Jd. Santangelo et al., CHARACTERIZATION AND EXPRESSION OF THE HYDROGENASE-ENCODING GENE FROMCLOSTRIDIUM-ACETOBUTYLICUM P262, Microbiology, 141, 1995, pp. 171-180
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
.