Rex in Clostridium kluyveri is a global redox-sensing transcriptional regulator.

Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H2. The genes encoding butyrate-producing enzymes, including electron-bifurcating butyryl-CoA dehydrogenase/electron transfer flavoprotein complex and NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase, form a cluster, which is preceded by a gene annotated as the transcriptional regulator Rex. Northern blotting and RT-PCR experiments indicated that the gene cluster forms a large transcriptional unit that possibly includes several small transcriptional units. The deduced Rex protein contains a winged helix DNA-binding domain and a Rossmann fold potentially interacting with NAD(H). Bioinformatics analysis revealed that Rex can bind the promoter regions of numerous genes, which are involved in carbon and energy metabolism, including NADH oxidation, hydrogen production, ATP synthesis, butyrate formation, and succinate metabolism. Rex may regulate the transcription of genes encoding certain transcriptional regulators and transporters. Electrophoretic mobility shift and isothermal titration calorimetry assays revealed that Rex specifically formed protein-DNA complexes with the promoter regions of target genes, which could be inhibited by NADH but restored by an excess amount of NAD(+). These results suggest that Rex plays a key role in the carbon and energy metabolism of C. kluyveri as a global transcriptional regulator in response to the cellular NADH/NAD(+) ratio.

Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33.

Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are ∼26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33.

Heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin.

Ferredoxins are small, acidic proteins containing iron-sulfur clusters that are widespread in living organisms. They play key roles as electron carriers in various metabolic processes, including respiration, photosynthesis, fermentation, nitrogen fixation, carbon dioxide fixation, and hydrogen production. However, only several kinds of ferredoxins are commercially available now, greatly limiting the investigation of ferredoxin-related enzymes and metabolic processes. Here we describe the heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin. Adding extra iron and sulfur sources to the medium in combination with using Escherichia coli C41(DE3) harboring pCodonplus and pRKISC plasmids as host greatly enhanced iron-sulfur cluster synthesis in the three ferredoxins. After induction for 12 h in terrific broth and purification by affinity chromatography and anion exchange chromatography, approximately 3.4 mg of streptavidin (Strep)-tagged and 3.7 mg of polyhistidine (His)-tagged clostridial 2[4Fe4S] ferredoxins were obtained from 1 l of culture. Excitingly, after induction for 24 h in terrific broth, around 40 mg of His-tagged clostridial [2Fe2S] and 23 mg of His-tagged agrobacterial [2Fe2S] ferredoxins were purified from 1 l of culture. The recombinant ferredoxins apparently exhibited identical properties and physiological function to native ferredoxins. No negative impact of two different affinity tags on ferredoxin activity was found. In conclusion, we successfully developed a convenient method for heterologous overproduction of the three kinds of ferredoxins with satisfactory yields and activities, which would be very helpful for the ferredoxin-related researches.