Transcriptome analysis of the WalKR regulon in Staphylococcus aureus
ABSTRACT: The WalKR regulon was studied by comparing genes expression in a strain producing a constitutively active WalR regulator (WalRc) versus the wild type strain carrying the empty expression vector. Hybridizations were performed with three independent biological replicates. Our data allowed to identify 165 genes differentially expressed in the walRc expressing strain with a P-value M-bM-^IM-$ 0.05 using Z-test and a threshold value of two-fold change in transcriptional levels. Among them, 108 genes are activated, and 57 genes are repressed. About 10% of these results were confirmed by quantitative real time PCR, revealing the high reliability of this overall study. Beyond the major effect of the WalKR system on genes expressing cell wall hydrolases, we have shown that it activates a large number of genes involved in virulence, and probably through its positive impact on the SaeRS two-component system, is a major activator of S. aureus virulence. Cells were grown in TSB rich medium added with 0.25 M-BM-5M CdCl2 to allow walRc expression until OD600nm of 1. Gene expression was compared between the HG001 wild type reference strain carrying the pCN51 empty vector (HG001/pCN51) and the HG001 strain carrying a pCN51 derivative with the walRc allele under the control of the Pcad inducible promoter (HG001/pSD3-12). Each condition was done in triplicate and the data were analyzed using the Arraystat software. Genes were considered as differentially expressed if their transcriptional level differed by at least two-fold with a Z-test P-value M-bM-^IM-$ 0.05 in at least two of the replicates.
Project description:The WalKR regulon was studied by comparing genes expression in a strain producing a constitutively active WalR regulator (WalRc) versus the wild type strain carrying the empty expression vector. Hybridizations were performed with three independent biological replicates. Our data allowed to identify 165 genes differentially expressed in the walRc expressing strain with a P-value ≤ 0.05 using Z-test and a threshold value of two-fold change in transcriptional levels. Among them, 108 genes are activated, and 57 genes are repressed. About 10% of these results were confirmed by quantitative real time PCR, revealing the high reliability of this overall study. Beyond the major effect of the WalKR system on genes expressing cell wall hydrolases, we have shown that it activates a large number of genes involved in virulence, and probably through its positive impact on the SaeRS two-component system, is a major activator of S. aureus virulence. Cells were grown in TSB rich medium added with 0.25 µM CdCl2 to allow walRc expression until OD600nm of 1. Gene expression was compared between the HG001 wild type reference strain carrying the pCN51 empty vector (HG001/pCN51) and the HG001 strain carrying a pCN51 derivative with the walRc allele under the control of the Pcad inducible promoter (HG001/pSD3-12). Each condition was done in triplicate and the data were analyzed using the Arraystat software. Genes were considered as differentially expressed if their transcriptional level differed by at least two-fold with a Z-test P-value ≤ 0.05 in at least two of the replicates.
Project description:A comprehensive expression analysis of Wnt signaling genes was performed in a panel of prostate cancer cell lines and tissue specimens using TaqMan low density arrays. The effect of DNA methylation on gene expression was investigated using DNMT inhibitor 5-Aza-CdR. Tissue specimens from a range of disease states were used to represent the stepwise progression of prostate cancer, including benign prostatic hyperplasia (BPH), histologically benign epithelium adjacent to tumor (HB), pre-invasive high-grade prostatic intraepithelial neoplasia (HGPIN) and primary localized tumors categorized into low- and high-grade disease. Fifteen Wnt signaling related genes were idenified with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumour (r=0.76) than to BPH (r=0.57) (P<0.001). Overall, the expression profile was highly similar between tumors of high (M-bM-^IM-%7) and low (M-bM-^IM-$6) Gleason scores. Pharmacological demethylation of PC-3 cells reactivated 38 genes (M-bM-^IM-%2-fold); 40% of which inhibit Wnt signaling. qPCR gene expression profiling using TLDA Human Wnt Gene set v1.0 microfluidic card (Applied Biosystems, Foster City, CA).The TLDA consisted of 4 identical 96-gene sets preconfigured in a 384-well format. Three different malignant cell lines were profiled LNCaP, 22Rv1 and PC3 (+/- 5-Aza-2M-bM-^@M-^YDeoxycytidine). One benign cell line was included for comparative puposes: PWR1E. Patient samples were obtained from FFPE tissue sections from men who underwent radical prostatectomy or transrectal resection of the prostate. To overcome the limited amount of RNA obtained from FFPE tissues, RNA samples were pooled. Five different pools were generated: high grade prostate cancer (Gleason score M-bM-^IM-%7), low grade prostate cancer (Gleason score M-bM-^IM-$6), HGPIN, HB and BPH. Each pool consisted of DNase-treated total RNA (100ng), isolated from microdissected tissue from 4 individual cases, selected on the basis of similar histological and clinical features and previous epigenetic characterization in our laboratory. Two Biological replicates for each pool were prepared. The high capacity cDNA Archive Kit (Applied Biosystems) was used to reverse transcribe pooled RNA (400ng). TaqManM-BM-. reactions were performed in duplicate on a 7900HT Sequence Detection System. RQ data from TLDAs were analyzed using Real Time StatMinerM-BM-. v3.1 (Integromics, Granada, Spain).
