Control of butanol formation in Clostridium acetobutylicum by transcriptional activation

Abstract

The sol operon of Clostridium acetobutylicum is the essential transcription unit for formation of the solvents butanol and acetone. The recent proposal that transcriptional regulation of this operon is controlled by the repressor Orf5/SolR (R. V. Nair, E. M. Green, D. E. Watson, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 181:319-330, 1999) was found to be incorrect. Instead, regulation depends on activation, most probably by the multivalent transcription factor Spo0A. The operon is transcribed from a single promoter. A second signal identified in primer extension studies results from mRNA processing and can be observed only in the natural host, not in a heterologous host. The first structural gene in the operon (adhE, encoding a bifunctional butyraldehyde/butanol dehydrogenase) is translated into two different proteins, the mature AdhE enzyme and the separate butanol dehydrogenase domain. The promoter of the sol operon is preceded by three imperfect repeats and a putative Spo0A-binding motif, which partially overlaps with repeat 3 (R3). Reporter gene analysis performed with the lacZ gene of Thermoanaerobacterium thermosulfurigenes and targeted mutations of the regulatory region revealed that the putative Spo0A-binding motif, R3, and R1 are essential for control. The data obtained also indicate that an additional activator protein is involved.

FIG. 1.

Purification of clostridial mature AdhE and the butanol dehydrogenase domain from the heterologous host E. coli. Separation was performed with an SDS-6 to 25% (wt/vol) polyacrylamide linear gradient gel and was followed by silver staining. Lane 1, marker proteins; lanes 2 and 8, crude extract from WL3(pGP1-2, pTWa4-2) (negative control); lanes 3 and 7, crude extract from WL3(pGP1-2, pTWa4-2::adhE6×His); lanes 4 and 6, fractions after affinity chromatography with Ni-NTA; lane 5, fraction after affinity chromatography with Reactive Green 19.

FIG. 2.

Plasmids constructed with targeted mutations in the sol operon regulatory region of C. acetobutylicum. Only the regulatory region and the first structural gene (adhE, altered by a six-histidine-encoding tail) are shown; these regions were inserted into the E. coli-C. acetobutylicum shuttle vector pIMP1. P₁ and P₂ are putative promoter structures deduced from primer extension studies. The hairpin symbol indicates a stem-loop structure predicted by the computer program MFold. For further details see the text.

FIG. 3.

Colorimetric detection of His-tagged AdhE and butanol dehydrogenase domain in recombinant C. acetobutylicum strains. Cell extracts were separated by SDS-polyacrylamide gel electrophoresis and were transferred onto a nitrocellulose membrane. Subsequently, His-tagged proteins were detected with Ni-AP conjugate. The molecular sizes of marker proteins are indicated on the left. Lane S, marker proteins; lane 1, C. acetobutylicum(pKLIMP7): lane 2, C. acetobutylicum(pKLIMP12); lane 3, C. acetobutylicum(pKLIMP17).

FIG. 4.

Secondary structure of the 5′ untranslated region of the sol operon transcript and effect of targeted mutations on transcription start mapping by primer extension. (A) Secondary structure of the beginning of the sol operon transcript, as determined by the program MFold and plotted by the program PlotFold. The positions of the second primer extension signal, which led to deduction of P₂, and the region used for targeted mutagenesis are indicated. RBS, ribosome-binding site. (B) Primer extension experiments showing signals related to P₁ (distal promoter) and P₂ (proximal start site). The results of sequence reactions, which were obtained by using the same oligodeoxynucleotides, are shown on the left in each gel. mut, mutated region; nat, natural sequence.

FIG. 5.

Regulatory motifs in the sol operon regulatory region. The top line schematically shows the end of the orf5 gene with its two transcriptional terminators (hairpins), the position of the P₁ promoter as the start point of the mRNA, and the beginning of the adhE gene. The second line indicates the fragment used to construct pACT1. The sequence at the bottom is the sequence of the DNA region comprising the three incomplete repeats (from left to right, R1, R2, and R3) and the Spo0A-binding site (box).

FIG. 6.

Targeted mutations in the sol operon regulatory region. The top sequence is the wild-type nucleotide sequence in the reporter gene plasmid pZF-sol; the positions of the three incomplete repeats and the Spo0A-binding site (shaded box) are indicated. The other sequences indicate the mutations in the various motifs (exchanged nucleotides are indicated by boldface type).

FIG. 7.

LacZ expression from wild-type and mutant regulatory regions of the sol operon in vivo. Growth (•), butanol formation (dotted line), and β-galactosidase activity were assayed during the bacterial growth cycle for strains carrying the plasmids shown in Fig. 6. Symbols: □, wild type (pZF-sol); ◊, pZF-sol-R1; +, pZF-sol-R2; ×, pZF-sol-R3; ○, pZF-solR1/R2; ∗, pZF-sol-spo0A; ▵, pZF-sol-act. Growth and butanol formation patterns were virtually identical in all experiments. For the sake of clarity, only the data obtained with pZF-solR1/R2 are shown.