Project description:Directional RNAseq analyses was undertaken in Klebsiella pneumoniae Ecl8 and isogenic mutants Ecl8delta ramA and Ecl8delta ramR to determine the RamA regulon. All samples were grown in Luria Bertani broth until OD600 approx 0.6 prior to RNA extraction. Differential expression was determined using DEseq upon pairwise comparisons of Ecl8 vs Ecl8delta ramA, Ecl8deltaramA vs Ecl8deltaramR, Ecl8 vs Ecl8deltaramR.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:RarA is an AraC-type regulator in Klebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both the acrAB and oqxAB efflux genes. Increased rarA expression has also been shown to be integral in the development of tigecycline resistance in the absence of ramA in K. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8?rarA/pACrarA-2 (rarA-expressing construct) and Ecl8?rarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences of K. pneumoniae MGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show that rarA overexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141, sdaCB, and leuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g., nuoA, narJ, and proWX) were found to be downregulated. Biolog phenotype analyses demonstrated that rarA overexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype of K. pneumoniae.
Project description:To get a high resolution understanding of the effect of Fur on global gene expression, we compared by high-resolution RNAseq the transcriptomes of a wild-type E. coli K-12 strain and its Fur deletion derivative grown in minimal medium with or without supplementation of iron. Three independent total RNA extraction and RNAseq assays were performed for each strain in each condition.
Project description:Investigation of whole genome gene expression level changes in E. coli O103:H25 in co-culture with Bacteroides thetaiotaomicron CCUG 10774 compared to when E. coli is cultured individually.
Project description:Regulation of EHEC gene expression by the gut commensal bacterium B. thetaiotaomicron. EHEC is a human pathogen that colonizes in the colon where B. thetaiotaomicron is a predominant commensal. We used microarrays to evaluate global regulation of EHEC when cultured with B. thetaiotaomicron.
Project description:Helicobacter pylori encounters a wide range of pH within the human stomach. In a comparison of H. pylori cultured in vitro under neutral or acidic conditions, about 15% of genes are26 differentially expressed, and corresponding changes are detectable for many of the encoded proteins. The ArsRS two-component system (TCS), comprised of the sensor kinase ArsS and its cognate response regulator ArsR, has an important role in mediating pH-responsive changes in H. pylori gene expression. In this study, we sought to delineate the pH-responsive ArsRS regulon and further define the role of ArsR in pH-responsive gene expression. We compared H. pylori strains containing an intact ArsRS system with an arsS null mutant or strains containing site32 specific mutations of a conserved aspartate residue (D52) in ArsR, which is phosphorylated in response to signals relayed by the cognate sensor kinase ArsS. We identified 178 genes that were pH-responsive in strains containing an intact ArsRS system but not in ∆arsS or arsR mutants. These constituents of the pH-responsive ArsRS regulon include genes involved in acid acclimatization (ureAB, amidases), oxidative stress responses (katA, sodB), transcriptional regulation related to iron or nickel homeostasis (fur, nikR), and genes encoding outer membrane proteins [including sabA, alpA, alpB, hopD (labA), and horA]. When comparing H. pylori strains containing an intact ArsRS TCS with arsRS mutants, each cultured at neutral pH, relatively few genes are differentially expressed. Collectively, these data suggest that ArsRS41 mediated gene regulation has an important role in H. pylori adaptation to changing pH conditions.
Project description:The Bradyrhizobium japonicum NtrC regulatory protein influences gene expression in response to changes in intracellular nitrogen status. Under conditions of low nitrogen, phosphorylation of NtrC results in up-regulation of a number of genes involved in nitrogen metabolism and nitrogen acquisition. To better define the exact nature of NtrC’s influence on gene expression, a ntrC mutation was created in B. japonicum and transcriptional profiling was performed by DNA microarray analysis of both the mutant and wild type strains.
Project description:The Bradyrhizobium japonicum NtrC regulatory protein influences gene expression in response to changes in intracellular nitrogen status. Under conditions of low nitrogen, phosphorylation of NtrC results in up-regulation of a number of genes involved in nitrogen metabolism and nitrogen acquisition. To better define the exact nature of NtrC’s influence on gene expression, a ntrC mutation was created in B. japonicum and transcriptional profiling was performed by DNA microarray analysis of both the mutant and wild type strains.