Project description:The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ?lytM ?ssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.
Project description:Nascent embryonic joints, interzones, contain a distinct cohort of progenitor cells responsible for the formation of the majority of articular tissues. However, to date the interzone has largely been studied using in situ analysis for candidate genes in the context of the embryo rather than using an unbiased genome wide expression analysis on isolated interzone cells, leaving significant controversy regarding the exact role of the intermediate and outer interzone layers in joint formation. Therefore, in this study, using laser capture microdissection (three biological replicates), we selectively harvested the intermediate and outer interzones of mouse embryos at gestational age 15.5 days, just prior to cavitation, when the differences between the layers should be most profound. Microarray analysis (Agilent Whole Mouse Genome Oligo Microarrays) was performed and the differential gene expression between the intermediate interzone cells and outer interzone cells was examined by performing a 2-sided paired student t-test and pathway analysis. 197 genes were differentially expressed (M-bM-^IM-% 2-fold) between the intermediate interzone and the outer interzone with a p-value M-bM-^IM-$ 0.01. Of these, 91 genes showed higher expression levels in the intermediate interzone and 106 were expressed higher in the outer interzone. Pathway analysis of differentially expressed genes suggests an important role for inflammatory processes in the interzone layers, especially in the intermediate interzone, and hence in joint and articular cartilage development. The high representation of genes relevant to chondrocyte hypertrophy and endochondral ossification in the outer interzone suggests that it undergoes endochondral ossification. 2 study groups (intermediate and outer interzone) with three biological replicates (1 biological replicate = 1 embryo)
Project description:P. syringae pv. phaseolicola, the causal agent of halo blight disease in bean, produces a toxin known as phaseolotoxin, whose synthesis involves the product of some of the genes found within the Pht region. This region, considered a pathogenicity island, comprises 23 genes arranged in five transcriptional units; two single-gene units (argK, phtL) and three arranged as operons (phtA, phtD, phtM), most with unknown function. In P. syringae pv. phaseolicola, maximal expression of most of the genes encoded in the Pht region and the synthesis of phaseolotoxin require the product of the phtL gene, which has been proposed to have a regulatory function. In order to evaluate the role of phtL gene in P. syringae pv. phaseolicola, we performed a comparative transcriptional analysis with the wild type and a phtL- mutant strains using microarrays. The microarray data analysis showed that PhtL regulates the expression not only of genes within the Pht region, but also alters the expression of genomic genes related with the iron-acquisition system, pathogenicity, oxidative stress and virulence. This study suggests the possible relation of the PhtL protein with the iron response genes and with the pathogenicity and or virulence of this bacterium. The microarrays used in this study correspond to an assembled DNA microarray of P. syringae pv. phaseolicola NPS3121 previously reported (GEO accession number GPL7115) (HernM-CM-!ndez-Morales et al., 2009). This microarray consist of 1911 probes, with an average length 2.4 Kbp, representing 1X the genome of P. syringae pv. phaseolicola NPS3121, as well as several PCR products corresponding to various known genes, which were printed as controls. Each microarray experiment was repeated six times (two technical replicates with the same RNA samples and three biological replicates using RNA isolated from a different culture). cDNA synthesis, labeling, hybridization, washing, and chip scanning were performed at the Microarray Core Facility at Cinvestav-Langebio. Hybridized microarray slides were scanned (GenePix 4000, Axon Instruments, Inc) at a 10 um resolution adjusting the laser and gain parameters to obtain similar levels of fluorescence intensity in both channels. The spot intensities were quantified using Axon GenePrix Pro 6.0 image analysis software. First, an automatic spot finding and quantification option of the software was used. Subsequently, all spots were inspected individually and in some cases, the spot diameters were corrected or the spots were removed from the