bLegionella pneumophila is a waterborne pathogen, and survival in the aquatic environment is central to its transmission to humans. Therefore, identifying genes required for its survival in water could help prevent Legionnaires' disease outbreaks. In the present study, we investigate the role of the sigma factor RpoS in promoting survival in water, where L. pneumophila experiences severe nutrient deprivation. The rpoS mutant showed a strong survival defect compared to the wild-type strain in defined water medium. The transcriptome of the rpoS mutant during exposure to water revealed that RpoS represses genes associated with replication, translation, and transcription, suggesting that the mutant fails to shut down major metabolic programs. In addition, the rpoS mutant is transcriptionally more active than the wild-type strain after water exposure. This could be explained by a misregulation of the stringent response in the rpoS mutant. Indeed, the rpoS mutant shows an increased expression of spoT and a corresponding decrease in the level of (p)ppGpp, which is due to the presence of a negative feedback loop between RpoS and SpoT. Therefore, the lack of RpoS causes an aberrant regulation of the stringent response, which prevents the induction of a successful response to starvation. L egionella pneumophila is the causative agent of Legionnaires' disease. It is widely distributed in natural freshwater systems (1) and readily colonizes man-made water systems such as cooling towers and water distribution systems (2). L. pneumophila persists in aquatic environments thanks to its ability to adapt to a variety of different ecological niches, either as an intracellular parasite of amoebae or ciliate protozoans, as a free-living member of complex biofilm communities, or as planktonic cells (3, 4). Amoebae support intracellular multiplication of L. pneumophila, and protect against suboptimal growth conditions and exposure to chlorine (5-7). Infection of HeLa cells and Tetrahymena tropicalis leads to the differentiation of L. pneumophila into mature infectious forms (MIFs), characterized by ultrastructural changes and accumulation of poly-␤-hydroxybutyrate (8-10). MIFs are highly infectious and are more resistant to antibiotics and detergent than other forms (9, 11). Increased resistance to antibiotics was also described after incubation of L. pneumophila in Acanthamoeba castellanii buffer for 16 h (12). MIFs are able to resist starvation better than stationary-phase forms in encystment buffer (11), but both forms show similar levels of resistance in distilled water (8).In the free-living state outside the amoebal host, L. pneumophila encounters stressful conditions, such as limited nutrient availability in aquatic systems (1, 13). Nevertheless, previous studies have shown that L. pneumophila is able to survive for long periods (up to 1.5 years) in sterilized tap, surface and estuarine waters (14-17). The genetic determinants underlying the ability of L. pneumophila to survive in its natural habitat of low-nutrient water for a...

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