Response to acetic acid stress in Candida glabrata: effect of the CgHaa1 transcription factor
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ABSTRACT: This transcriptomics analysis was undertaken to elucidate the effect of acetic acid stress in the reprogramming of C. glabrata KUE100 genomic expression. For this cells were cultivated in MM growth medium (at pH 4) either or not supplemented with a sub-lethal inhibitory concentration of acetic acid (30 mM). Furthermore it was also examined the response to the same concentration of acetic acid of a mutant devoid of the CgHaa1 transcription factor
Project description:Primary graft dysfunction (PGD) continues to be a major cause of early death after lung transplantation. Moreover, there remains a lack of accurate pre-transplant molecular markers for predicting PGD. To identify distinctive gene expression signatures associated with PGD, we profiled human donor lungs using microarray technology prior to the graft implantation. The genomic profiles of 10 donor lung samples from patients who subsequently developed clinically defined severe PGD were compared with 16 case-matched donor lung samples from those who had a favorable outcome without PGD. Matched factors used were: recipient age (± 10 years), recipient gender, recipient lung disease, and type of transplantation (single or bilateral). Keywords: Observational case-control study Matched case-control observational study: 10 primary graft dysfunction cases vs 16 Good outcome cases. One replicate per array.
Project description:The species Campylobacter jejuni is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by dns, inhibits natural transformation in C. jejuni. In the present study, genetic factors coding for DNase activity in absence of dns were identified. DNA arrays indicated that nonnaturally transformable dns-negative strains contain putative DNA/RNA non-specific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on C. jejuni integrated element 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative Escherichia coli and C. jejuni strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable C. jejuni strain resulted in a decreased efficiency of natural transformation. Modelling suggests that the C. jejuni DNases belong to the Serratia nuclease family. Overall, the data indicate that the acquisition of prophage encoded DNA/RNA non-specific endonucleases inhibits the natural transformability of C. jejuni through hydrolysis of DNA. The genomic diversity of 15 naturally competent or nonnaturally transformable Campylobacter jejuni strains were examined by microarray-based comparative genomic indexing (CGI) analysis. The CGI analysis allowed the assessment of CDS content for each C. jejuni strain relative to the C. jejuni DNA microarray, which comprises ORFs from strains NCTC 11168, RM1221. ORFs were spotted in duplicate. Genomic DNA from strains NCTC 11168/RM1221 were used as a reference DNA and competitively hybridized with genomic DNA from each of the other C. jejuni strains. Two replicates for each strain were performed. Data normalization was performed as in Parker et al. J Clin Microbiol 2006, 44(11):4125-4135.
Project description:Transcriptional profiling of E. coli DH5alpha cells comparing control untreated cells with cells exposed to sublethal concentrations of reuterin (3-hydroxypropionaldehyde). Two-condition experiment, reuterin-treated vs. untreated control E. coli DH5alpha cells. 5 biological replicate sets of reuterin-treated vs. untreated cells which were independently grown and harvested. Each array is probed with 1 replicate set.
Project description:Invasion of host tissue by the human fungal pathogen, Candida albicans is an important step during many forms of candidosis. However, not all C. albicans strains possess the same invasive and virulence properties. It is known for example that the two clinical isolates SC5314 and ATCC10231 differ in their ability to invade into host tissue and to cause infections. Strain SC5314 is invasive whereas strain ATCC10231 is non-invasive and strongly attenuated in virulence as compared to SC5314. In this study we compare the in vitro transcriptional profiles and the genotypic profiles of these two widely used laboratory strains in order to determine the principal biological and genetic properties which may govern the different potential for invasiveness and virulence. Keywords: transcriptional profiling, comparative genomic hybridisation, invasive vs. non-invasive C. albicans strain Genomic DNA from C. albicans strains SC5314 and ATCC10231 hybridisations were done in duplicate including one dye swap. Total RNA from C. albicans strains SC5314 and ATCC10231 strains were compared in triplicate including one biological replicate and one dye swap.
Project description:Sulfolobus solfataricus P2 was grown aerobically, with O2 concentrations ranging from 1.5 to 26 % (v/v; gas phase). To gain some insight in control of the respiratory system, transcriptomes of the strain cultivated in different O2 concentrations (1.5 % vs 21 %, 1.5 % vs 26 %) were compared using a DNA microarray approach. Two-condition experiments: 1.5% O2 vs two other O2 concentrations (21 or 26% O2). Biological replicates independently grown. One replicate per array. Dye swaps.
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.
