Figure 3.

In vitro analysis of D-[¹¹C]Met in bacteria. (A) Workflow of high-throughput in vitro assay. Bacteria is grown overnight in LB broth, then subcultured in F12 media in 50 mL centrifuge tubes in the presence of a radiotracer. After the designated time, the subculture is aliquotted into 1.5 mL spin filters equipped with a 0.22 micron nylon filter. The aliquot is centrifuged and washed. The spin filter is separated and each portion analyzed on a gamma counter. (B) Sensitivity analysis of D-[¹¹C]met using a broad panel of human pathogens indicating avid incorporation (up to approximately 7 Bq incorporation of radiolabel) D-[¹¹C]Met by S. aureus and P. aeruginosa. (C) Dynamic cellular uptake of D-[¹¹C]Met in E. coli, S. aureus, and P. aeruginosa in exponential-phase cultures at 30,60,90, and 120 minutes. No radioactivity was incorporated into heat-killed organisms (P < 0.05 for live versus heat-killed, all organisms at all time points). (D) Accumulation of D-[¹¹C]Met in the presence of increasing concentrations of unlabelled D-methionine. In all cases blocked uptake suggests specific incorporation.