FIGURE 7.

Francisella tularensis LVS takes up less Gm conjugated to Texas Red (Tr-Gm) at 26°C. F. tularensis LVS showed an increase in Tr-Gm fluorescence at mammalian body temperature (37°C) compared to ambient temperature (26°) (A). Bacteria were suspended in TSBc and treated with Tr-Gm (at an amount equivalent to 128 μg/ml Gm) at the indicated temperature. Bacteria were centrifuged, the pellets were washed twice and then suspended in PBS at the indicated times. The fluorescence was determined using an Eppendorf plate reader with a 535/595 filter. Plotted values represent mean relative fluorescence units ±SE. Data were analyzed using a two-way ANOVA and a Sidak’s multiple comparisons test. ^∗P < 0.05 37°C vs. 26°C at 2 h; 37°C at 2 h vs. 37°C 15 min or 1 h. 26°C 2 h vs. 26°C 15 min or 26°C 1 h, not significant. To show that Tr-Gm was taken up through the Gm transporter (B), bacteria were incubated with Tr-Gm and increasing concentrations of Gm for 15 min at 37°C. Bacteria were centrifuged, washed in PBS, and then fluorescence was determined using an Eppendorf plate reader with a 535/595 filter. Plotted values represent mean relative fluorescence units ±SE. Data were analyzed using a one-way ANOVA and a Dunnetts’s multiple comparisons test. ^∗P = 0.0150, 0 μg/ml vs. all other conditions.