Dataset Information

2D-LC/MS/MS analysis of Escherichia coli during exposure to an abrupt temperature downshift

ABSTRACT: 2D-LC/MS/MS analysis was used to examine time-dependent changes in proteome of E. coli O157:H7 strain Sakai upon an abrupt downshift in temperature (i.e., from 35°C to 14°C). Cell cultures were harvested at 30, 90, 160, and 330 min post-temperature downshift. It also should be noted that these time points were chosen with the aims to characterize the physiology of E. coli during dynamic changes in growth kinetics induced by an abrupt temperature downshift. Specifically, the samples taken at time 30 and 90 min were obtained during adaptation period, whereas the samples at time 160 and 330 min reflected the physiological state of E. coli during growth after the shift. 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%.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Escherichia Coli

SUBMITTER: Chawalit Kocharunchitt

LAB HEAD: Tom Ross

PROVIDER: PXD000573 | Pride | 2019-11-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	E.coliat160minafterTempshift-MEM-Rep1.mzXML	Mzxml
	E.coliat160minafterTempshift-MEM-Rep1.pep.xml	Pepxml
	E.coliat160minafterTempshift-MEM-Rep1.prot.xml	Xml
	E.coliat160minafterTempshift-MEM-Rep2.mzXML	Mzxml
	E.coliat160minafterTempshift-MEM-Rep2.pep.xml	Pepxml

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Publications

Global genome response of Escherichia coli O157∶H7 Sakai during dynamic changes in growth kinetics induced by an abrupt temperature downshift.

King Thea T Kocharunchitt Chawalit C Gobius Kari K Bowman John P JP Ross Tom T

PloS one 20140613 6

Escherichia coli O157∶H7 is a mesophilic food-borne pathogen. We investigated the growth kinetics of E. coli O157∶H7 Sakai during an abrupt temperature downshift from 35°C to either 20°C, 17°C, 14°C or 10°C; as well as the molecular mechanisms enabling growth after cold stress upon an abrupt downshift from 35°C to 14°C in an integrated transcriptomic and proteomic analysis. All downshifts caused a lag period of growth before growth resumed at a rate typical of the post-shift temperature. Lag and ...[more]

PMID: 24926786

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Project description:Escherichia coli O157 presents a number of specific problems in terms of food safety and public health. It has been found that E. coli O157 is more resistant to a number of the stresses encountered during food production such as heat, pH and osmotic shock. This greater resistance is thought to contribute to the low infectious dose of E. coli O157 (<100 organisms). Moreover, E. coli O157 is associated with debilitating conditions such as haemorrhagic colitis and haemoytic uraemic syndrome, particularly in children and the elderly. We have been studying the stress responses of E. coli O157:H7 (Sakai) and comparing with a commensal strain of E. coli K-12, MG1655. We found that E. coli O157 (Sakai) is more sensitive to oxidative stress than MG1655. A microarray study of these strains treated with sub-lethal concentrations (0.5mg/ml) of menadione revealed big differences in their responses. In E. coli O157 (Sakai), 540 genes responded significantly to the treatment compared to 121 genes in MG1655. One surprising finding from the microarray data was the observation that many iron-transport genes were up-regulated in E. coli O157 (Sakai) whereas relatively few were induced in MG1655 despite the fact that the bacteria were grown in a medium containing ample iron. We speculated that the induction of iron transport genes in an iron-rich medium might have contributed to the enhanced killing of E. coli O157 (Sakai) through triggering of a Fenton reaction. We speculated that the difference in sensitivity to oxidative stress might be due to differences in the intracellular iron content of E. coli O157 and MG1655. We found that E. coli O157 contains ~50% more iron than MG1655 and believe that during oxidative stress, this iron is released by damaged proteins. The greater levels of free iron in E. coli O157 will trigger a greater Fenton reaction that can damage the ferric uptake regulator (Fur), resulting in unregulated iron transport. In MG1655, the lower iron content results in a smaller Fenton reaction, enabling the cellular protection systems to limit damage and protect Fur.