FIG. 3.

Effect of growth medium on yadA expression and entry of Y. pseudotuberculosis into mammalian cells. (A) Strain YP28 (yadA-phoA) was grown in LB broth and MMA supplemented with 0.2% glucose and 0.2% Casamino Acids under the growth conditions indicated. Stationary-phase (stat.) bacteria had an OD₆₀₀ of 4.0 to 4.5 and exponential-phase (exp.) cultures were harvested at an OD₆₀₀ of 0.6 when alkaline phosphatase activity was determined. Each value represents the average ± the standard deviation of three different experiments performed in triplicate. (B) Strains YPIII (inv⁺ yadA⁺), YP29 (inv⁺ yadA::Cm^r), YP30 (inv::Kan^r yadA⁺), YP31 (inv::Kan^r yadA::Cm^r), and YP32 (inv::Kan^r pIB1⁻) were grown in LB broth at 25°C or in minimal medium at 37°C overnight. About 5 × 10⁶ bacteria were used to challenge 5 × 10⁴ HEp-2 cells, which were incubated for 1 h at 20°C to determine cell attachment or at 37°C to monitor invasion efficiency. The total numbers of adherent and internalized bacteria were determined as described in the legend to Fig. 1. Each value represents the mean of three independent assays done in triplicate. wt, wild type.