Akinobu NAKAMURA

Quantification of hydrogenase gene expression of anaerobic propionate-oxidizing bacterium by Real-Time PCR

Akiyoshi OHASHI, Hiroyuki IMACHI, Hideki HARADA

Anaerobic, thermophilic, propionate-oxidizing bacterium (Pelotomaculum thermopropionicum strain SI) can grow only under syntrophic condition with hydrogenotrophs such as hydrogen-utilizing methanogens. Hydrogenase plays an important role in the production and/or reduction of hydrogen in the interspecies hydrogen transfer. Thus monitoring the hydorogenase gene expression could provide us some useful informations to understand unwilling propionic accumulation. This study aims to identify the hydrogenase gene sequence of strain SI, to investigate optimum method of RNA extraction and reverse transcription (RT) reaction efficiency, and finally to establish quantification method of gene expression of strain SI by Real-Time PCR.
The 1,449bp length DNA sequence obtained from strain SI by cloning techniques and PCR suggested that it must be a gene of [NiFe]-hydrogenase large subunit. Of the three examined RNA extraction methods, the acid-phenol one was the highest in the proportional relationship between cell number and amount of extracted RNA regarding strain SI, which means that quantifications of mRNA and rRNA can be estimated more precisely. Therefore, RNA extractions by the acid-phenol method were employed in subsequent experiments. RT reaction efficiencies were significantly dependent on temperature. The optimal temperatures were determined as 51 and 52�� for rRNA and mRNA, respectively. In the batch experiments of pure culture of SI and pure co-culture with ��H, the quantification of 16S rRNA as well as hydrogenase mRNA were successfully by using Real-Time PCR.