CHARACTERIZATION AND EXPRESSION OF THE HYDROGENASE-ENCODING GENE FROMCLOSTRIDIUM-ACETOBUTYLICUM P262

The hydrogenase enzyme of Clostridium acetobutylicum plays a pivotal r ole in controlling electron flow, and hence carbon flow, during the co mplex biphasic fermentation of carbohydrates to the neutral solvents a cetone and butanol. We report here the cloning and molecular character ization of the hydrogenase-encoding gene (hydA) from C. acetobutylicum P262. This gene was isolated by colony hybridization. using the Clost ridium pasteurianum hydrogenase-1 gene as a probe. The DNA sequence en coding the hydA gene from C. acetobutylicum was determined. and reveal ed an ORF (1722 bp) encoding a 574 amino-acid protein. This C. acetobu tylicum hydrogenase protein product has 82% similarity and 67% identit y with the C. pasteurianum hydrogenase-l protein. Northern blot analys is of RNA isolated from C. acetobutylicum indicates that the C. acetob utylicum hydrogenase protein product is translated from a monocistroni c operon, RNA was isolated from the different morphological and physio logical stages of a batch C. acetobutylicum fermentation, and further Northern blot analyses revealed no differences in the expression of th e gene during acidogenesis as opposed to solventogenesis. Primer exten sion experiments confirmed these results and identified the 5' start o f the mRNA transcript. These results correlated well with the physiolo gical need for this organism to dispose of excess reducing equivalents .