Transcriptomic response of Enterococcus faecalis to iron excess
ABSTRACT: To further investigate the homeostatic response of E. faecalis to Fe exposure, we examine the whole-genome transcriptional response of wild-type (WT) exposed to non toxic Fe excess. This experiment correspond the work titled Transcriptomic response of Enterococcus faecalis to iron excess (work in preparation) A four chip study using total RNA recovered from four separate wild-type cultures of Enterococcus faecalis OG1RF, two controls samples (N medium growth) and two iron samples (N medium gowth with 0.5 mM Fe-NTA). Each chip measures the expression level of 3,114 genome genes from Enterococcus faecalis strain V583 (A7980-00-01).
Project description:The microarrays experiments was performed with the purpose of identify transcriptional networks activated by copper. This experiment correspond the work tituled Enterococcus faecalis reconfigure the activation of its transcriptional regulatory networks under different copper exposure levels (work in preparation).Mauricio Latorrea,b, Jessica Galloway-Peñac,d,e, Jung Hyeo Rhoc,d, Marko Budinichf, Barbara E. Murrayc,d,e, Alejandro Maassb,f, Mauricio Gonzáleza,b,f*. a INTA, Laboratorio de Bioinformática y Expresión Génica, INTA, Universidad de Chile, Santiago, Chile. b Center for Genome Regulation (Fondap 15090007), University of Chile, Santiago, Chile. c Division of Infectious Disease, Department of Medicine, University of Texas Medical School, Houston, Texas, United States of America. d Center for the Study of Emerging and Reemerging Pathogens, University of Texas Medical School, Houston, Texas, United States of America. e Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas, United States of America f Mathomics, Center for Mathematical Modeling (UMI2807CNRS), Santiago, Chile. * Corresponding author. Address: El Líbano 5524, Santiago 11, Chile. Fax: +56 (2) 2214030. A eight chip study (two technical replicates) using total RNA recovered from four separate cultures of: Enterococcus faecalis OG1RF (N medium growth), Enterococcus faecalis OG1RF copΔ mutant strain (N medium growth), Enterococcus faecalis OG1RF copΔ mutant low copper treatment (N medium growth + 0.05 mM CuSO4) and Enterococcus faecalis OG1RF copΔ mutant low copper treatment (N medium growth + 0.5 mM CuSO4). Each chip measures the expression level of 3,114 genome genes from Enterococcus faecalis strain V583 (A7980-00-01).
Project description:Gene content in various Enterococcus faecalis strains compared to E. faecalis V583. Strains have been compared to the V583 strain by comparative genomic hybridization using genome-wide PCR-based microarrays representing the V583 genome. Genes have been deemed "present" or "divergent" in the various strains.
Project description:Diatoms, which are responsible for up to 40% of the 45 to 50 billion metric tons of organic carbon production each year in the sea, are particularly sensitive to Fe stress. Here we describe the transcriptional response of the pennate diatom Phaeodactylum tricornutum to Fe limitation using a partial genome microarray based on EST and genome sequence data. Processes carried out by components rich in Fe, such as photosynthesis, mitochondrial electron transport and nitrate assimilation are down-regulated to cope with the reduced cellular iron quota. This retrenchment is compensated by nitrogen (N) and carbon (C) reallocation from protein and storage carbohydrate degradation, adaptations to chlorophyll biosynthesis and pigment metabolism, removal of excess electron s by mitochondrial alternative oxidase (AOX), augmented Fe-independent oxidative stress responses, and sensitized iron capture mechanisms. Keywords: Marine phytoplankton, pinnate diatom Wild-type Phaeodactylum tricornutum was grown under Fe replete (10,000 nM) and Fe limiting (5nM) conditions. Partial genome gene expression analysis of iron-inducible genes was conducted using a two-color competitive hybridization microarray.
Project description:Analysis of changes in gene expression in Enterococcus faecalis OG1 delta-EF2638 mutant compared to wild-type OG1 strain. The deletion mutant has a growth defect when grown with aeration The mutant presented in this study is described and characterized in Vesic, D. and Kristich, C.J. 2012. A Rex-family transcriptional repressor influnces H2O2 accumulation by Enterococcus faecalis. (submitted for publication) Microarray analysis was done using RNA isolated from two independent cultures of wild-type Enterococcus faecalis OG1 and two independent cultres of Enterococcus faecalis OG1 delta-EF2638 mutant; each RNA sample was subjected to triplicate hybridization (technical replicates) . Microarrays were custom designed to investigate expression of ORFs in Enterococcus faecalis OG1RF genome. The arrays were designed based on the OG1RF annotation generated with the Rapid Annotation Using Subsystem Technology (RAST) server (Aziz et. al. 2008. BMC Genomics 9:75), as described in Frank et al (2012) Infect. Immun. 80:539. The aim was eighteen probe pairs per ORF, each of which is present in triplicate.
