Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis

Abstract

A DNA region of Clostridium acetobutylicum contiguous with the adc operon has been cloned and sequenced. Structural genes encoding the acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (ctfB and ctfA) and an alcohol/aldehyde dehydrogenase (adhE) could be identified. These three genes together with a small open reading frame (ORF) of unknown function (upstream of adhE) formed an operon (sol operon), as shown by mRNA analyses. The complete sol operon was transcriptionally induced or derepressed before the onset of solventogenesis, thus confirming earlier results of Northern hybridizations with a ctfB gene probe (U. Gerischer and P. Dürre, J. Bacteriol. 174:426-433, 1992). Upstream of the sol operon, we identified two putative promoters that were located in regions with possible stem-loop structures formed by several inverted repeats. The distal promoter P1 showed only minor transcription initiation in solventogenic C. acetobutylicum cells but was recognized in Escherichia coli, presumably because of its high similarity to the sigma 70 consensus sequence. The adhE-proximal promoter P2 directed the major transcription start point in solventogenic C. acetobutylicum but was not recognized in E. coli. The clostridial AdhE showed high similarity to a novel family (type III) of alcohol dehydrogenases. Two other ORFs (ORF 5 and ORF 6) were found on the cloned DNA region that showed no significant similarity to sequences in various available data bases. mRNA studies revealed that ORF 5 formed a monocistronic operon and showed increased expression before onset of solventogenesis.