Abstract
An integrated transcriptomic and proteomic analysis was undertaken to determine the physiological response of Escherichia coli O157:H7 Sakai to steady-state conditions relevant to low temperature and water activity conditions experienced during meat carcass chilling in cold air. The response of E. coli during exponential growth at 25 °C a(w) 0.985, 14 °C a(w) 0.985, 25 °C a(w) 0.967, and 14 °C a(w) 0.967 was compared with that of a reference culture (35 °C a(w) 0.993). Gene and protein expression profiles of E. coli were more strongly affected by low water activity (a(w) 0.967) than by low temperature (14 °C). Predefined group enrichment analysis revealed that a universal response of E. coli to all test conditions included activation of the master stress response regulator RpoS and the Rcs phosphorelay system involved in the biosynthesis of the exopolysaccharide colanic acid, as well as down-regulation of elements involved in chemotaxis and motility. However, colanic acid-deficient mutants were shown to achieve comparable growth rates to their wild-type parents under all conditions, indicating that colanic acid is not required for growth. In contrast to the transcriptomic data, the proteomic data revealed that several processes involved in protein synthesis were down-regulated in overall expression at 14 °C a(w) 0.985, 25 °C a(w) 0.967, and 14 °C a(w) 0.967. This result suggests that during growth under these conditions, E. coli, although able to transcribe the required mRNA, may lack the cellular resources required for translation. Elucidating the global adaptive response of E. coli O157:H7 during exposure to chilling and water activity stress has provided a baseline of knowledge of the physiology of this pathogen.
Highlights
From the ‡Food Safety Centre, Tasmanian Institute of Agricultural Research, School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart TAS 7001, Australia; §Commonwealth Scientific and Industrial Research Organization (CSIRO) Food and Nutritional Sciences, PO Box 52, North Ryde NSW 1670, Australia; ¶CSIRO Food and Nutritional Sciences, PO Box 745, Archerfield BC QLD 4108, Australia
The present study employed both transcriptomic and proteomic analysis to investigate the physiology of E. coli O157:H7 Sakai grown under steady-state conditions of chill and/or osmotic stress, with the aim to obtain a comprehensive insight into the adaptation of this pathogen under the conditions mimicking that experienced on meat carcases during cold-air chilling
To enable comparison of transcriptomic and proteomic data, changes in gene and protein expression profiles of O157:H7 Sakai under four steady-state conditions of 25 °C aw 0.985, 14 °C aw 0.985, 25 °C aw 0.967, and 14 °C aw 0.967 were determined relative to the baseline response of a reference culture grown at near-optimal conditions for rapid growth (35 °C aw 0.993)
Summary
Division of Experimental Work—Transcriptomic and proteomic studies were conducted in parallel at the Commonwealth Scientific and Industrial Research Organization (CSIRO) Food and Nutritional Sciences (Brisbane, Australia) and Food Safety Centre, University of Tasmania (Hobart, Australia), respectively. Preparation of Cell Extracts for Proteomic Analysis—Parallel to the transcriptomic analysis, a 25 ml sample of each secondary culture at an OD600 of 0.1 Ϯ 0.01 was harvested by centrifugation at 4500 ϫ g for 10 min in a Hettich Zentrifugen EBA 12 centrifuge (Tuttlingen, Germany) operating at RT. The SpI method is based on the SpC data derived from each set of replicates (i.e. control and treatment) and the number of replicates for detectable peptides (i.e. number of replicates in which the peptides are detected) These features employ the robust statistical power of SpI to account for the variation in absolute number of SpC obtained in different 2D-LC/MS/MS runs, and those proteins with low spectral counts, which exhibit greater relative variability [27]. All proteins with a spectral index significant at the 95% confidence level were considered to be differentially expressed
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