Characterization and expression of the hydrogenase-encoding gene from Clostridium acetobutylicum P262

The hydrogenase enzyme of Clostridium acetobutylicum plays a pivotal role in controlling electron flow, and hence carbon flow, during the complex biphasic fermentation of carbohydrates to the neutral solvents acetone and butanol. We report here the cloning and molecular characterization of the hydrogenase-encoding gene (hydA) from C. acetobutylicum P262. This gene was isolated by colony hybridization, using the Clostridium pasteurianum hydrogenase-1 gene as a probe. The DNA sequence encoding the hydA gene from C. acetobutylicum was determined, and revealed an ORF (1722 bp) encoding a 574 amino-acid protein. This C. acetobutylicum hydrogenase protein product has 82% similarity and 67% identity with the C. pasteurianum hydrogenase-1 protein. Northern blot analysis of RNA isolated from C. acetobutylicum indicates that the C. acetobutylicum hydrogenase protein product is translated from a monocistronic operon. RNA was isolated from the different morphological and physiological stages of a batch C. acetobutylicum fermentation, and further Northern blot analyses revealed no differences in the expression of the gene during acidogenesis as opposed to solventogenesis. Primer extension experiments confirmed these results and identified the 5' start of the mRNA transcript. These results correlated well with the physiological need for this organism to dispose of excess reducing equivalents.