(A) Representative fluorescence micrographs (20X) of REMI mutants after 72 hr. incubation with L. pneumophila. AX4 is the parental wild type strain and RI4, RI11, RI18, RI10, RI6 are REMI mutants. Arrows show representative L. pneumophila phagosomes. (B) REMI mutants alter the number of Legionella-containing vacuoles (LCVs) compared to wild type amoebae. Images (20X) of noted REMI mutants challenged with L. pneumophila-GFP for 72 hrs. were captured as in (A), and fluorescent foci were quantitated (Experimental Procedures). Each fluorescent focus was defined as a single LCV. Shown are the mean ± SD for triplicate infections. (C). REMI mutants show alterations in yield of intracellular L. pneumophila. Images grabbed in panel (B) were used to determine the yield of bacteria. (D). Intracellular growth of wild-type L. pneumophila in REMI mutants. The number of viable bacteria was determined by plating supernatants of lysed D. discoideum onto CYE plates at the denoted times and counting CFU (Experimental Procedures). Plotted is the mean CFU from triplicate samples ± SD.