mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis

Abstract

By using primer extension analysis, we located the transcription start point of the acetoacetate decarboxylase (adc) gene of Clostridium acetobutylicum 90 nucleotides upstream from the initiation codon with A as the first transcribed nucleotide. From this site the promoter structure TTTACT(18 bp)TATAAT was identified; it shows high homology to the consensus sequences of gram-positive bacteria and Escherichia coli. Northern blot experiments revealed a length of 850 bases for the transcript of the adc gene. It thus represents a monocistronic operon. Transcription of adc was induced by conditions necessary for the onset of solvent formation. Induction occurred long before the respective fermentation product (acetone) could be detected in the medium. Transcription of the operon containing the genes for acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (designated ctf) downstream of the adc gene but divergently transcribed is also induced by conditions necessary for the onset of solvent formation. The length of the respective RNA transcript, 4.1 kb, indicates additional coding capacity, since the genes for the two subunits of the coenzyme A transferase cover only approximately 1.5 kb. No distinct transcripts for the other open reading frames of the adc gene region, ORF1 and ORF2, could be detected. Computer analysis indicated that ORF1, which showed significant similarity to the alpha-amylase gene of Bacillus subtilis (U. Gerischer and P. Dürre, J. Bacteriol. 172:6907-6918, 1990), probably is indeed a coding region. ORF2, however, does not seem to have a coding function.