Project description:Transcript abundance in Escherichia coli O157:H7 was determined in the presence or absence of pulsed expression of the small RNA, AsxR. AsxR was cloned under the control the arabinose inducible promoter Para. Escherichia coli O157:H7 str. TUV93-0 with pAsxR or empty vector was cultured in MEM-HEPES media to an OD600 of 0.8 and 0.2% arabinose added. 10min after addition of arabinose 10ml of cells were harvested and and pellets resuspended in 1ml of Trizol and total RNA isolated. RNAs were labelled using the SuperScript Plus indirect cDNA labelling System. Triplicate control RNAs were pooled and hybridised to seperate AsxR test RNAs on three microarays. Arrays were hybridised using the Maui hybridisation platform and Scann using and Axon Autoloader Scanner. GenePix software was used to analyse images and GPR files were analysed using Genespring 7.3.1.
Project description:Cinnamaldehyde is a natural antimicrobial and has been found to be effective against many foodborne pathogens including Escherichia coli O157:H7. Although its antimicrobial effects have been well investigated, limited information is available on its effects at the molecular level. Sublethal treatment at 200 mg/l cinnamaldehyde inhibited growth of E. coli O157:H7 at 37oC and for ≤ 2 h caused cell elongation, but from 2 to 4 h growth resumed and cells reverted to normal length. To understand this transient behaviour, genome-wide transcriptional analysis of E. coli O157:H7 was performed at 2 and 4 h exposure to cinnamaldehyde. Drastically different gene expression profiles were obtained at 2 and 4 h. At 2 h exposure, cinnamaldehyde induced overexpression of many oxidative stress-related genes, reduced DNA replication, and synthesis of protein, O-antigen and fimbriae. At 4 h, many cinnamaldehyde-induced repressive effects on E. coli O157:H7 gene expressions were reversed and oxidatve stress genes were nolonger differentially expressed. Duplicate E. coli O157:H7 cultures with or without 200 mg/l cinnamaldehyde were incubated at 37°C for ≤ 4 h. Cinnamaldehyde-induced changes in gene expression profiles were compared at 2 and 4 h using Affymetrix Ginechip 2.0 microarrays.
Project description:Deletion of yhaO was found to signifcantly decrease type three secretion in EHEC O157:H7. Transcriptional profiles of Escherichia coli O157: H7 and the isogenic yhaO mutant were generated and compared. Overall design: RNA was extracted from biological tripicate flasks and labelled with Cy3 or Cy5. Reference RNAs (w.t. TUV93-0) were pooled and divided into 80pmol aliquots for hybridisation. 80pmol of test Cy3 labelled cDNA was mixed with 80 pmol of pooled reference Cy5 labelled cDNA and hybrised to a single micorarray. Triplicate microarrays were scanned and data analysed using Genespring GX 7.3
Project description:Deletion of yedL was found to signifcantly decrease type three secretion in EHEC O157:H7. Transcriptional profiles of Escherichia coli O157: H7 and the isogenic yedL mutant were generated and compared. Overall design: RNA was extracted from biological tripicate flasks and labelled with Cy3 or Cy5. Reference RNAs (w.t. TUV93-0) were pooled and divided into 80pmol aliquots for hybridisation. 80pmol of test Cy3 labelled cDNA was mixed with 80 pmol of pooled reference Cy5 labelled cDNA and hybrised to a single micorarray. Triplicate microarrays were scanned and data analysed using Genespring GX 7.3
Project description:Leafy green vegetables, such as lettuce, have been increasingly implicated in outbreaks of foodborne illnesses due to contamination by Escherichia coli O157:H7. While E. coli can survive in soils, colonize plants, and survive on produce, very little is known about the interaction of E. coli with the roots of growing lettuce plants. In these studies, a combination of microarray analyses and surface enhanced Raman spectroscopy (SERS) were used to gain a comprehensive understanding of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots and compared to E. coli K12 using a hydroponic system (HS) which we have reported in the previous studies. Using microarray, after three days of interaction with lettuce roots, 94 and 109 genes of E. coli O157:H7 were significantly up-regulated and down-regulated at least 1.5 fold, respectively. Only 8 genes were also found in the E. coli K12 up-regulated genes. No genes were found in the down-regulated genes clusters between those two strains. For E. coli O157:H7, forty out of the 94 up-regulated genes (43%) were involved in protein synthesis and were highly repressed compared to 40 out of 193 (23%) E. coli K12 up-regulated genes associated with protein synthesis. The wildtype of E.coli O157:H7 colonized two log CFU per root less compared to E. coli K12. Genes involved in biofilm modulation (bhsA and ybiM) were significantly up-regulated in E. coli O157:H7 and curli production (crl and csgA) were found important for E. coli K12 to attach to lettuce roots in the previous studies. BhsA mutant of E. coli O157:H7 was impaired in the colonization of lettuce roots. The SERS spectra of E. coli K12 and O157 controls (cells without interacting with roots) were very similar. The spectra of E. coli K12 and O157 exposed to the hydroponic system (HS) showed some differences in the nucleic acid, protein, and lipid regions compared with controls. The spectra of E. coli K12 HS cells exhibited significant differences compared to spectra from E. coli O157 HS cells in the RNA and protein regions. The overall band intensity of amide regions declined for E. coli O157 HS cells, while it increased for E. coli K12 HS cells. The intensity of the RNA bands of E. coli K12 HS cells were also found much higher than those of E. coli O157 HS cells. These findings were in agreement to our Microarray data. Our microarray and SERS data showed that E. coli K12 and O157:H7 behavior dramatically differently in colonizing on lettuce roots. Compared to K12, E. coli O157:H7 colonized less efficiently on lettuce roots. Escherichia coli O157:H7 strains were grown in the lettuce rhizosphere for three days. Transcriptional profiling of E. coli was compared between cells grown with and without rhizosphere . Three biological replicates of each treatment were prepared, and six microarray slides were used.
