The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in @entity2114 . To analyze GFP expression in @entity39812 , transcriptional fusions of gfp with the @entity39812 -specific mip (Macrophage Infectivity Potentiator) promoter (P(mip)) and the sod ( @entity224 Dismutase) promoter (P(sod)) derived from @entity5125 were constructed. Following transformation into the virulent @entity2114 JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1ten-fold higher intensity. No fluorescence was observed in @entity2114 transformed with the promoterless gfp gene. Comparison of fluorescence yields between various @entity2114 strains that differ in their virulence characteristics and were transformed with the P(mip)-gfp carrying plasmid revealed no differences in GFP expression. Infection studies using @entity16762 as host and recombinant @entity2114 strains carrying the P(mip)-gfp and P(sod)-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by. @entity2114 strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent @entity39812 was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin D, @entity1880 , and @entity8776 on the uptake of @entity39812 by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in @entity39812 can be followed in real time and allows analysis of promoter activities in @entity39812 and monitoring of the infection process.

Expression and use of the green fluorescent protein as a reporter system in XXXX .

@entity1880 :: ('MESH:D003513', 'Chemical') :: ['cycloheximide'] @entity5125 :: ('1639', 'Species') :: ['Listeria monocytogenes'] @entity39812 :: ('400673', 'Species') :: ['L. pneumophila strain Corby', 'Legionella'] @entity16762 :: ('5755', 'Species') :: ['Acanthamoeba castellanii'] @entity2114 :: ('446', 'Species') :: ['L. pneumophila strain', 'Legionella pneumophila', 'L. pneumophila'] @entity8776 :: ('MESH:C027451', 'Chemical') :: ['methylamine'] @entity224 :: ('MESH:D013481', 'Chemical') :: ['SuperOxide']
@entity2114 :: (446,Species) :: ['Legionella pneumophila']
