Molecular Characterization of the Glycerol-Oxidative Pathway of Clostridium butyricum VPI 1718

Abstract

The glycerol oxidative pathway of Clostridium butyricum VPI 1718 plays an important role in glycerol dissimilation. We isolated, sequenced, and characterized the region coding for the glycerol oxidation pathway. Five open reading frames (ORFs) were identified: dhaR, encoding a putative transcriptional regulator; dhaD (1,142 bp), encoding a glycerol dehydrogenase; and dhaK (995 bp), dhaL (629 bp), and dhaM (386 bp), encoding a phosphoenolpyruvate (PEP)-dependent dihydroxyacetone (DHA) kinase enzyme complex. Northern blot analysis demonstrated that the last four genes are transcribed as a 3.2-kb polycistronic operon only in glycerol-metabolizing cultures, indicating that the expression of this operon is regulated at the transcriptional level. The transcriptional start site of the operon was determined by primer extension, and the promoter region was deduced. The glycerol dehydrogenase activity of DhaD and the PEP-dependent DHA kinase activity of DhaKLM were demonstrated by heterologous expression in different Escherichia coli mutants. Based on our complementation experiments, we proposed that the HPr phosphoryl carrier protein and His9 residue of the DhaM subunit are involved in the phosphoryl transfer to dihydroxyacetone-phosphate. DhaR, a potential regulator of this operon, was found to contain conserved transmitter and receiver domains that are characteristic of two-component systems present in the AraC family. To the best of our knowledge, this is the first molecular characterization of a glycerol oxidation pathway in a Gram-positive bacterium.Glycerol can be utilized as a carbon source by bacteria via several metabolic pathways that convert glycerol to dihydroxyacetone-phosphate (DHAP) before DHAP enters the glycolytic pathway. Under aerobic conditions, Escherichia coli phosphorylates glycerol using an ATP-dependent glycerol kinase (19), and glycerol-3-phosphate then is oxidized to DHAP by a membrane-bound FAD-dependent glycerol-3-phosphate dehydrogenase (35). Under anaerobic conditions, Klebsiella pneumoniae and Citrobacter freundii oxidize glycerol using a soluble NAD ϩ -dependent glycerol dehydrogenase (9, 13), and dihydroxyacetone (DHA) then is phosphorylated by a DHA kinase to DHAP (9,13,21). DHA kinases can be grouped into two structurally related families according to the source of the high-energy phosphate: ATP or phosphoenolpyruvate (PEP). ATP-dependent DHA kinases are single-polypeptide two-domain proteins (37), while the PEP-dependent DHA kinases consist of three subunits: DhaK, DhaL, and DhaM (18). DhaK and DhaL are homologous to the amino-terminal K domain and the carboxy-terminal

Glycerol Dehydrogenase Plays a Dual Role in Glycerol Metabolism and 2,3-Butanediol Formation in Klebsiella pneumoniae

“…The dhaD gene is located in the dha regulon and is 1,089 bp long, with a deduced amino acid sequence that shares 58% identity with GDH of E. coli (13), 50% identity with GDH of Geobacillus stearothermophilus (14), and 46% identity with GDH of Clostridium butyricum (20). The gldA gene is 1,104 bp long, and its deduced amino acid sequence has 60 and 89% identities with DhaD of K. pneumoniae ATCC 25955 and GDH of E. coli, respectively (13).…”

“…During the past few decades, GDHs from different sources have been characterized (5,(12)(13)(14)(15)(16). Recently, the glycerol surplus produced by the tremendous growth of the biodiesel industry stimulated intensive research efforts focused on glycerol metabolism and related enzymes and pathways, especially in efficient glycerol utilizers, such as Klebsiella and Clostridium (17)(18)(19)(20). In K. pneumoniae, glycerol is metabolized in a dismutation process via two branches, reductive and oxidative (21) (Fig.…”

From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae

“…Similar to the case in the bacteria Citrobacter freundii and C. butyricum, which metabolize glycerol reductively, DhaR serves as a positive regulator of genes mediating the reductive metabolism of glycerol (47,48). Using the BLAST algorithm (41), a maximum identity of 81% to DhaR of C. freundii was detected.…”

Novel Listerial Glycerol Dehydrogenase- and Phosphoenolpyruvate-Dependent Dihydroxyacetone Kinase System Connected to the Pentose Phosphate Pathway

“…Divalent cations were necessary for both activities of Lin0362, and the Mg 2ϩ and Mn 2ϩ ions had similar stimulating effects, whereas the enzyme was inactive in the presence of Zn 2ϩ . Similar to the dha operon in most enterococci and clostridia (6,40), the L. innocua lin0359-lin0369 and L. monocytogenes lmo0341-lmo0351 operons contain a gene encoding a protein with NAD ϩ -dependent glycerol dehydrogenase activity. However, while the enterococcal and clostridial enzymes exhibit between 40 to 60% sequence identity compared to glycerol dehydrogenase (GldA) from firmicutes (31) or enterobacteria (46), the listerial protein is, to the best of our knowledge, the first member of the short-chain dehydrogenase/reductase family exhibiting glycerol dehydrogenase/dihydroxyacetone reductase activity.…”

“…Enterococci and clostridia also contain in the dha operon a gene encoding a glycerol dehydrogenase resembling the E. coli glycerol dehydrogenase GldA (13). Glycerol is converted by GldA into dihydroxyacetone, which is subsequently phosphorylated to Dha-P (6,40). Enterococci possesses a glycerol dehydrogenase/DhaKLM system as well as GlpK (48) and the glycerol-3-P oxidase GlpO (8).…”