Project description:While significant advances have been made in EHEC pathogenesis, we still do not fully understand the impact of environmental stress on EHEC virulence. During the course of infection, EHEC must evade or overcome several biological barriers, the first of which is the gastric acidity encountered during passage through the stomach. EHEC is remarkable in its ability to tolerate this acidity. There are four different acid resistance systems that provide E. coli O157:H7 protection against exposure to low pH (2-2.5). Interestingly, EHEC uses these acid resistance systems differentially for survival in foods versus the bovine intestinal tract. The glutamate-dependent acid-resistance system is thought to offer the best protection below pH 3. Since the infectious dose of EHEC is so low (50-100 organisms), acid resistance becomes an important virulence trait. Studies of EHEC response to acid stress have focused primarily on levels of acid tolerance and the molecular basis of tolerance. However, the impact of acid stress on EHEC virulence is less well understood. In the related pathogen, EPEC, the plasmid-encoded regulator, Per, that regulates expression of many EPEC virulence factors, is regulated negatively at pH 5.5 and positively at pH 8.0, suggesting that virulence gene expression is repressed during mild acid stress and enhanced in alkaline pH typical of the small intestine. Expression of EPEC type III secreted factors involved in A/E lesion formation has been shown to be influenced by factors including culture media, iron and calcium levels. Protein secretion was inhibited at pH 6 and 8. In a third study, a gadE (encoding acid resistance regulator) mutation resulted in increased adhesion of E.coli O157:H7 to colonic epithelial cells, suggesting negative regulation of one or more adhesins. Other studies have reported that shiga toxin production is sensitive to culture conditions including pH. However, there are no studies of EHEC virulence changes after more severe acid stress nor studies linking stressed EHEC virulence phenotype with transcriptional changes. The goal of this study was to determine how acid stress affects EHEC virulence properties and through microarray analysis, define the genetic basis for these changes. Understanding how acid stress modulates the virulence potential of this pathogen is essential for delineating the pathogenesis of disease caused by EHEC infection and may offer novel approaches to prevent and treat EHEC infections. Bacteria were grown in LB broth overnight, then subcultured into DMEM and grown at 37C, 5%Co2. Bacteria were then subjected to one of three acid stress protocols: 1) UA30: growth in DMEM pH 7.4 followed by growth in DMEM pH 3.0 for 30 minutes; 2) AA30: growth in DMEM pH 5.0 (adaptation) followed by growth in DMEM pH 3.0; 3) UA15: growth in DMEM pH 7.4 followed by growth in DMEM pH 3.0 for 15 minutes. DMEM was supplemented with 25 mM MES (pH 5.0) and in the case of the control (unadapted, unshocked) 25 mM MOPS (pH 7.4) and the adaptation step was again carried out at 37C and 5% CO2. Acid shocking was done at pH 3.0 (unbuffered) at room temperature for all treatments
Project description:In this project we developed phosphopeptides functionalized with methacrylate ester warheads installed on a cysteine residue, which bind covalently to the protein 14-3-3 sigma. We prepared complexes of the purified protein with the peptides and used trypsin digestion and LC-MSMS to elucidate the binding site of the peptides, by comparison of compound-treated complexes to DMSO-treated protein and observing the disappearance on non-modified peptides. The file names are based on internal names used for the project and are distinct from the names given to the peptides in the final publication. Names are as follows: 4IEA - peptide 3 in the final publication 128C - peptide 8 in the final publication 132C - peptide 11 in the final publication
Project description:We would like to know how symbiotic molecules such as Nod Factors (NF) influence lateral root (LR) development in M. truncatula. We have preliminary evidence that this action is through early stages of root development. Auxin is the major phytohormone controlling LR development and we also have evidence that NF interfere with auxin for the control of LR development. This transcriptomic study aims at finding new molecular targets that would be responsive to auxin and NF treatment, even at a higher level by the combination of both auxin and NF. Such targets could be involved in the control or early development stages of LR that would be controlled both by auxin and NF treatment. - 2 day old M. truncatula (accession A17) plantlets grown on M medium were transferred for 2 days on M medium+ 10-5M NPA (1-N-Naphthylphthalamic acid, auxin transport inhibitor) then transferred for 10 h on M medium containing 10-6 M NAA (naphthalene-1-acetic acid, permeant auxin analog) or 10-7 M Nod factors (NF) or a combination of both (10-6 M NAA + 10-7 M NF). These are compared to mock treated (solvent only) plants. 12plex_med_2013_04 12 dye-swap - treated vs untreated comparison