Characterisation of salt and cold stress response mechanisms in Listeria monocytogenes as revealed by whole genome microarray analysis

This study was conducted to investigate the genetic adaptation of Listeria monocytogenes to hyperosmotic (created by high NaCl concentrations) conditions and low-temperature stress. L. monocytogenes is an opportunistic pathogenic bacterium capable of causing serious systemic illness, with a high mortality rate in susceptible individuals. Its environmental abundance significantly increases the likelihood of carry-over contamination in food processing surroundings leading to possible contamination of finished products. In addition frequent exposure to environmental stresses during its saprophytic existence renders L. monocytogenes able to overcome stresses often used in food production to limit microbial proliferation and to extend shelf-life. Therefore a more detailed understanding of molecular adaptations in this organism to stresses it encounters as a saprophyte may provide insights to minimise proliferation in foods. To broaden the understanding of L. monocytogenes stress resistance a population study, unique in its magnitude, was conducted on growth parameters obtained for a large assortment of isolates of diverse origin, subjected to hyperosmotic stress and, independently, to cold-temperature. On the basis of generation times, no direct correlation (r=0.4) between tolerance to either hyperosmotic- or cold-stress, was evident between isolates, but strains of animal origin were on average found to grow faster when exposed to ether of the two stresses. Strains of serotype 4b appeared to have shorter average generation times of 6.6±0.25 h in BHI at 12.5% (w/v) NaCl and 25°C, whereas serotype 4a strains had an average generation time of 19.4±1.0 h at 4°C in 0.5% (w/v) NaCl BHI, which was faster than other serotypes examined. Hyperosmotic and cold stress factors, though quite different in terms of physiochemical stress on bacterial cells, revealed many parallels in terms of gene expression in three strains of L. monocytogenes studied. The results suggest that a broadly similar genetic regulatory mechanism could be operating in response to cold and hyperosmotic stresses. Osmoadaptation of four L. monocytogenes strains, possessing different tolerances to NaCl, revealed a clear pattern in terms of genomic expression. Prolonged exposure to high levels of NaCl was coupled with activation of genes associated with the bacterial cell envelope, DNA repair and protein synthesis. Repression of genes associated with carbohydrate up-take and metabolism was evident in osmo-adapted cells reflecting the overall suppression of cellular metabolism characterised by reduced growth rate of these cells. In addition the initial stage of osmo-adaptation, was examined in strain ATCC 19115 (serotype 4b) post short exposure to 10.0% (w/v) NaCl to investigate continuous spectrum of gene expression in response to osmotic stress in this organism. Gene enrichment analysis revealed a prominent, almost reverse gene expression profile in response to shock, compared to the adaptive response to the same concentration of NaCl. This study is the first to strongly highlight such distinction in gene expression between phases of hyperosmotic adaptation, further emphasising the complexity of this response. Cold adaptation in three L. monocytogenes strains, possessing distinctly different growth rates at 4°C, lead to activation of gene sets associated with ribosomes, fatty acid and peptidoglycan biosynthesis and cell division and suppression of carbohydrate transport and metabolism related genes. Cold adapted strains did not activate a SigB or PrfA regulatory responses, suggesting that SigB-mediate stress responses are not closely involved in low temperature adaptation. A similar response was observed during hyperosmotic adaptation in L. monocytogenes. Strain specific response to stress factors has been overlooked in previous whole genome analyses, and although this study suggests a clear correlation in overall response to cold and hyperosmotic stress in this species, strain specific and phase specific responses are also crucial in understanding fully its mechanisms of stress tolerance, survival and persistence. The importance of strain-specific variations in genetic response to stress should be considered particularly if genetic targeting is to be applied to controlling L. monocytogenes proliferation in food products. This study is the first to assess genetic stress adaptation in such depth, significantly contributing to the understanding of L. monocytogenes physiology.