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UV-crosslinking and high throughput sequencing of cDNAs (CRAC) of the RNA chaperone Hfq in enterohaemorhaggic E. coli

ABSTRACT: UV-crosslinking and high througput sequencing of cDNAs (CRAC) was used to map the binding sites Hfq in enterohaemorhaggic E. coli (EHEC). Hfq was tagged with a His-FLAG dual affintiy tag and UV crosslinked after growth in the MEM-HEPES media essentailly as per Granneman et al (2009) PNAS. We additionally crosslinked Hfq in non-pathogenic E. coli K12 str. MG1655 grown in LE media. Hfq-RNA complexes were purified and trimmed using RNase A/T1. RNA fragments were isolated and converted to cDNA, PCR amplified and sequenced using Illumina Solexa GAxII and HiSeq2000 platforms. His-FLAG tagged Hfq and untagged controls were cultured to an OD of 0.8 and crosslinked with UV-C. Five replicates of tagged EHEC Hfq and 2 replicates of untagged Hfq were crosslinked. We have additionally crosslinked 2 replicates each of E. coli K12 tagged and untagged Hfq.

ORGANISM(S): Escherichia coli O157:H7 str. Sakai

SUBMITTER: Jai Tree

PROVIDER: E-GEOD-46118 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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UV-crosslinking and high throughput sequencing of cDNAs (CRAC) of the RNA chaperone Hfq in enterohaemorhaggic E. coli

Project description:UV-crosslinking and high througput sequencing of cDNAs (CRAC) was used to map the binding sites Hfq in enterohaemorhaggic E. coli (EHEC). Hfq was tagged with a His-FLAG dual affintiy tag and UV crosslinked after growth in the MEM-HEPES media essentailly as per Granneman et al (2009) PNAS. We additionally crosslinked Hfq in non-pathogenic E. coli K12 str. MG1655 grown in LE media. Hfq-RNA complexes were purified and trimmed using RNase A/T1. RNA fragments were isolated and converted to cDNA, PCR amplified and sequenced using Illumina Solexa GAxII and HiSeq2000 platforms.

2014-05-22 | GSE46118 | GEO

2D-LC/MS/MS analysis of Escherichia coli during exposure to abrupt downshifts in temperature and water activity

Project description:2D-LC/MS/MS analysis was used to examine time-dependent changes in proteome of E. coli O157:H7 strain Sakai upon abrupt downshifts from 35°C aw 0.993 to 14°C aw 0.967. Bacterial cells were harvested before abrupt downshifts in both temperature and aw (i.e. control), or at 0 (i.e. immediately after the shift), 60, 250, 1605, 4,070, 5,700, 9,900 or 18,565 min after the shifts.

2016-09-29 | PXD004803 | Pride

Cell Surface Proteome during Enterohaemorrhagic E. coli infection

Project description:Proteins on the cell surface are crucial for the interaction of a cell with its surrounding environment. Viral infection is known to remodel to the host cell surface to the benefit of the pathogen, but relatively little is known about how bacterial pathogens alter the host cell surface. Enterohaemorrhagic E. coli(EHEC) infects the apical surface of gut epithelium, and maniplates multiple aspects of host physiology. Here we used quantitative cell surface proteomics to investigate EHEC-induced changes to the host cell surface and show that the complement regulatory protein CD55 is cleaved from epithelial surfaces by the EHEC metalloprotease StcE. As a consequence of this, neutrophil attachment to the apical surface of epithelial cells is increased. This study is the first to apply quantitaive cell surface proteomics to EHEC infection and reveals a novel mechanism by which EHEC manipulates the host immune system.

2018-09-10 | PXD010361 | Pride

2D-LC/MS/MS analysis of Escherichia coli under optimal growth condition

Project description:2D-LC/MS/MS analysis was used to examine the proteome of E. coli O157:H7 strain Sakai during exponential growth under optimal condition (i.e., from 35°C aw 0.993). MS/MS data obtained from each protein sample were processed by the Computational Proteomics Analysis System (CPAS), a web-based system built on the LabKey Server (v9.1, released 02.04.2009). The experimental mass spectra produced were subjected to a semi-tryptic search against the combined databases of E. coli O157:H7 Sakai (5,318 entries in total) downloaded from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, downloaded 25.11.2008) using X!Tandem v2007.07.01. These databases included the E. coli O157:H7 Sakai database (5230 entries, NC_002695.fasta) and two E. coli O157:H7 Sakai plasmid databases, plasmid pO157 (85 entries, NC_002128.fasta) and plasmid pOSAK1 (three entries, NC_002127.fasta). The parameters for the database search were as follows: mass tolerance for precursor and fragment ions: 10 ppm and 0.5 Da, respectively; fixed modification: cysteine cabamidomethylation (+57 Da); and no variable modifications. The search results were then analyzed using the PeptideProphet and ProteinProphet algorithms from the Trans Proteomic Pipeline v3.4.2. All peptide and protein identifications were accepted at PeptideProphet and ProteinProphet of ≥0.9, corresponding to a theoretical error rate of ≤2%.

