Transcription profiling of Salmonella enterica (I) serovar Typhi Ty2 wild type and ompR null strains in a strand-specific manner
ABSTRACT: Analysis of transcriptome in a strand-specific manner to further refine previous genome annotation; RNA-seq was also combined with microarray and proteome analysis to further define the S. Typhi ompR regulon and identify novel ompR regulated transcripts.
Project description:The Sfh protein is encoded by self-transmissible plasmids involved in human typhoid and is closely related to the global regulator H-NS. We have found that Sfh provides a stealth function that allows the plasmids to be transmitted to new bacterial hosts with minimal effects on their fitness. Introducing the plasmid without the sfh gene imposes a mild H-NS- phenotype and a severe loss of fitness due to titration of the cellular pool of H-NS by the A+T-rich plasmid. This stealth strategy seems to be used widely to aid horizontal DNA transmission and has important implications for bacterial evolution.
Project description:GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1 and the two kinases form part of a signalling pathway that responds to extracellular cAMP. We identify potential cellular effectors for the two kinases by analysing their null mutants. There are proteins and mRNAs that are altered in abundance in only one or other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and micro-array analysis respectively identified 3 proteins and 37 genes that are similarly mis-regulated in both mutant strains. The positive correlation between the array data and the proteomics data is consistent with the Zak1-GskA signalling pathway functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterised markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed.
Project description:We describe the serendipitous discovery and first characterization of a new resistant cell type from Dictyostelium, which we propose to call the aspidocyte (aspida: Greek for shield). These cells are induced from amoebae by a range of toxins including heavy metals and antibiotics, and were first detected by their striking resistance to detergent lysis. Aspidocytes are separate, rounded or irregular shaped cells that are immotile but remain fully viable; once the toxic stress is removed, they revert to amoeboid cells within an hour. Induction takes a few hours and is completely blocked by the protein synthesis inhibitor cycloheximide. Aspidocytes lack a cell wall and their resistance to detergent lysis is active, requiring continued energy metabolism, and may be assisted by a complete cessation of endocytosis, as measured by uptake of the dye FM1-43. Microarray analysis shows that aspidocytes have a distinct pattern of gene expression with a number of genes up-regulated that are predicted to be involved in lipid metabolism. Aspidocytes were initially detected in a hypersensitive mutant, in which the AMP deaminase gene is disrupted, suggesting that the inductive pathway involves AMP levels or metabolism. Since aspidocytes can also be induced from wild-type cells and are much more resistant than amoebae to a membrane disrupting antibiotic, it is possible that they are an adaptation allowing Dictyostelium cells to survive a sudden onslaught of toxins in the wild.
Project description:We have performed microarray hybridization studies on forty clinical isolates from twelve common serovars within Salmonella enterica subspecies I (sspI) to identify the conserved gene pool present.
Project description:Microarray based CGH was conducted over a group of 29 strains of S. Enteritidis spanning different epidemiological periods in Uruguay, plus 6 other S. Enteritidis strains isolated from distant geographical regions. We also included 9 Salmonella enterica strains of other serovars isolated in Uruguay. A S. Enteritidis dispensable genome of 233 chromosomal genes and high extent of variation in virulence plasmid was found. Strains isolated before the epidemic show the highest genomic differences as compared with the PT4 reference strain. Comparison with the gene content of other serovars demonstrate extensive horizontal gene transfer between circulating strains beyond serovar definition. Our results show that the epidemic of S Enteritidis in Uruguay was produced by the introduction of strains closely related to PT4, and corroborate the extensive genetic homogeneity among S. Enteritidis isolates worldwide. Phage SE14 emerges as the only specific region for S. Enteritidis. Genetic differences detected in pre-epidemic strains, mainly associated with the absence of phage SE20, suggest that genetic features encoded in this phage may be related to particular epidemiological behavior.