Project description:Comparative genomic hybridization analysis of diverse Enterococcus faecalis strains to determine core species genome.<br>An additional data file, Gilmore_M-Genomotyping_Tables_12-21-2006.xls, containing information about data processing is available for download from <a href="ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1090/">ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1090/</a>
Project description:Essential metals such as iron are required for healthy plant growth. Fe is an important cofactor and catalytic element in many biological processes. Fe and other metals can also be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalisation. The NAS genes are supposed to play an important role in Fe homeostasis. They are coding for enzymes called nicotianaminesynthase (NAS), which synthesize nicotianamine (NA) by a one-step condensation reaction of three molecules S-adenosyl-methionine. NA acts as a chelator for Fe, Cu, Ni and Zn and might be involved in the transport and allocation of Fe throughout the plant. We generated quadruple T-DNA insertion mutant nas plants to investigate NA function as described in Klatte et al., 2009, Plant Physiol. The nas4x-1 plants show an interveinal leaf chlorosis when turning from vegetative to reproductive stage, which intensifies when growing under Fe deficiency conditions. nas4x-1 plants have strongly reduced NA contents and show an elevated Fe deficiency response in roots. By performing microarray experiments we want to reveal global changes on transcriptional level in roots and leaves of nas4x-1 mutant compared to wild type plants grown under Fe supply and Fe deficiency conditions, respectively. The loss of NAS genes has a strong impact on the regulation of other metal homeostasis genes and allows to draw conclusions about nicotianamine function in metal homeostasis of A.thaliana. For this study, four-week old nas4x-1 mutant and wild type plants were exposed for 7 days to plant medium with and without Fe supply. These conditions have been established previously and have resulted in a reproducibly strong interveinal leaf chlorosis of nas4x-1 plants compared to wild type, especially upon Fe deficiency conditions. The experiment was repeated three times in consecutive weeks to obtain three independent biological repetitions. Rosette leaves and roots of five week-old plants were harvested, RNA was isolated and microarray hybridization was performed. 24 Total samples were analyzed. We generated the following pairwise comparisons: WT + Fe vs. – Fe, nas4x-1 + Fe vs. – Fe, + Fe WT vs. + Fe nas4x-1, - Fe WT vs. - Fe nas4x-1, roots and leaves
Project description:Enterococcus (E.) faecalis is a commensal in healthy humans, frequently found in a variety of fermented foods, and can serve as a probiotic. However, it has also been recognized as a pathogen causing diseases such as endocarditis, bacteremia and urinary tract infections. As known virulence factors are not limited to clinical isolates but widespread in many strains, additional fitness determinants should influence E. faecalis behavior in the host. We have performed a transcriptomic in vivo study with E. faecalis in the intestine of living mice to identify novel latent and adaptive fitness determinants within E. faecalis. The transcriptomic data derived from E. faecalis strain OG1RF monoassociated with wild type mice provide a first insight in the genes used to live as a commensal in the intestinal tract. Clear changes are observed as compared to growth under laboratory conditions (BHI broth) in the expression of genes involved in energy metabolism (e.g. dhaK and glpK pathway), transport and binding mechanisms (e.g. phosphoenolpyruvate carbohydrate PTS) as well as fatty acid metabolism (fab genes). This knowledge can be used to help explain its persistence in this environment, which is a prerequisite to cause infection in a compromised or inflamed host and possibly develop improved treatment strategies of the so far hard to cure infections. Overall design: Comparison of transcriptome data from Enterococcus faecalis growing in BHI broth and monoassociated mice
Project description:au10-14_fer - response of ein3eil1 mutants to fe deficiency - Response of ein3eil1 mutants to Fe deficiency - Wild type seedlings and ethylene insensitive ein3eil1 seedlings were germinated and grown in the presence of 50 µM Fe or absence of Fe (0 µM) on Hoagland medium agar plates until the age of 6 days. Under these growth conditions symptoms of Fe deficiency develop in the 0 Fe plants. Ethylene is known to promote Fe acquisition responses. Whole seedlings were harvested for transcriptome analysis, in a total of three biological replicates. 12 dye-swap - gene knock out,treated vs untreated comparison
Project description:The ability of Geobacter species to readily donate electrons to extracellular electron acceptors makes the study of their physiology not only important for the understanding of environmental processes, but also for industrial applications such as bioelectronics and electrosynthesis. Studies in G. sulfurreducens have shown that outer surface components, such as c-type cytochromes and conductive type IV pili play an important role in direct electron transfer to extracellular electron acceptors such as Fe(III) oxides and electrodes. However, many of these thoroughly studied outer surface components, including c-type cytochromes, are not well conserved among Geobacter species. In order to better understand which components are involved in extracellular electron transfer in Geobacter species other than G. sulfurreducens, studies were conducted with its close relative G. metallireducens. Whole-genome microarray analysis revealed that 23 of the 91 putative c-type cytochromes encoded in the G. metallireducens genome were upregulated at least 2-fold in cells grown with Fe(III) oxide compared to cells in which Fe(III) citrate was provided as the terminal electron acceptor. Protein identification with liquid-chromatography/mass spectrometry detected 6 c-type cytochromes that were more abundant in the outer surface cell fraction of cells that were grown with Fe(III) oxide as the terminal electron acceptor compared to cells grown on Fe(III) citrate. 22 genes encoding c-type cytochromes were chosen for gene deletion. Deletion of 6 genes encoding for c-type cytochromes, a gene encoding for a lipopolysaccharide biosynthesis-associated protein, and a gene encoding for a NHL- repeat containing protein inhibited growth when Fe(III) oxide was provided as the electron acceptor. This study suggests that there are different roads for extracellular electron transfer in Geobacteraceae since homologous c-type cytochromes have different functions from one species to the other, and novel components not previously found to be essential for extracellular electron transfer were identified. An eight-chip study using total RNA recovered from four separate cultures of Geobacter metallireducens GS-15 grown with acetate (10mM)-Fe(III) oxide (100 mmol l-1) (experimental condition) or with acetate (10 mM)-Fe(III) citrate (55mM) (control condition) during exponential growth. Each chip measures the expression level of 3,627 genes from Geobacter metallireducens GS-15 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.