| Until recently, little has been known about anaerobic toluene metabolism in bacteria. Through the study of three bacterial strains (Thauera aromatica strain T1, Thauera aromatica strain K172, and Azoarcus sp. strain T), the initial step of this anaerobic pathway has been well studied and documented. There is less known about downstream steps of the toluene degradation pathway, and thus this work focuses on the cloning and characterizing of the genes important in these downstream steps.; Differential display (RFDD-PCR) was originally used to identify novel toluene regulated genes in T. aromatica T1; however, results were nonrepresentative of differentially expressed messages from toluene grown cells. Only twelve differentially expressed messages were amplified, and several of the amplified messages were from rRNA genes. Only one fragment resulted in a novel toluene regulated message.; During experimentation with RFDD-PCR, a report was made that the genes responsible for downstream events of anaerobic toluene degradation, bbs genes, had been cloned from T. aromatica K172. The nucleotide sequences of the bbs genes were used as a reference to design primers for PCR amplification of these same genes in T. aromatica T1. Five of the nine genes, bbsA, bbsB, bbsC, bbsE, and bbsH, were successfully PCR amplified, while the other four genes were not identified. Northern analysis showed that the bbs genes of T. aromatics T1 were toluene regulated; however they are transcriptionally organized into several transcriptional units rather than one 8.5kb operon that is present in T. aromatica K172.; A bbsB- mutant was created to determine the importance of the bbs genes in anaerobic toluene degradation. HPLC analysis showed that the mutant was not able to degrade toluene and there was no accumulation of pathway intermediates. Further study of this mutant has shown that the bbs genes are not induced in the same manner as previously identified toluene regulated genes of T. aromatica T1; however, they may be regulated by a similar mechanism. |