Project description:Salmonella enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104) has caused significant morbidity and mortality in humans and animals for almost three decades. We have completed the full DNA sequence of one DT104 strain, NCTC13348 and show that the main differences between the genome of this isolate and the previously sequenced S. Typhimurium LT2 lie in integrated prophage elements and the Salmonella Genomic Island 1 encoding antibiotic resistance genes. Thirteen isolates of S. Typhimurium DT104 with different pulsed field gel electrophoresis (PFGE) profiles were analyzed by multi locus sequence typing (MLST), plasmid profiling, hybridization to a Pan-Salmonella DNA microarray and prophage-based multiplex PCR. All the isolates belonged to a single MLST type ST19. Microarray data demonstrated that the 13 DT104 isolates were remarkably conserved in gene content. The PFGE band-size differences in these isolates could be explained to a great extent by changes in prophage and plasmid content. Thus, here the nature of variation in different S. Typhimurium DT104 isolates is further defined at the genome level illustrating how this phage type is evolving over time.
Project description:Fission yeast cells belong to one of two specialized cell types, M or P. Specific environmental conditions trigger sexual differentiation, which leads to an internal program starting with pheromone signalling between M and P cells, followed by mating, meiosis and sporulation. The initial steps of this process are controlled by Ste11p, a master transcriptional regulator that activates the expression of cell type-specific genes (only expressed in either M or P cells) as well as genes expressed in both M and P cells. <br><br> Pheromone signalling is activated by Ste11p-dependent transcription, and in turn enhances some of this transcription in a positive feedback. To obtain a genome-wide view of Ste11p target genes, their cell-type specificity, and their dependence on pheromone, we used DNA microarrays along with different genetic and environmental manipulations of fission yeast cells.We directly compared the transcriptome of homothallic wild-type cells (h90 fus1) with that of ste11 delta mutants under conditions that induce sexual differentiation. To allow for indirect effects of the ste11 delta mutation, we took advantage of the fact that ectopic expression of ste11 can drive cells into sexual differentiation and therefore is expected to cause the expression of Ste11p targets. We thus defined Ste11p targets as those genes whose expression was significantly reduced in a ste11 delta mutant and significantly increased when ste11 is overexpressed in vegetative cells.<br> <br> This study looked at the effect of deletion or overexpression of ste11, and changes in gene expression after nitrogen starvation of h plus, h minus, h90fus1 and h90ste11 cells.
Project description:Dosage compensation ensures that males and females, despite unequal number of X chromosomes, equalize for X linked gene expression. In Drosophila, it is achieved by a two-fold up-regulation of most of the genes present on the male X chromosome, and requires the association of the Dosage Compensation Complex (DCC) on the X chromosome. One of the main intriguing aspects of dosage compensation is how this complex is able to target specifically hundreds of sites only on the X chromosome in order to ensure dosage compensation. In order to better understand the targeting of the DCC and the dosage compensation mechanism, we have then decided to analyze the distribution of the DCC as well as the expression levels in male and female in a more complete and precise manner, using microarrays. In this experiment, we present the data used to analyse the distribution of the MSL-1 and MSL-3 protein (part of the DCC complex) on the X chromosome in WT embryos aged from 0H-14H, as well as the distribution of MSL-1 in embryos aged from 4-6H and in male III instar salivary glands. The DNA amplified from the specific immunoprecipitation (MSL-1 or MSL-3 IP) were labelled using Cy5-dCTP and hybridized against DNA amplified from a non specific immunoprecipitation (mock IP), labeled with Cy3 dye. In parralel we present the data used to analyse the expression profile of X-linked genes in WT male or female III instar larvae salivary glands. The cDNA amplified from the RNA extracted from the male or female salivary glands were labelled using Cy5-dUTP and hybridized against the reference sample, labelled with Cy3-dUTP (pool RNA from ON embryos, adultes, and salivary glands mixed at a ratio 1:1:1.and amplified as the RNA from salivary glands). We used for this study a cDNA array developed by the Genecore facility in EMBL, covering the DGC1 and DGC2 cDNA libraries from the Berkeley Drosophila Genome Project, which represents more than 70% of the coding Drosophila genome.