An investigation into the global cellular responses of listeria monocytogenes during the transition from exponential to stationary growth phase

Contamination of foodstuffs by microbial pathogens is a major ongoing concern in the food industry. Consequently, an understanding of the biological mechanisms that permit microorganisms to survive in food environments designed to preclude their growth is highly desirable. The aim of this study was to use a state-of-the-art proteomic approach to identify those cellular processes that enable Listeria monocytogenes to exhibit enhanced resistance to stress whilst in stationary phase. The following investigation relates functional competence (survival of the organism in food environment) to concerted regulation of gene expression via systematic identification of key changes in expression of specific genes or related groups of genes during entry into stationary phase. A defined medium was shown to support the growth of Listeria monocytogenes Scott A to stationary phase. Monitoring amino acid and glucose utilisation within the medium has differentiated, exponential growth, stationary phase and growth arrest. Flow cytometry has been used as a means of defining the point of onset of stationary phase by monitoring the growth phase specific effects of a nisin challenge. The point at which an identifiable change in growth rate could be attributed to the onset of stationary phase occurred at an OD600 of 0.75. Using a proteomic platform with 2-D SDS-PAGE as the core technology, consistent 'coverage' and protein resolution of the Listeria proteome at pH 4 - 7 separating range has been achieved. With this system up to 1200 discrete proteins were routinely observed. A comparison of functional gene (protein) expression during exponential growth and transition of the organism into stationary phase at specific time points has been undertaken. Analysis of the resulting gel images has revealed global changes in gene expression during that transition Overall, 64% of all proteins visualised showed a change in expression indicating that Listeria monocytogenes cells undergo a radical physical adaptation in preparation for stationary phase and growth arrest. Ten proteins showing major quantitative change throughout growth have been identified using MALDI MS. The expression of each identified at fixed time points throughout growth and stationary phase has been demonstrated and shows good correlation with the expected patterns of expression. Additional experimental evidence supporting the role for quorum sensing in the onset of stationary phase of Listeria monocytogenes batch cultures is presented. The combined growth characterisation, medium utilisation and flow data also indicated the presence of subpopulations of cells responding to changing medium conditions and population growth. This may be a general indication that the culture is demonstrating phase shift behaviour. The identification and characterisation of further growth phase specific proteins should lead to a greater understanding of the physiology of the stress response and a possible strategy for more effective microbial control strategies. This investigation has highlighted several gene products important to the early onset of stationary phase and shown that subpopulations may be present that expand the range of environmental conditions in batch culture over which the organism may survive