analysis. The mean of the signals and the median of backgrounds were used for further analysis. Raw data were imported into the R.2.2.1 software. Background signals were subtracted using the Robust Multichip Analysis (RMA) whereas the normalization of the signal intensities within slides was carried out using M-bM-^@M-^\print-tip loessM-bM-^@M-^] method and the LIMMA package. Normalized data were log2 transformed and then fitted into mixed model ANOVAs using the Mixed procedure. The p-values of the phtL effects were adjusted for by the False Discovery Rate Method M-bM-^@M-^\FDRM-bM-^@M-^]. Changes in signal intensity of M-BM-11.5-fold or higher/lower between phtL- mutant and wild type were highly significant (FDR, p-value M-bM-^IM-$0.05), however we focused only in differential expressed genes that fell within the more traditional criteria, which is the cut-off threshold for up-regulated (M-bM-^IM-%1.8) and down-regulated genes (M-bM-^IM-$0.50).
Project description:A screening process was used to determine hyperosmotic as well as cold stress tolerance of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at sub lethal levels of NaCl and 4M-BM-0C. Analysis of gene expression in cells adapted to cold and hyperosmotic stress revealed significant strain specificity in terms of individual gene expression profiles though general patterns of expression within functional gene subsets were largely similar. . Strain ATCC19115 revealed 150 significantly up- and 146 down-regulated genes in cells adapted to both hyperosmotic and cold stress, 82 up- and 56 down-regulated genes were matching in response to both stresses in strain 70-1700, whereas ScottA, a relatively stress-tolerant strain, showed up-regulation of 77 genes whilst 106 genes were significantly down-regulated. Among these homologously expressed genes only 22 were found to be significantly up-regulated and only 7 were down-regulated in all three strains adapted to hyperosmotic stress and cold temperature, suggesting a dominating strain specific response. Overall adaptation to hyperosmotic and low temperature growth conditions in three L. monocytogenes strains were found to have many parallels, especially when examined using a statistically-based ontological approach. Evidence was presented for strong activation of genes associated with protein synthesis, in particular those coding proteins of the ribosome and involved directly in transcription. Genes associated with DNA maintenance; modification to cell envelope, and cell division were also up-regulated. On the other hand strong suppression of genes associated with carbohydrate metabolism and transport as well as flagella assembly was evident in stress adapted cells most likely due to energy preservation via CodY regulon. This suggests that adaptation to these adverse conditions engages similar mechanisms to cope with the induced stressful environments. The microarray component of the experiments had the aim of determining: i) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic salt resistances following adaptation to hyperosmotic stress. ii) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic cold tolerances following adaptation to cold stress. iii) To evaluate common pathways of adaptation to hyperosmotic and cold stresses Control cultures (four biological replicates; exception strain ATCC19115 for the 10%NaCl stress which had three biological replicates) were labeled with Cy3 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains. Sample cultures (four biological replicates; exception strain ATCC19115 for the 10%NaCl which had three biological replicates) were labeled with Cy5 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains. Condition one M-bM-^@M-^Ssub-lethal hyperosmotic stress: Control culture (Cy3-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) - strains were grown to mid exponential growth phase at 25M-BM-0C (in a shaking water bath) in brain-heart infusion broth. Test cultures (Cy5-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) M-bM-^@M-^S strains grown to mid exponential growth at 25M-BM-0C phase (in a shaking water bath) in BHI broth supplemented with 8% additional NaCl (strain 701700), 10% additional NaCl (strain ATCC19115) or 12% additional NaCl (ScottA). Condition two M-bM-^@M-^S cold stress: Control culture (Cy3-labelled RNA) - strains were grown to mid exponential growth phase at 25M-BM-0C (in a shaking water bath) in brain-heart infusion broth. Test cultures (Cy5-labelled RNA) M-bM-^@M-^S strains grown to exponential growth phase at 4M-BM-0C in BHI broth.
