The Rnf Complex Is an Energy-Coupled Transhydrogenase Essential To Reversibly Link Cellular NADH and Ferredoxin Pools in the Acetogen Acetobacterium woodii

Abstract

The Rnf complex is a respiratory enzyme that catalyzes oxidation of reduced ferredoxin to the reduction of NAD, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic, acetogenic bacteria the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, , which enabled us to delete the genes and to test their role. The mutant did not grow on H+CO, neither did it produce acetate or ATP from H+CO, and ferredoxin:NAD oxidoreductase activity as well as Na translocation was also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low energy substrates such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of ferredoxin but only of NAD, and we speculated that the growth phenotype is caused by a loss of reduced ferredoxin, indispensable for biosynthesis and CO reduction. The electron bifurcating hydrogenase of reduces ferredoxin and indeed, addition of H to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex which renders the Rnf complex essential also for growth on low energy substrates. Ferredoxin and NAD are key electron-carriers in anaerobic bacteria, but energetically they are not equivalent since the redox-potential of ferredoxin is lower than that of the NADH/NAD couple. We describe by mutant studies in that the main function of Rnf is to energeticallylink cellular pools of ferredoxin and NAD When ferredoxin>NADH, exergonic electron flow from ferredoxin to NAD generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH>ferredoxin, the Rnf works in reverse. This reaction is essential for growth on low energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO reduction. Our studies put a new perspective on the cellular function of the membrane-bound, ion-translocating Rnf complex widespread in bacteria.

Regulation of lactate metabolism in the acetogenic bacterium Acetobacterium woodii

“…To this end, the genes Awo_c08720 ( lctC ) coding for the electron transferring subunit of the lactate dehydrogenase (EtfA), Awo_c08730 ( lctD ) coding for the major subunit of the lactate dehydrogenase, Awo_c08740 ( lctE ) coding for a potential l ‐lactate permease (LctP) and Awo_c08750 ( lctF ) coding for a potential lactate racemase were deleted from the chromosome of A. woodii. The mutagenesis was performed in a pyrE deletion mutant (Westphal et al ., ) by double homologous recombination using a suicide plasmid (pMTL‐JPB24, plasmid sequence S1) containing homology arms (HA) with the upstream and downstream region of the lctCDEF genes leaving 6 bp behind the start codon of lctC and 3 bp in front of the stop codon of lctF. After homologous recombination, the strain was tested for plasmid loss through thiamphenicol sensitivity and the 5565 bp deletion was confirmed by a flanking polymerase chain reaction (PCR) using primers AW_lct_FlankF and AW_lct_FlankR (Supporting Information Table S1) followed by Sanger sequencing.…”

“…The suicide plasmid (pMTL-JPB24, Plasmid sequence S1) for the in-frame deletion of the lctCDEF genes without deletion of lctB in A. woodii was constructed in E. coli HB101 (Promega). Plasmid pMTL-JPB24 is an allelic exchange KO vector which lacks a Gram-positive replicon but carries the Clostridium perfringens catP marker (specifying resistance to thiamphenicol) and a heterologous pyrE (cac_0027) from Clostridium acetobutylicum ATCC 824 that is used as a counter selectable marker (Westphal et al, 2018). For construction of the deletion cassette, HA were designed to flank the A. woodii lctC and lctF genes of 997 bp (left HA) (NC_016894.1, 1004845-1005841) and 982 bp (right HA) (NC_016894.1, 1011407-1012388), respectively, thus retaining the three starting codons of lctC and two end codons of lctE without affecting the 5 0 -untranslated region (UTR) and 3 0 -UTR.…”

The defining genomic and predicted metabolic features of the Acetobacterium genus

“…Generation of a transmembrane gradient is utilized by an F 1 F 0 ATP synthase for ATP synthesis and relies upon either the Rhodobacter nitrogen fixation (Rnf) complex or cytochromes depending upon the species. The Acetobacterium genus appears to lack cytochromes and therefore Acetobacterium woodii has served as a model organism for understanding the mechanisms of energy conservation in acetogens lacking cytochromes [22,[60][61][62][63][64].…”

“…The Rnf complex is an energy-coupled transhydrogenase responsible for transfer of electrons from reduced ferredoxin to NAD + , which generates a transmembrane sodium ion gradient to drive ATP generation via sodium-dependent ATP synthase. This process is reversible when the concentration of NADH is greater than ferredoxin [64]. The Rnf cluster is common across many Clostridia including C.…”

Combinatorial assembly of ferredoxin‐linked modules in Escherichia coli yields a testing platform for Rnf‐complexes

“…Based on sequences and homology models, RnfB (polyferredoxin) could be the Fd docking site, whereas RnfC contains the NAD binding site. Despite much speculation, the electron route is hitherto unsolved (Westphal, Wiechmann, Baker, Minton, & Müller, ). Flavin cofactors have been identified in homologs of RnfD and RnfG (Backiel et al, ; Suharti, Wang, de Vries, & Ferry, ) and predicted in RnfC, whereas RnfB and RnfC harbor arrays of iron–sulfur clusters (Suharti et al, ).…”