MOLECULAR CHARACTERIZATION AND TRANSCRIPTIONAL ANALYSIS OF THE PUTATIVE HYDROGENASE GENE OF CLOSTRIDIUM-ACETOBUTYLICUM ATCC-824

A 2.8-kbp DNA region of Clostridium acetobutylicum ATCC-824 containing the putative hydrogenase gene (hydA) was cloned and sequenced, The 1, 745-bp hydA encodes a 64,415-Da protein and presents strong identity w ith the [Fe] hydrogenase genes of Desulfovibrio and Clostridium specie s. The level of the putative hydA mRNA was high in cells from an acido genic or an alcohologenic phosphate-limited continuous culture, while it was comparatively very low in cells from a solventogenic phosphate- limited continuous culture, These results were in agreement with the h ydrogenase protein level, indicating that expression of hydA. is regul ated at the transcriptional level. Primer extension analysis identifie d a major transcriptional start site 90 bp upstream of the hydA start codon. The position of a putative rho-independent transcription termin ator immediately downstream of the termination codon is in agreement w ith the size of the hydA transcript (1.9 kb) determined by Northern (R NA) blot experiments and confirms that the gene is transcribed as a mo nocistronic operon, Two truncated open reading frames (ORFs) were iden tified downstream and upstream of hydA and in opposite directions, The amino acid sequence deduced from ORF2 presents strong identity with o rtho phosphoribosyl transferases involved in pyrimidine synthesis. The amino acid sequence deduced from ORF3 presents no significant similar ity to any sequence in various available databases.