Functional Genomics and physiology of growth initiation in Salmonella
Abstract Lag phase is a period of bacterial adaptation that occurs prior to cell division. The aim of this project was to characterise the processes used by Salmonella enterica serovar Typhimurium to escape from lag phase, and determine whether these processes are dependent on the bacterial ‘physiological history’. The lag phase transcriptomic response at 25 °C of stationary phase cells that had been held for twelve days at 2 °C was compared with that of stationary phase cells not subjected to this cold storage treatment. Cold-stored cells showed significant changes in expression of 78 % genes during lag phase, with 875 genes altering their expression ≥2-fold within the first four minutes of inoculation into fresh medium. Functional categories of genes that were significantly up-regulated included those encoding systems involved with metal ion uptake, stress resistance, phosphate uptake, ribosome synthesis and cellular metabolism. Genes in the OxyR regulon were induced earlier in cold-stored cells, a response coupled with a delay in the expression of Fe2+ acquisition genes, and down-regulation of genes encoding central metabolic enzymes. Together, these findings with physiological tests demonstrated that Salmonella held in cold storage exhibited an increased sensitivity to oxidative stress in midlag phase, although the lag time was not increased. Despite an oxidative stress response at the transcriptomic level during lag phase under both experimental conditions, deletion of the OxyR and SoxRS systems did not lead to an increased lag time during aerobic growth at 25 °C. The intracellular concentration of metal ions was quantified using ICP-MS, and changes observed during lag phase confirmed the transcriptomic data. Metal ions specifically accumulated during lag phase included Mn2+, Fe2+, Cu2+ and Ca2+, with the latter being the most abundant metal ion. The intracellular concentration of Zn2+ and Mg2+ remained the same as for stationary phase cells, and Ni2+, Mo2+ and Co2+ were expelled from the cell during lag phase. Metal homeostasis was determined to be a critical process, highlighted by growth in the presence of a chelator causing an extended lag time. Overall, lag phase was found to be a robust and reproducible adaptation period which was not perturbed by the mutagenesis approaches utilised in this study.
Networked Digital Library of Theses and Dissertations