The molecular characterization of a B12-independent glycerol dehydratase from Clostridium butyricum has recently been reported [Raynaud, C., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5010-5015]. In this work, we have further characterized this system by biochemical and crystallographic methods. Both the glycerol dehydratase (GD) and the GD-activating enzyme (GD-AE) could be purified to homogeneity under aerobic conditions. In this form, both the GD and GD-AE were inactive. A reconstitution procedure, similar to what has been reported for pyruvate formate lyase activating enzyme (PFL-AE), was employed to reconstitute the activity of the GD-AE. Subsequently, the reconstituted GD-AE could be used to reactivate the GD under strictly anaerobic conditions. We also report here the crystal structure of the inactive GD in the native (2.5 A resolution, Rcryst = 17%, Rfree = 20%), glycerol-bound (1.8 A resolution, Rcryst = 21%, Rfree = 24%), and 1,2-propanediol-bound (2.4 A resolution, Rcryst = 20%, Rfree = 24%) forms. The overall fold of the GD monomer was similar to what has been observed for pyruvate formate lyase (PFL) and anaerobic ribonucleotide reductase (ARNR), consisting of a 10-stranded beta/alpha barrel motif. Clear density was observed for both substrates, and a mechanism for the dehydration reaction is presented. This mechanism clearly supports a concerted pathway for migration of the OH group through a cyclic transition state that is stabilized by partial protonation of the migrating OH group. Finally, despite poor alignment (rmsd approximately 6.8 A) of the 10 core strands that comprise the barrel structure of the GD and PFL, the C-terminal domains of both proteins align well (rmsd approximately 0.7 A) and have structural properties consistent with this being the docking site for the activating enzyme. A single point mutation within this domain, at a strictly conserved arginine residue (R782K) in the GD, resulted in formation of a tight protein-protein complex between the GD and the GD-AE in vivo, thereby supporting this hypothesis.

The genes encoding the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum VPI1718 were characterized from a molecular and a biochemical point of view. This operon is composed of three genes, dhaB1, dhaB2, and dhaT. When grown in a vitamin B12-free mineral medium with glycerol as carbon source, Escherichia coli expressing dhaB1, dhaB2, and dhaT produces 1,3-PD and high glycerol dehydratase and 1,3-PD dehydrogenase activities. dhaB1 and dhaB2 encode, respectively, a new type of glycerol dehydratase and its activator protein. The deduced proteins DhaB1 and DhaB2, with calculated molecular masses of 88,074 and 34,149 Da, respectively, showed no homology with the known glycerol dehydratases that are all B12 dependent but significant similarity with the pyruvate formate lyases and pyruvate formate lyases activating enzymes and their homologues. The 1,158-bp dhaT gene codes for a 1,3-PD dehydrogenase with a calculated molecular mass of 41,558 Da, revealing a high level of identity with other DhaT proteins from natural 1,3-PD producers. The expression of the 1,3-PD operon in C. butyricum is regulated at the transcriptional level, and this regulation seems to involve a two-component signal transduction system DhaAS͞DhaA, which may have a similar function to DhaR, a transcriptional regulator found in other natural 1,3-PD producers. The discovery of a glycerol dehydratase, coenzyme B12 independent, should significantly influence the development of an economical vitamin B12-free biological process for the production of 1,3-PD from renewable resources.

The ubiGmccBA operon of Clostridium acetobutylicum is involved in methionine to cysteine conversion. We showed that its expression is controlled by a complex regulatory system combining several RNA-based mechanisms. Two functional convergent promoters associated with transcriptional antitermination systems, a cysteine-specific T-box and an S-box riboswitch, are located upstream of and downstream from the ubiG operon, respectively. Several antisense RNAs were synthesized from the downstream S-box-dependent promoter, resulting in modulation of the level of ubiG transcript and of MccB activity. In contrast, the upstream T-box system did not appear to play a major role in regulation, leaving antisense transcription as the major regulatory mechanism for the ubiG operon. The abundance of sense and antisense transcripts was inversely correlated with the sulfur source availability. Deletion of the downstream promoter region completely abolished the sulfur-dependent control of the ubiG operon, and the expression of antisense transcripts in trans did not restore the regulation of the operon. Our data revealed important insights into the molecular mechanism of cis-antisense-mediated regulation, a control system only rarely observed in prokaryotes. We proposed a regulatory model in which the antisense RNA controlled the expression of the ubiG operon in cis via transcriptional interference at the ubiG locus.

The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum, a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep-tag fusion protein and purified to homogeneity also demonstrated NADHdependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2, are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824.Clostridium acetobutylicum is a gram-positive spore-forming anaerobic bacterium capable of converting different sugars and polysaccharides to organic acids (acetate and butyrate) and solvents (acetone, butanol, and ethanol). Acetone is produced from acetoacetyl coenzyme A (acetoacetyl-CoA) in a two-step process that involves a CoA transferase and an acetoacetate decarboxylase. The CoA transferase consists of two different subunits encoded by the ctfA and ctfB genes, which are part of the sol operon (13). The acetoacetate decarboxylase gene (adc), adjacent but not part of the sol operon, is transcribed in the opposite direction from its own promoter. Both the sol operon and the adc gene are located on the pSOL1 megaplasmid (9). Ethanol and butanol are produced from acetyl-CoA and butyryl-CoA, respectively, in two reductive steps catalyzed by aldehyde dehydrogenases and alcohol dehydrogenases (ADHs). The aad gene of C. acetobutylicum ATCC 824 (referred to as adhE in strain DSM 792) is part of the sol operon, and it encodes a bifunctional aldehyde/alcohol dehydrogenase (AADH) (13,29). This enzyme is mainly involved in butanol formation. Two NADH-dependent butanol dehydrogenases (BDH) (BDH I and BDH II) have been purified, and their genes (bdhA and bdhB) have been cloned (43). These genes are adjacent on the chromosome but are each transcribed by their own promoters.In continuous culture, C. acetobutylicum can be maintained in three different stable metabolic states (15): acidogenic (production of acetic and butyric acids) when grown at neutral pH on glucose, solventogenic (production of acetone, butanol, and ethanol) when grown at low pH on glucose, and alcohologenic (formation of butanol and ethanol but not acetone) when grown at neutral pH under conditions of high NAD(P)H availability. When solventogenesis is induced by lowering the pH of a continuous culture, transcription ...