intermediary metabolism, and several hypothetical proteins. This complement of different responses could potentially alter phenotypes resulting in different cell envelope properties and metabolic properties that effect accumulation of acetate anion. The data demonstrates that amongst different L. monocytogenes strains acetate has differing effects that may ultimately influence growth efficiency under stressful conditions relevant to acidic foods and the gastrointestinal environment. The microarray component of the experiments had the aim of determining: i) To examine the affect of elevated levels of unprotonated sodium diacetate (21 mM sodium diacetate added to cultures at pH 5.0, resulting in ~7.7 mM unprotonated acetate) compared to mild acid stress at pH 5.0 in which lower levels of acetate accumulates through natural end-product formation. ii) To examine the gene expression responses of two L. monocytogenes strains that had different intrinsic acid resistances, including an organic acid resistant strain FW04/0025 and a more sensitive reference strain EGD on which genome the microarray oligonucleotide set is based. Two to four biologically replicated control cultures were labelled with Cy3 for each treatment sample with two sets of experimental conditions investigated for two different L. monocytogenes strains . Condition 1) â Mildly acidic stress: Control culture (Cy3-labelled RNA) - strains were grown to early- mid exponential growth phase at 25°C (in a shaking water bath) in brain-heart infusion broth at pH 7.3. Test cultures (Cy5-labelled RNA) â strains grown to early- mid exponential growth at 25°C phase in BHI broth adjusted to pH 5.0 (in a shaking water bath). Condition 2) â Organic acid (sodium diacetate) stress: Control culture (Cy3-labelled RNA) - strains were grown to exponential growth phase at 25°C (in a shaking water bath) in BHI broth at pH 5.0. Test cultures (Cy5-labelled RNA) â strains grown to exponential growth at 25°C phase in BHI broth adjusted to pH 5.0 and amended with 21 mM (0.3% w/v)sodium diacetate (in a shaking water bath).
Project description:5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localisation of 5hmC. The first approach, termed GLIB (GLucosylation, perIodate oxidation, Biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites (TSS). 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a likely role in transcriptional regulation, and suggest a model in which 5hmC contributes to the M-bM-^@M-^\poisedM-bM-^@M-^] chromatin signature found at developmentally-regulated genes in ES cells. Mapping of 5-hydroxymethylcytosine in ES cells by GLIB and anti-CMS methodologies
Project description:The physiological basis of the hypothesis that temperature is the primary governing factor of bacterial cell inactivation under otherwise non-growth permissive conditions was investigated. Application of simultaneous low pH (pH 3.5) and low water activity (aw = 0.9; 2.5 M NaCl) conditions, applied to L. monocytogenes strains Scott A and FW03/0035, and increasing incubation temperature from 25°C up to 45°C resulted in increased permeability to ethidium homodimer-1 and corresponded to accelerated declines in esterase activity and ATP basal levels but did not result in autolysis. Triphasic survival curves were readily observable when sufficiently large cell populations were inactivated at 25°C and 35°C; indicative of a mixture of sensitive and resistant sub-populations. Enrichment-based recovery experiments however indicate that the stress conditions eventually lead to complete loss of reproductive capacity, potentially corresponding to an irreversible collapse of pH homeostasis. Transcriptomic analyses were used to further obtain insights into the physiology of the inactivation process occurring at 25°C. RT-PCR, rifampin-enforced decay and microarray experiments revealed transcripts of tufA and other genes become substantially more stable during inactivation during exposure to combined low pH/aw and during non-growth permissive temperature exposure. Gene transcripts were delineated through K-means clustering that appear to be important for initial survival of combined low pH/aw and include an overrepresentation of SigB-activated genes, the response of which fades with increasing time of inactivation exposure. The microarray component of the experiments had the aim of determining: i) Gene expression responses of L. monocytogenes strain Scott A when exposed to a non-growth permissive environment consisting of a broth system acting as a food simulated environment adjusted to low pH (pH 3.5) and low water activity (2.5 M NaCl). ii) To examine the trend in gene expression over time under inactivating (killing) conditions by applying gene set (functional and regulatory) expression trend analysis and K-means cluster analysis. iii) To correlate this data to other physiological and mRNA quantification (real-time-PCR) data. Experimental design: Two biologically replicated control cultures (each with two technical replicate sper chip) were labelled with Cy3 for each treatment sample for a total of 4 pairs of biological replicates. The treatments consisted of treated (inactivated in brain-heart infusion broth at pH 3.5 and water activity 0.9) L. monocytogenes cells with two biological replicates (each with two technical replicates per chip) labelled with Cy5. The time course (incubation time under inactivating conditions) of treatments was âinitialâ (20 minutes), 24 h, 48 h, and 72 h.