2019-12-04 | PXD000583 | Pride

Global gene expression analysis of Yersinia pestis Ype AHL quorum sensing at 30Â°C

Project description:Quorum sensing is a cell to cell communication process that involves chemical signaling. Yersinia pestis, the agent of plague, has two functional AHL quorum sensing systems Ysp and Ype. For several reasons, it was not clear what effect AHL pathways have on virulence gene expression and survival in the two different hosts, flea and human. To investigate to what effect AHL quorum sensing has on gene expression, we conducted microarray studies comparing Yersinia pestis CO92 (â??pgm) to a double AHL mutant strain (â??pgm Î?ypeIR) at 30Â°C. Six independent RNA samples from Y. pestis CO92 Î?pgm Î?ypeIR cultures were paired with six independent RNA samples from control Y. pestis CO92 R88 cultures for hybridization to six two-color microarrays. For three arrays, the control RNA sample was labeled with Alexa 555 dye and the experimental RNA sample was labeled with Alexa 647 dye; the dyes were reversed for the other three arrays to account for any dye bias.

2016-07-03 | E-GEOD-30332 | biostudies-arrayexpress

Global gene expression analysis of Yersinia pestis Ysp AHL quorum sensing at 37Â°C

Project description:Quorum sensing is a cell to cell communication process that involves chemical signaling. Yersinia pestis, the agent of plague, has two functional AHL quorum sensing systems Ysp and Ype. For several reasons, it was not clear what effect AHL pathways have on virulence gene expression and survival in the two different hosts, flea and human. To investigate to what effect Ysp AHL quorum sensing has on gene expression, we conducted microarray studies comparing Yersinia pestis CO92 (âpgm) to a single AHL mutant strain (âpgm ÎyspI) at 37Â°C. Six independent RNA samples from Y. pestis CO92 Îpgm ÎyspI cultures were paired with six independent RNA samples from control Y. pestis CO92 R88 cultures for hybridization to six two-color microarrays. For three arrays, the control RNA sample was labeled with Alexa 555 dye and the experimental RNA sample was labeled with Alexa 647 dye; the dyes were reversed for the other three arrays to account for any dye bias.

2016-07-03 | E-GEOD-30328 | biostudies-arrayexpress

Global gene expression analysis of Yersinia pestis Ysp AHL quorum sensing at 30Â°C

Project description:Quorum sensing is a cell to cell communication process that involves chemical signaling. Yersinia pestis, the agent of plague, has two functional AHL quorum sensing systems Ysp and Ype. For several reasons, it was not clear what effect AHL pathways have on virulence gene expression and survival in the two different hosts, flea and human. To investigate to what effect Ysp AHL quorum sensing has on gene expression, we conducted microarray studies comparing Yersinia pestis CO92 (âpgm) to a single AHL mutant strain (âpgm ÎyspI) at 30Â°C. Six independent RNA samples from Y. pestis CO92 Îpgm ÎyspI cultures were paired with six independent RNA samples from control Y. pestis CO92 R88 cultures for hybridization to six two-color microarrays. For three arrays, the control RNA sample was labeled with Alexa 555 dye and the experimental RNA sample was labeled with Alexa 647 dye; the dyes were reversed for the other three arrays to account for any dye bias.

2016-07-03 | E-GEOD-30329 | biostudies-arrayexpress

Transcriptional analysis of ypeIR mutation in Yersinia pestis at 37Â°C

2016-07-03 | E-GEOD-30341 | biostudies-arrayexpress

Global gene expression analysis of Yersinia pestis AHL quorum sensing at 37Â°C

Project description:Quorum sensing is a cell to cell communication process that involves chemical signaling. Yersinia pestis, the agent of plague, has two functional AHL quorum sensing systems Ysp and Ype. For several reasons, it was not clear what effect AHL pathways have on virulence gene expression and survival in the two different hosts, flea and human. To investigate to what effect AHL quorum sensing has on gene expression, we conducted microarray studies comparing Yersinia pestis CO92 (âpgm) to a double AHL mutant strain (âpgm ÎypeIR ÎyspIR) at 37Â°C. Six independent RNA samples from Y. pestis CO92 R114 AHL deficient cultures were paired with six independent RNA samples from control Y. pestis CO92 R88 cultures for hybridization to six two-color microarrays. For three arrays, the control RNA sample was labeled with Alexa 555 dye and the experimental RNA sample was labeled with Alexa 647 dye; the dyes were reversed for the other three arrays to account for any dye bias.

2016-07-03 | E-GEOD-30331 | biostudies-arrayexpress

Yersinia pestis uses AHL-based quorum sensing to regulate metabolic functions likely to be important for flea colonization

Project description:The etiologic agent of bubonic plague, Yersinia pestis, senses cell density-dependent chemical signals to synchronize transcription between cells of the population in a process named quorum sensing. Though the closely related enteric pathogen Y. pseudotuberculosis uses quorum sensing system to regulate motility, the role of quorum sensing in Y. pestis has been unclear. In this study we performed transcriptional profiling experiments to identify Y. pestis quorum sensing regulated functions. Our analysis revealed that acyl-homoserine lactone based quorum sensing controls the expression of several metabolic functions. Maltose fermentation and the glyoxylate bypass are induced by acyl-homoserine lactone signaling. This effect was seen to be temperature conditional. Metabolism is unresponsive to quorum sensing regulation at mammalian body temperature, indicating a potential role for quorum sensing regulation of metabolism specifically during colonization of the flea vector. It is proposed that utilization of alternative carbon sources may enhance growth and/or survival during prolonged flea colonization, contributing to maintenance of plague in nature. Six independent RNA samples from Y. pestis CO92 R114 AHL deficient cultures were paired with six independent RNA samples from control Y. pestis CO92 R88 cultures for hybridization to six two-color microarrays. For three arrays, the control RNA sample was labeled with Alexa 555 dye and the experimental RNA sample was labeled with Alexa 647 dye; the dyes were reversed for the other three arrays to account for any dye bias.

2016-07-03 | E-GEOD-29240 | biostudies-arrayexpress

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