FIG. 4.

Intracellular behavior of EGDΔlmo2537/pLivlmo2537. (A) The intracellular growth of the conditional mutant was compared to that of EGD-e in J774 macrophages. Invasion and intracellular multiplication was monitored in J774 macrophages at a bacterium/macrophage ratio of 10:1 over a 6-h period in the presence of gentamicin at 50 μg/ml (final concentration) as described previously (27). At selected intervals, infected J774 cells were lysed, and the titer of viable bacteria was determined by spreading cells onto BHI plates containing (in all cases) 1 mM IPTG. Mut+IPTG:cells+IPTG, EGDΔlmo2537/pLivlmo2537 was grown in BHI containing 1 mM, and infection was performed in the presence of 1 mM IPTG. Mut+IPTG:cells−IPTG, EGDΔlmo2537/pLivlmo2537 was grown in BHI containing 1 mM, and infection was performed in the absence of IPTG. Mut-IPTG:cells−IPTG, EGDΔlmo2537/pLivlmo2537 was grown in BHI containing 10 μM IPTG, and infection was performed in the absence of IPTG. (B) Fluorescence analyses. Infections of J774 macrophage-like cells were performed at a bacterium/macrophage ratio of 10:1. At 30 min (a) or 4 h (b) postinfection, J774 macrophages infected by EGD-e expressing GFP under plmo2537 promoter control were collected and processed for fluorescence analysis. (C) Real-time PCR. The induction ratio of lmo2537 transcription in EGD-e was quantified under two growth conditions: (i) in infected J774 macrophage-like cells after 30 min and 4 h of infection or (ii) in DMEM or BHI broth.