Project description:We did transcription profiling on the effect of RCK1 over-expression. rck1 mutant strain was transformed with empty high copy vector pRS425 empty vector), or with RCK1 cloned into pRS425 (the RCK1-overexpressing strain: RCK1 cloned into pRS425). Analysis was performed with rck1 (Empty vector, pRS425) mutant and RCK1 over-expressed cells (pRS425-RCK1). Yeast cells were grown on SD-leu medium. Yeast cells were grown overnight at 30M-BM-0C . The culture was refreshed to 0.2 O.D and grown at 30M-BM-0C for 2h 30min. Cell wall stress was applied by addition of zymolyase to a final concentration of 5 unit/ml . Cells were collected at 2 hours of growth and processed for RNA extraction.
Project description:Long non-coding RNAs (lncRNAs) have emerged recently as key regulatory molecules with diverse roles at almost every level of the regulation of gene expression. The roles of these RNAs in the pathogenesis of cystic fibrosis (CF); a lethal multisystem, autosomal recessive disorder have yet to be explored. Our aim was to examine the expression profile of lncRNA, in the airway epithelium of people with CF. We examined the expression of 30,586 lncRNAs by microarray (Human LncRNA Array v3.0, Arraystar, Inc), in vivo in bronchial cells isolated from endobronchial brushings obtained from CF and non-CF individuals. In total, we identified 1,063 lncRNAs with differential expression between CF and non-CF individuals (fold changeM-bM-^IM-%3, pM-bM-^IM-$0.001). The microarray also contained probes for ~26,109 protein coding transcripts, of which 720 were differentially expressed between CF and non-CF brush samples (fold changeM-bM-^IM-%3, pM-bM-^IM-$0.001). Confirmation of a selection of differentially expressed coding mRNA and lncRNA transcripts such as TLR8 and XIST was achieved using qRT-PCR. Gene ontology bioinformatics analysis, highlighted that many processes over-represented in the CF bronchial epithelium are related to inflammation. These data show a significantly altered lncRNA and mRNA expression profile in CF bronchial cells in vivo. Dysregulation of some of these lncRNAs may play important roles in the chronic infection and inflammation that exists in the lungs of people with CF. RNA was extracted from bronchial epithelial cells obtained from bronchial brushings at bronchoscopy. Three individual biological replicates from each group (Non-CF and CF) were profiled by microarray.
Project description:- Identification of proteins whose abundance was altered by two DNA methyltransferases (XvDMT1 and XvDMT2) in Xanthomonas campestris pv. vesicatoria strain 85-10. - Shotgun proteomic analysis was used - Three strains were used with three biological replicates (total 9 samples). W+V: the wild-type strain carrying an empty vector. 2165OE: XvDMT1-overexpressing strain. 2405OE: XvDMT2-overexpressing strain.
Project description:In order to gain a better understanding of the effects of Streptomyces coelicolor A3(2) wild type strain exposed to simulated microgravity condition by clinorotation, we constructed a whole genome microarray to screen simulated microgravity sensitive genes using the Agilent eArray 5.0 program. The genechip was custom designed according to the manufacturerM-bM-^@M-^Ys recommendations (Agilent, 8*15K). The genome sequences were downloaded from: http://www.ncbi.nlm.nih.gov/genome?Db =genome&Cmd = Search&Term = NC_003888, NC_003903.1, NC_003904.1. Each gene was represented by one 60-nt oligonucleotide probe, and 680 genes associated with morphological differentiation and secondary metabolism out of the total 8116 genes were replicated 9 times each. Cells of Streptomyces coelicolor A3(2) wild type strain were harvested from the colonies grown in JCM42 agar medium at 28M-BM-0C for 96h (4d) under simulated microgravity and 1g control conditions. Total RNA was extracted to conduct the microarray hybridization experiments. Three independent experiments were performed in which ASM_1A, ASM_2A, ASM_3A were the test group samples and ANG_1A, ANG_2A, ANG_3A were the control group samples. Differentially expressed genes were selected with PM-oM-<M-^\0.05, FCM-bM-^IM-%2.0 by T-test methods using the independent three sets data.