Transcriptome of Staphylococcus aureus Response to Spermine
ABSTRACT: To explore the Spermine(Spm)-based antibacterial targets in S. aureus, time course-dependent transcriptome analysis was conducted on Mu50 (MRSA) in the absence and presence of Spm. We conducted five independent microarray experiments in the absence (control) and the presence (experimental) of Spm. We calculated fold change as the ratio between the signal of untreated (control) and Spm-treated (experimental) cultures for 15, 30 and 60 min exposures.
Project description:Arabidopsis thaliana polyamine oxidase 5 gene (AtPAO5) functions as a thermospermine (T-Spm) oxidase. Aerial growth of its knock-out mutant (Atpao5-2) is significantly repressed by low dose(s) of T-Spm but not by other polyamines. Massive analysis of 3’-cDNA ends (MACE) was performed. Cell wall, lipid and secondary metabolisms were dramatically affected in low dose T-Spm-treated Atpao5-2. Intriguingly Fe-deficient responsive genes and drought stress-induced genes were up-regulated, suggesting that vascular system loses the function. Histological observation showed that vascular system of the joint part between stem and leaves was structurally destroyed. The results indicate that T-Spm homeostasis by a balance of synthesis and catabolism, catalysed by AtPAO5 in Arabidopsis, is important for maintaining vascular system. Phylogenetic analysis showed that PAOs from vascular plants are classified into four clades (I-IV) and AtPAO5 belongs to the clade III. Clade III members show high identity to metazoan PAOs and are not found in non-vascular plants. Furthermore, all the clade III genes are intron-less or contain a single intron whereas the other three clade genes usually contain 7 to 9 introns. The data suggest the occurrence of a horizontal gene transfer of ancestral clade III PAO gene(s) from primitive animals. Fine tuning of T-Spm metabolism is critical for vascular plants and its catabolic gene was acquired from a certain Metazoan to equip the vascular system. 8 Samples analyzed by MACE (Massive Analysis of cDNA ends)
Project description:Novel anti-infective agents targeting Staphylococcus aureus and capable of increasing S. aureus susceptibility towards antibiotics are needed. One alternative approach is targeting the bacterial quorum sensing (QS) system. QS is a process by which bacteria produce and detect signal molecules and thereby coordinate their behaviour, virulence and biofilm formation in a cell-density-dependent manner. Hamamelitannin (HAM) was previously suggested to target the S. aureus QS system, thereby increasing the susceptibility of S. aureus biofilms towards vancomycin. However, mechanistic insights are still lacking. For this reason, we evaluated the effect of Hamamelitannin, vancomycin and combination treatment of Hamamelitannin and vancomycin on gene expression in S. aureus Mu50 biofilms.
Project description:Box C/D-type small nucleolar RNAs (snoRNAs) are functional RNAs responsible for mediating 2’-O-ribose methylation of ribosomal RNAs (rRNAs) within the nucleolus. Previously, in relation to a novel chromosomal translocation in a human B-cell lymphoma, we identified U50HG, a non-protein-coding gene that hosted a box C/D-type U50 snoRNA within its intron. To investigate the physiological importance of the U50 snoRNA and its involvement in tumorigenesis, we generated a mouse model deficient in mouse U50 (mU50) snoRNA expression without altering the expression of mouse mU50 host-gene, mU50HG-b. The established mU50 snoRNA-deficient mice showed a significant reduction of mU50 snoRNA expression and the corresponding target rRNA methylation in various organs. Lifelong phenotypic monitoring showed that the mU50-deficient mice looked almost normal without accelerated tumorigenicity; however, a notable difference was the propensity for anomalies in the lymphoid organs. B-cells were isolated from spleens of DmU50(HG-b) mice or wild-type C57BL/6J with antibody-conjugated magnetic beads system (Myltenyi Biotec). Total RNA was purified with QIAGEN RNeasy Micro kit. Affymetrix GeneChip® Microarrays (Mouse Expression 430 2.0 Array) were used.
Project description:Investigation of mRNA expression level changes in a Staphylococcus aureus Mu50 delta-SAV1322 mutant, compared to the wild-type strain. A comparison of the wild-type and the mutant transcription profiles Overall design: Total RNA recovered from four separate wild-type culture of Staphylococcus aureus Mu50 and four separate culture of a mutant strain, Mu50 delta-SAV1322, in which 689bp deleted of SAV1322 locus. Each chip measures the expression level of 967 genes from Staphylococcus aureus Mu50 with 11 probe pairs per genes (total probes 10,637).
