Project description:The role of rpoS gene in the formation of Escherichia coli biofilms were investigated. The gene expression was compared among E. coli MG1655 wild type strain and rpoS knock-out strain in the biofilms, the planktonic exponential phase, and the planktonic stationary phase. The analysis revealed that the wild type bilfilms (WBF) showed similar pattern of gene expression with the WT planktonic stationary phase (WS), whereas the rpoS knock-out biofilms (MBF) showed similar pattern of gene expression with the wild type planktonic exponential phase (WE). Genes involved in the energy metabolism and the flagella synthesis showed higher expression in the rpoS knock-out biofilms (MBF), but not in the wild type biofilms (WBF). Moreover, genes involved in the stress responses showed higher expression in the wild type biofilms (WBF), but not in the rpoS knock-out biofilms (MBF). Keywords: cell type comparison (biofilms vs planktonic cells, wild type vs rpoS knock-out strains)
Project description:We investigate sigma factor binding regions for each sigma factor under various enviromental and/or genetic conditions. To measure sigma factor binding at a genome scale, we employed a ChIP-chip method to derivative strains of E. coli K-12 MG1655 wild type and its isogenic rpoS and rpoN knock-out strains under various conditions.
Project description:The role of rpoS gene in the formation of Escherichia coli biofilms were investigated. The gene expression was compared among E. coli MG1655 wild type strain and rpoS knock-out strain in the biofilms, the planktonic exponential phase, and the planktonic stationary phase. The analysis revealed that the wild type bilfilms (WBF) showed similar pattern of gene expression with the WT planktonic stationary phase (WS), whereas the rpoS knock-out biofilms (MBF) showed similar pattern of gene expression with the wild type planktonic exponential phase (WE). Genes involved in the energy metabolism and the flagella synthesis showed higher expression in the rpoS knock-out biofilms (MBF), but not in the wild type biofilms (WBF). Moreover, genes involved in the stress responses showed higher expression in the wild type biofilms (WBF), but not in the rpoS knock-out biofilms (MBF). Experiment Overall Design: Affymetrix E. coli antisense genome array was used to compare the gene expression among E. coli wild type and rpoS konck-out strains in the biofilms, the planktonic exponential phase, and the planktonic stationary phase. All samples were grown in MOPS minimal media with 0.2% glucose. Biofilms were grown for 72 hours on glass surface in flow cells (1x4x40 mm), and planktonic cells were grown for 8 hours (exponential phase) and 12 hours (stationary phase). Experiments were repeated 3 times, which resulted in 3 replicates of 6 different samples.
Project description:In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E. coli (EAEC)-specific virulence factors. Here, we identified an E. coli O104:H4 isolate that carried a single nucleotide polymorphism (SNP) in the start codon (ATG>ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG>ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E. coli O104:H4 Dstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG>ATA and DrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E. coli O104:H4 virulence, primarily through an AggR-dependent mechanism.
Project description:We investigate sigma factor binding regions for each sigma factor under various enviromental and/or genetic conditions. To measure sigma factor binding at a genome scale, we employed a ChIP-chip method to derivative strains of E. coli K-12 MG1655 wild type and its isogenic rpoS and rpoN knock-out strains under various conditions. A 45 ChIP-chip study under 4 separate culture conditions. The high-density oligonucleotide tiling arrays used were consisted of 371,034 oligonucleotide probes spaced 25 bp apart (25-bp overlap between two probes) across the E. coli genome.
Project description:This study is to identify the RpoS regulon in MG1655 in early exponential growth. RNA samples from wild type or rpoS mutants were extracted using acidic hot phenol method and hybridized to Affymetrix E. coli Antisense Genome Array. Keywords: Define regulon of RpoS
Project description:RpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in Escherichia coli and other γ-proteobacteria. RpoS is also involved in virulence of many pathogens including Salmonella and Vibrio species. Though well characterized in non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. E. coli O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of E. coli K12, and many of the additional genes are required for virulence. The genomic difference may result in substantial changes in RpoS-regulated gene expression. To test this, we compared the transcriptional profile of wild type and rpoS mutants of the E. coli O157:H7 EDL933 type strain. The rpoS mutation had a pronounced effect on gene expression in stationary phase, and more than 1,000 genes were differentially expressed (two-fold, p<0.05). By contrast, we found 11 genes expressed differently in exponential phase. Western blot analysis revealed that, as expected, RpoS level was low in exponential phase and substantially increased in stationary phase. The defect in rpoS resulted in impaired expression of genes responsible for stress response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler, espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the rpoS mutants under LEE induction conditions. Our results show that RpoS has a profound effect on global gene expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS regulon, including many EDL933-specific genes, differs substantially from that of laboratory K12 strains. In this study, we characterized the RpoS regulon of E. coli O157:H7 strain EDL933 using microarray analysis.
Project description:Investigation of whole genome gene expression level changes in a Escherichia coli MG1655 K-12 ?fnr mutant, compared to the wild-type strain. The mutations engineered into this strain produce a strain lacking the FNR protein. WT strains were grown under aerobic and anaerobic growth conditions.
Project description:Expression profiling of wild type and purR deletion strains of E. coli K-12 MG1655 under both M9 minimal media and addition of adenine.
Project description:ChIP-seq to identify sigma38 binding sites in wild-type and delta ssrS (6S RNA knockout) strains of E. Coli K-12 MG1655, during stationary phase