The bacterium Pseudomonas stutzeri KC is the only known denitrifying organism ever isolated that is capable of degrading carbon tetrachloride without producing common toxic intermediate chloroform. This capability makes it highly attractive for bioremediation of CT contaminated sites. The physiology of this bacterium has been characterized and its mode of degrading carbon tetrachloride determined. Pseudomonas stutzeri KC produces and secretes the metal chelating agent pyridine-2,6-bis thiocarboxylate (PDTC) which degrades and detoxifies carbon tetrachloride. The reactive species responsible for transforming CT to carbon dioxide is actually a PDTC-Cu complex (see Criddle's Publications, Lee et al, 1995, Lewis et al., 2001).
Our current effort focuses on the investigation of the physical, kinetic and genetic characterization of the carbon tetrachloride transformation by this organism. We have accurately determined the transformation capacity of the PDTC-Cu complex for CT transformation using synthetic PDTC and also demonstrated that PDTC-Cu complex is both a reactant and a catalyst in carbon tetrachloride transformation. To further characterize the PDTC production process by KC, a highly sensitive and specific detection method for PDTC was developed using LS/MS/MS to quantitatively measure the real-time production of this compound by Pseudomonas stutzeri KC. The measurement of PDTC production from an actively growing Pseudomonas stutzeri KC culture indicated also that PDTC production was evidently growth-associated. Previous studies also led us to believe that the PDTC-mediated transformation of carbon tetrachloride can be inhibited by secreted proteins. These proteins apparently compete for copper. To identify and size these proteins, we developed assays based on specific binding interactions with metals and used surface enhanced laser desorption ionization mass spectrometry (SELDI-MS). Several supernatant proteins bind specifically with surface-bound ferric iron as well as other surface-bound metals such as copper and nickel. We are currently purifying these proteins.
By understanding the nature of these proteins, we expect to be better able to develop a complete model for the transformation of carbon tetrachloride by Pseudomonas stutzeri KC and thus improve engineering design and bioremediation using this organism.
Researchers working on this project: Shiang Fu