Project description:Six isolates of PT21/28 and six of PT32 were analysed by CGH using UBECarray3 microarrays (containing probes for E. coli K-12 str. MG1655 and O157:H7 str. EDL933 and Sakai) to define genotypic differences between phage types. gDNA from E.coli O157 str. Sakai was hybridised to all arrays to provide a universal control channel on all arrays.
Project description:Comparative analysis of EHEC O157:H7 Sakai vs. TW14359 secreted and cytosolic protein using iTRAQ and mass spectrometry on an LTQ Orbitrap. Samples were fractionated through strong cation exchange and HPLC before analsyis. For TW14359 sepD secretome analysis, strains were similarly prepared and analyzed. All samples were searched against a TW14359 specific protein database (Uniprot) coupled with randomized protein sequences.
Project description:Six isolates of PT21/28 and six of PT32 were analysed by CGH using UBECarray3 microarrays (containing probes for E. coli K-12 str. MG1655 and O157:H7 str. EDL933 and Sakai) to define genotypic differences between phage types. gDNA from E.coli O157 str. Sakai was hybridised to all arrays to provide a universal control channel on all arrays. gDNA from 12 PT 21/28 & 32 isolates were labelled with Cy5 and control gDNA from str. Sakai was labelled with Cy3. Test and control gDNA was hybridised to UBECarray3 microarrays. The LOWESS normalised relative signal to the Sakai control channel was used to compare between samples.
Project description:Food-borne illness arising for Shiga-toxigenic Escherichia coli is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. Attachment of the bacteria to host tissue is one of the first steps in the interaction, and, as with mammalian hosts, has shown to be mediated by a combination of non-specific and specific adhesin-mediated interactions. We took a high-throughput positive-selection approach to investigate adherence mechanisms for E. coli O157:H7 isolate Sakai by inoculating a BAC clone library onto spinach, which was quantified by microarray hybridisation and gene loci enrichment measured using a Bayesian hierarchical model. The screen involved four successive rounds of adherence to spinach roots, resulting in 115 CDS credible candidates, covered by seven contiguous genomic regions. Two candidates regions selected for functional assessment included a chaperone-usher fimbrial gene cluster (loc6) and the type two secretion system (T2SS). The TS22 was found to significantly enhance binding to spinach roots and leaves, demonstrated with a BAC-T2SS clone and by mutagenesis of the secretin protein, EtpD. Both etpD and the inner membrane anchor protein gene etpC were expressed at 18 degree celsius, and expression of etpD was demonstrated for STEC (Sakai) resident in the apoplastic spaces in spinach leaf tissue. Together, these data indicate a novel function for STEC T2SS in adherence to plant tissue. Experiment 1: screening E coli O157:H7 Sakai genes for adherence to spinach roots. A BAC library of Sakai clones in an E coli DH10B background (which has poor root adherence) defined as the 'Input pool', was incubated with spinach roots for 4 rounds of enrichment, defined as the 'Output pool'. Control samples (defined as 'Input control' & 'Output control') were cultures of pV41 vector only. DNA extractions from test pools were labelled with Cy3 throughout. DH10B DNA was used for grid alignment and labelled with Cy5 throughout.
Project description:Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of glutamate decarboxylase (GAD) system, the most efficient acid resistance mechanism in E. coli. The full contribution of GadE to the acid resistance and virulence of pathogenic E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles with that of wild type in exponential and stationary phases of growth using microarrays containing 6088 ORFs from three E. coli genomes. gadE inactivation significantly altered the expression of 60 genes independent of growth phase and 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly down-regulated the expression of gadA, gadB, gadC and many acid fitness island genes in a growth phase-dependent manner. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Altogether, our data indicate that GadE is critical for acid resistance of E. coli O157:H7 and plays an important role in virulence by down-regulating expression of LEE.