Project description:Escherichia coli O157:H7 can cause haemorrhagic colitis and haemolytic uremic syndrome (HUS) in humans. This pathogen has been implicated in large food-borne outbreaks all over the world. By investigating the implicated salted salmon roe, Makino et al. (2000) suggested that E. coli O157:H7 in the viable but nonculturable (VBNC) state should be the culprit of the outbreak in Japan. High pressure CO2 (HPCD), one of the non-thermal pasteurization techniques, is an effective means to inactivate microorganisms. But in the previous study, we have demonstrated for the first time that HPCD could induce E. coli O157:H7 into the VBNC state, which poses a potential health risk to HPCD-treated products. In order to explore the potential formation mechanisms of VBNC E. coli O157:H7 induced by HPCD, the high-throughput Illumina RNA-seq transcriptomic analysis was conducted for E. coli O157:H7 cells treated at 5 MPa and 25 ℃ for 40 min (VBNC cells) and exponential-phase cells (the control). Finally, 97 genes that differentially transcribed between VBNC state and the control were obtained, with 22 genes up-regulated and 75 genes down-regulated in VBNC cells. These differentially expressed genes were classified in a variety of functional categories, including central metabolic processes, gene replication and expression, cell division, general stress response, respiration, membrane biosynthesis and transport and pathogenicity. Based on these differentially expressed genes, we suggest putative formation mechanisms of VBNC cells induced by HPCD. The finding will provide theoretical foundation for restraining the VBNC state formation under HPCD processing. Overall design: Whole mRNA profiles of Escherichia coli O157:H7 in the VBNC state and the exponential phase were generated using Illumina sequencing technology and differentially expressed genes were anylyzed.
Project description:The transcriptome of Escherichia coli K-12 has been widely studied over a variety of conditions for the past decade while such studies involving E. coli O157:H7, its pathogenic cousin, are just now being conducted. To better understand the impact of intracellular life within a ruminant and environmental protozoan on E. coli O157:H7, global transcript levels of strain EDL933 cells inside Acanthamoeba were compared to cell grown in the protozoan media (ATCC PYG712) by microarray. Overall design: Nine independent RNA samples from cell from within Acanthamoeba were paired with seven independent RNA samples from control cultures for hybridization to nine two-color microarrays. For four arrays, the control RNA sample was labeled with Cy3 dye and the experimental RNA sample was labeled with Cy5 dye, the dyes were reversed for the other five arrays to overcome dye bias.
Project description:Pathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents. To identify non-toxic biofilm inhibitors for enterohemorrhagic Escherichia coli O157:H7, indole-3-acetaldehyde was used and reduced E. coli O157:H7 biofilm formation. Global transcriptome analyses revealed that indole-3-acetaldehyde most repressed two curli operons, csgBAC and csgDEFG, and induced tryptophanase (tnaAB) in E. coli O157:H7 biofilm cells. Electron microscopy showed that indole-3-acetaldehyde reduced curli production in E. coli O157:H7. Together, this study shows that Actinomycetales are an important resource of biofilm inhibitors as well as antibiotics. For the microarray experiments, E. coli O157:H7 EDL933 was inoculated in 100 ml of LB in 250 ml flasks with overnight cultures that were diluted at 1:100. Cells were shaken with 10g of glass wool at 250 rpm and 37°C for 7 hrs. Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80°C. RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA). To eliminate DNA contamination, Qiagen RNase-free DNase I was used to digest DNA. RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).
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:The transcriptome of Escherichia coli K-12 has been widely studied over a variety of conditions for the past decade while such studies involving E. coli O157:H7, its pathogenic cousin, are just now being conducted. To better understand the impact of an anaerobic environment on E. coli O157:H7, global transcript levels of strain EDL933 cells grown aerobically were compared to cells grown anaerobically using microarrays. Overall design: Eight independent RNA samples from aerobic cultures were paired with eight independent RNA samples from anaerobic cultures for hybridization to eight two-color microarrays. For four arrays, the control RNA sample was labeled with Cy3 dye and the experimental RNA sample was labeled with Cy5 dye, the dyes were reversed for the other six arrays to overcome dye bias.