Project description:Background: Based on 32 Escherichia coli and Shigella genome sequences, we have developed an E. coli pan-genome microarray. Publicly available genomes were annotated in a consistent manor to define all currently known genes potentially present in the species. The chip design was evaluated by hybridization of DNA from two sequenced E. coli strains, K-12 MG1655 (a commensal) and O157:H7 EDL933 (an enterotoxigenic E. coli). A dual channel and single channel analysis approach was compared for the comparative genomic hybridization experiments. Moreover, the microarray was used to characterize four unsequenced probiotic E. coli strains, currently marketed for beneficial effects on the human gut flora. Results: Based on the genomes included in this study, we were able to group together 2,041 genes that were present in all 32 genomes. Furthermore, we predict that the size of the E. coli core genome will approach ~1,560 essential genes, considerably less than previous estimates. Although any individual E. coli genome contains between 4,000 and 5,000 genes, we identified more than twice as many (11,872) distinct gene groups in the total gene pool (âpan-genomeâ) examined for microarray design. Benchmarking of the design based on sequenced control strain samples demonstrated a high sensitivity and relatively low false positive rate. Moreover, the array was highly sufficient to investigate the gene content of apathogenic isolates, despite the strong bias towards pathogenic E. coli strains that have been sequenced so far. Our analysis of four probiotic E. coli strains demonstrate that they share a gene pool very similar to the E. coli K-12 strains but also show significant similarity with enteropathogenic strains. Nonetheless, virulence genes were largely absent. Strain-specific genes found in probiotic E. coli but absent in E. coli K12 were most frequently phage-related genes, transposases and other genes related to mobile DNA, and metabolic enzymes or factors that may offer colonization fitness, which together with their asymptomatic nature may explain their nature. Conclusion: This high-density microarray provides an excellent tool for characterizing either DNA content or gene expression from unknown E. coli strains. Factorial design: Each of four test samples (G 1/2, G3/10, G 4/9, G5) are co-hybridized with two control strain samples (K-12 MG1655 and O157:H7 EDL933). Additional replicate co-hybridizations are included of the two control strain samples (O157:H7 EDL933 vs. K-12 MG1655).

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. A precise rpoS deletion mutant of EDL933 was constructed and employed in this study. EDL933 wild type and rpoS mutants were inoculated in triplicate into LB media at a starting OD of 0.0001 and grown aerobically at 37C. Cultures were harvested at OD600 = 0.3 in exponential phase and OD600=1.5 in stationary phase. For RNA extraction, cultures were mixed directly with a boiling lysis buffer containing SDS and EDTA followed by acidic hot phenol to minimize RNA degradation. RNA samples were hybridized to Affymetrix E. coli Genome 2.0 Array according to Affymetrix's standard protocols.

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.

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Global transcriptional and phenotypic analyseis of Escherichia coli O157:H7 strain Xuzhou21 and its pO157_Sal cured mutant

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