Gene expression analysis of an anaerobic benzene degrading consortium using differential display

Differential gene expression analysis provides a powerful tool to decipher metabolic processes of complex cellular systems. A restriction fragment differential display reverse transcription-PCR technique (RFDD RT-PCR) was employed to compare mRNA expressed in a nitrate-reducing, anaerobic culture degrading benzene, toluene or benzoate. The latter two substrates are suspected downstream metabolites for the as-yet unknown benzene degradation pathway. Total RNA from each culture was analyzed using the display PROFILE(TM) kit (QBIOgene). The RFDD RT-PCR products represent a snapshot of the total RNA expressed in each culture, including potential differentially expressed mRNA. The products were run on a polyacrylamide sequencing gel and visualized using P33-based autoradiography. Differentially expressed bands were excised, re-amplified, cloned and sequenced. Six differentially expressed sequences were obtained. Further analysis of those six sequences will confirm suspected differential expression. Using this novel RFDD RT-PCR technique for prokaryotes will help elucidate the metabolic pathway of benzene oxidation under anaerobic nitrate-reducing conditions.