Legionella pneumophila, the etiologic agent of Legionnaires' disease, is a major cause of both community-acquired an nosocomial pneumonia and can account for up to 30% of all pneumonias. This gram-negative bacterium is a facultative intracellular parasite of both human alveolar macrophages and fresh water protozoa. The intracellular replication of this bacterium is highly dependent upon iron acquisition. However, the mechanisms used by Legionella to scavenge intracellular iron remain essentially unknown. Thus, we recently embarked upon a "three-pronged' genetic analysis of Legionella intracellular iron acquisition. First, using transposon mutagenesis, we isolated five L. pneumophila mutants (designated as iraA-E mutants) which are defective for both iron acquisition and macrophage infection. These mutants, although generally resistant to streptonigrin, display a range of sensitivities to iron acquisition and macrophage infection. These mutants, although generally resistant to streptonigrin, display a range of sensitivities to iron chelators and exhibit different intracellular growth characteristics, suggesting that they bear mutations in different iron acquisition functions. Second, while attempting to clone a proteinaeous iron chelator of Legionella, we discovered that L. pneumophila and E. coli recA can promote the transcription of iron acquisition genes; i.e., the enterobactin operon. These data suggest that Rec A may be a physiologically-relevant modulator of iron acquisition. Third, we identified a fur gene in L. pneumophila and have isolated a Legionella Fur- mutant. In other bacteria, Fur is a regulator (repressor) of genes involved in iron acquisition and virulence. To gain insight into the effectors of intracellular iron acquisition, we propose to further characterize the iraA-E, ii) assessing their relative infectivities for iron-depleted macrophages and protozoa and experimental animals, and iii) examining them for changes in specific iron-scavenging activities; e.g., (radiolabeled) iron uptake. Second, to determine whether or not RecA can promote intracellular iron acquisition, we will assess the infectivity of a Legionella RecA mutant for macrophages containing differing amounts of iron. Third, to identify Fur-regulated, iron acquisition genes that are specifically required in the intracellular environment, we will, using random mutagenesis, identify strains bearing insertions in Fur-regulated genes and then screen for those mutants that are defective for intracellular iron acquisition but not extracellular iron acquisition. Taken together, these studies should enhance significantly our understanding of L. pneumophila pathogenesis, intracellular parasitism, and bacterial iron acquisition.