Project description:Ctbp2 regulates exit from pluripotency via silencing of embryonic stem cell active genes during differentiation. We mapped the genome-wide occupancy of Ctbp2 by ChIP-seq in mESC (E14).
Project description:Fruits of transgenic tomato (Solanum lycopersicum) plants engineered with ripening-induced, yeast S-adenosylmethionine decarboxylase (ySAMdc) gene, accumulate the higher polyamines spermidine (Spd) and spermine (Spm) and demonstrate ameliorated phytonutrient content, juice quality, and prolonged vine life. Enhanced nitrogen-carbon interactions were revealed by comprehensive Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolite profiling of the transgenics, suggesting that Spd and Spm are perceived as nitrogenous metabolites by the fruit cells (Mattoo et al., 2006). The recent work by our colleagues identified the effects of Spd/Spm accumulation on various functional classes of tomato genes affected during ripening by probing 1522 ESTs on a custom-made array (Srivastava et al., 2007). In this study we monitored alterations of genome-wide transcriptional patterns in pericarp of Spd/Spm-accumulating tomatoes by means of direct comparison with azygous controls using DNA-microarray technology. Consistent with the ySAMdc expression pattern, very minor transcriptional alterations were detected in mature green developmental stage. For both breaker and red stages, large mutual and unique gene sets displayed altered levels of transcript. Ontological term analysis of up- and down-regulated transcript groups revealed processes in cell metabolism that are regulated by increased levels of Spd/Spm in ripening tomato fruits. These processes mainly involve carbohydrate and amino acids metabolism and protein synthesis. Additionally, transcript levels of representative genes encoding structural enzymes for related biosynthetic pathways show strong relationship to specific metabolites that were identified as regulated in Spd/Spm-accumulating transgenics. Plants of both genotypes were grown in randomized blocks (3 blockes 8-10 plants each). At each of 3 developmental stages (mature green, breaker and pink-red) a single fruit was harvested from a randomly chosen plant of each genotype within each block. In each harvest different plants in each block were taken to represent each genotype. Following harvest, equal pericarp samples were cut from the equator region of each fruit. These samples were pooled according to genotype prior to RNA extraction, allowing equal representation of each block in the final sample. A total of 3 harvests taken from each genotype were eventually analyzed– each harvest representing a biological repeat.
Project description:Spo11-mediated DNA double strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To shed light on this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S-phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea (HU) treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thereby we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation. Keywords: ChIP-chip • The goal of the experiment Genome-wide localization of Spo11, Mre11, Rec8, and DSB sites on meiotic chromosomes in Saccharomyces cerevisiae • Keywords Meiosis, Meiotic homologous recombination, Premeiotic DNA replication, cohesin, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI), Spo11, Mre11, Rec8, DSB (Double strand break) • Experimental factor Distribution of Spo11, Mre11, and Rec8 in wild type in early meiosis (1.5 hrs, 2 hrs, 3 hrs, 4 hrs, and 5 hrs in sporulation medium) Distribution of Spo11 in rec8delta cells in early meiosis (1.5 hrs, 2 hrs, 3 hrs, 4 hrs, and 5 hrs in sporulation medium) Distribution of Spo11 in wild type in the presence of HU (2hrs and 4 hrs in sporulation medium containing HU) Distribution of DSB sites in rad50S mutant cells at 7 hrs in sporulation medium Distribution of DSB sites in rec8delta rad50S mutant cells at 7 hrs in sporulation medium • Experimental design ChIP analyses: SK1 background cells expressing FLAG tagged protein were used for the ChIP using anti-FLAG M2 antibody. ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI and rikDACF for chromosome VI) were used. Mapping of DSB sites: DSB rich fraction was concentrated by ChIP of Spo11-FLAG in rad50S mutant without crosslinking. In the mutant, DSBs ramain unrepaired with covalently attached Spo11.Meiotic cells (at 7 hours in sporulation medium) were used for the analyses. • Quality control steps taken Confirmation of several loci by quantitative real time PCR. Southern blotting of several DSB sites.