Figure 4.

Autoradiogram comparing the intracellular accumulation of radioactive intermediates by whole cells of wild-type R. palustris and the badE and badF mutants during a 2 min anaerobic incubation with 7-¹⁴C benzoate (50 μM). Cells were grown anaerobically with 3 mM cinnamate as carbon source. Following anaerobic incubation with labeled benzoate, cells were suspended in boiling water to release soluble intracellular contents, which were concentrated and then separated by thin layer chromatography before exposure to x-ray film (30). 7-¹⁴C-benzoyl-CoA was identified by co-migration with an unlabeled standard purchased from Sigma. Cyclohex-1,4-dienecarboxyl-CoA, prepared enzymatically with cyclohexanecarboxylate-CoA ligase (11) had a lower R_f than benzoyl-CoA (26).