The objective of this project is to develop molecular tools for detecting anaerobic alkybenzene-degrading bacteria in natural samples. This includes the identification of the genes involved, as well as the fabrication of a DNA microarray carrying probes for the key enzymes involved in anaerobic alkylbenzene degradation. We and others have cloned the genes encoding the key enzyme for anaerobic toluene mineralization, benzylsuccinate synthase, however, the genes facilitating anaerobic ethylbenzene degradation to benzoate are unknown.
Remediation of the many fuel hydrocarbon-contaminated sites by traditional physical-chemical processes amounts to enormous costs to the industry and society. Because aromatic hydrocarbons can be degraded biologically, it is in principle possible to predict whether or not these compounds can be mineralized at a given site and what the estimated rate would be. Furthermore, knowledge of whether a site has the potential for rapid or slow intrinsic degradation of aromatic hydrocarbons is crucial for taking action to enhance the intrinsic rate. Traditionally, microcosm studies with samples obtained from the contaminated site have been employed to determine the potential and the rate of biodegradation. These studies are time and labor intensive. With knowledge of the biochemical pathways and the genes involved, more rapid and less expensive tests can be developed. The principle of a molecular test that we envision is based on whether the genes encoding the key enzymes involved in these processes are present in contaminated environments. Using those genes that encode the key enzymes will provide highly specific probes, which unequivocally will query for the presence of the first unique biochemical steps involved in anaerobic degradation of the alkylbenzenes.
We are studying anaerobic toluene, m-xylene, and ethylbenzene-degrading denitrifying bacteria (Azoarcus strain T and Azoarcus strain EB1, respectively), and have so far identified the genes encoding the key enzymes involved in anaerobic toluene and, most likely, in m-xylene mineralization. These genes will provide the basis for developing probes for detecting the presence of these enzymes. However, the genes encoding the key enzymes of ethylbenzene degradation are unknown. We therefore will isolate those genes. Preliminary data obtained from 2-D protein gel electrophoresis indicated several polypeptides that are specifically expressed in cells of Azoarcus strain EB1 when grown on ethylbenzene but not when grown on benzoate. Using reverse genetics, the corresponding genes will be cloned, and probes derived from these genes will be printed on a DNA microarray together with the benzylsuccinate synthase genes.