Bacillus sphaericus JG-A12 is a natural isolate recovered from a uranium mining waste pile near the town of Johanngeorgenstadt in Saxony, Germany. The cells of this strain are enveloped by a highly ordered crystalline proteinaceous surface layer (S-layer) possessing an ability to bind uranium and other heavy metals. Purified and recrystallized S-layer proteins were shown to be phosphorylated by phosphoprotein-specific staining, inductive coupled plasma mass spectrometry analysis, and a colorimetric method. We used extended X-ray absorption fine-structure (EXAFS) spectroscopy to determine the structural parameters of the uranium complexes formed by purified and recrystallized S-layer sheets of B. sphaericus JG-A12. In addition, we investigated the complexation of uranium by the vegetative bacterial cells. The EXAFS analysis demonstrated that in all samples studied, the U(VI) is coordinated to carboxyl groups in a bidentate fashion with an average distance between the U atom and the C atom of 2.88 +/- 0.02 A and to phosphate groups in a monodentate fashion with an average distance between the U atom and the P atom of 3.62 +/- 0.02 A. Transmission electron microscopy showed that the uranium accumulated by the cells of this strain is located in dense deposits at the cell surface.