Project description:Legionella pneumophila Philadelphia-1 strain was grown to stationary phase in AYE broth and starved in freshwater for 2 hours and RNA was harvested with or without sublethal heat shock via immersion in a 55 degree C hot water bath for 5 minutes
Project description:Legionella pneumophila (Lp) is an opportunistic pathogen and its survival in water is critical for human infection. Therefore, identifying the genes of Lp that are required for survival in water may help devise strategies to prevent Legionella outbreaks. In this study, we exposed Lp in rich medium and in an artificial freshwater medium (Fraquil) for 2, 6 and 24 hours to uncover the global transcriptomic changes of Lp in water. The repression of major metabolic pathways, such as division, transcription and translation, suggests that Lp enters a dormant state in water. The induction of the flagellar associated genes (flg, fli and mot), enhance entry genes (enh) and some Icm/Dot effectors suggests that Lp may be waiting to establish intracellular replication in suitable host. Moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that Lp may be more tolerant to environmental stresses in water. Indeed, Lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than those cultured in rich medium. Apart from this, the gene bdhA involved in the degradation of the intracellular energy storage compound poly-hydroxybutyrate is highly expressed in water. Further characterization shows that bdhA is positively regulated by RpoS during short-term exposure to water. The deletion mutant of bdhA had a survival defect in water at 37°C, demonstrating that this gene is important for maintaining the long-term survivorship of Lp in water. Other identified genes highly induced upon exposure to water could also be necessary for Lp to survive in water. Legionella pneumophila Philadelphia-1 strain JR32 was grown in AYE broth at 25°C shaking to OD600 of 1 in triplicate. Samples were taken for analysis; this is the control. Then the cultures were washed three times in Fraquil and resuspended in Fraquil to an OD600 of 1 and transfered to vessels of bioreactior (Biostat Q-plus). Samples were taken after 2h, 6h and 24h.