Chromatin immunoprecipitation of Escherichia coli HNS-FLAG pchABC, pchBC and Ler mutants to determine Pch and H-NS distribution
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ABSTRACT:
ORGANISM(S): Escherichia coli
SUBMITTER: Toru Tobe
PROVIDER: E-TABM-818 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Chromatin immunoprecipitation of EHEC to determine Ler and H-NS distribution
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Project description:Enterohaemorrhagic Escherichia coli (EHEC) is an emerging pathogen that causes diarrhea and heamolytic uremic syndrome. Much of the genomic information that affects virulence is acquired via horizontal transfer. Genes necessary for attaching and effacing lesions are located in the LEE pathogenicity island. LEE gene transcription is positively regulated by Ler, which is also encoded by the LEE, and by Pch regulators, which are encoded at other loci. We identified genes whose transcription profiles were similar to those of the LEE genes, by comparing the effects of altering ler and pch transcript levels. We assigned these genes into two classes, according to their transcription profiles. 13 sets of comparison between transcription profiles in strains with different pch or ler genotypes. Labelling of cDNA and hybridization were performed twice with independently prepared RNAs.
2010-05-26 | E-GEOD-9752 | biostudies-arrayexpress
Project description:Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked AW1.7 or GGG10 cultures was performed to compare gene expression of these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR (q-PCR). DnaK, 30S and 50S risobomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50M-BM-0C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Gene expression of a heat resistant strain, E. coli AW1.7, was compared to gene expression in a heat sensitive strain, E coli GGG10. RNA was isolated from late exponential cultures, or from late exponential cells heat-shocked by exposure to 50M-BM-0C for 15 min. Three independent biological repeats were analyzed, and technical repeats (dye-swap) were performed for two of three biological repeats.
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Project description:Among the most important regulators of gene expression in bacteria are 'nucleoid-associated proteins'. These proteins alter the topology of the bound DNA by bending, wrapping or bridging it, thus having multiple effects, including transcriptional regulation, on the bacterial cell. Among the best-studied nucleoid proteins are H-NS and Fis, which bind to specific sequences on the DNA. H-NS is a global repressor of gene expression. Fis alters the global conformation of the DNA by introducing branched structures in it; but its effect on gene expression on a genomic scale remains largely unclear.<br><br>Several bacterial transcriptional regulators including H-NS and Fis have been studied using ChIP-chip. However, the higher resolution and dynamic range offered by ChIP-Seq have not been exploited for any bacterial species. By performing ChIP-Seq of these two proteins, we present the first such study in a bacterium. In addition to providing a proof-of-principle for the use of this technology for bacteria, we perform our study at multiple time-points during growth in rich medium, thus generating new insights into how these proteins function under different cellular conditions. Further, by analysing our data in conjunction with newly-generated gene expression and RNA polymerase-chromosome interaction data we provide new interpretation of the genome-scale patterns of the interactions of these proteins to the DNA.
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Project description:We report that inhibition of rho by treatment with bicyclomycin, a potential inhibitor of rho affects H-NS binding to chromosome across most of the binding sites. For the current study, we have flag tagged H-NS by homologous recomination method. Cells were grown in the presence and absence of antibiotic bicylomyin. Chromatin immunoprecipitation was performed and anti flag antibody was used for immunoprecipitation. Immunoprecipitated samples both from BCM- and BCM + conditions were sequenced using HiSeq 1000 model. Input DNA was used as control for this experiment which was also sequenced. The 50-mer reads obtained were then mapped to Escherichia coli Mg1655 K-12 genome. Further analysis was carried out to calculate the ChIP signal score. We observed a drop in ChIP signal for bcm+ condition as compared to bcm-. ChIP-seq data generated by Hiseq 1000 model of samples with bcm+ and bcm- in biological duplicates along with Input DNA as control
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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.
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