B-293. Isolation and Characterization of Legionella pneumophila DotL Mutants

M. C. Sutherland, V. Tseng, T-L. Nguyen, J. P. Vogel;
Washington Univ., St Louis, MO.

Legionella pneumophila is a Gram negative bacterium that is the causative agent of a bacterial form of pneumonia called Legionnaires’ disease. In the environment L. pneumophila replicates within protozoan hosts, whereas in humans it can replicate within alveolar macrophages within the lung. L. pneumophila is able to survive and replicate within these phagoctyic cells by injecting a large number of bacterial substrates into the host cells’ cytoplasm. Export of these substrates is mediated by a type IVB secretion system encoded by twenty-six dot/icm genes. Previously, we have performed a large scale mutagenesis on DotB, an ATPase that is required for the assembly of the type IV system and confers specificity to export of different substrates. Here we describe the isolation of a large number of mutants in a second putative ATPase of the L. pneumophila T4SS, DotL. DotL is a member of the type IV coupling protein (T4CP) family, which functions as inner-membrane receptors for substrates in type IV secretion systems. In contrast to other T4CPs, DotL is required for the viability of the L. pneumophila strain Lp02 under all conditions including growth on bacteriological media. In order to define DotL’s role in L. pneumophila, we performed two separate screens. In the first screen, we identified mutants that failed to complement the viability of a L. pneumophila dotL deletion for growth on bacteriological media. In the second screen, dotL mutants were identified that could complement a dotL deletion on plates, but were defective for growth within host cells. Characterization of these mutants has revealed several novel findings about the function of DotL in the assembly of the Dot/Icm complex and in export of specific substrates.