Pyruvate Metabolism of De s ulfovibrio desulfuricans strain G20

1. Absorbance (600nm) Hour Cells were grown with 60 mM pyruvate and 20mM SO4 at 370 C. At 55 h final protein, H2 and CO2 determinations were made and culture filtrates harvested for HPLC analysis. Absorbance (600nm) CycA- Hour Cultures were grown with 60 mM pyruvate and handled as described above. WT Absorbance (600nm) Absorbance (600nm) Hour Cytochrome c3 is not essential for growth of D. desulfuricans G20 in lactate sulfate medium. CycA- grew at the same rate, but yielded only 85% of the cell protein as compared to WT. About 50% of the reducing equivalents in the lactate used by the CycA- are accumulated as H2. Without a functional c3 these electrons are not available for further oxidation. Hour Composite of two previous graphs. The c3 mutant is not reducing the sulfate when pyruvate is the electron donor, but rather is growing fermentatively. Data presented in table further details this difference between CycA- mutant and WT. Concentration (mM) of compounds in culture filtrate was determined by HPLC; H2 and CO2 production by GC analysis. *<indicates the minimum detectible concentration of compound. Pyruvate Metabolism of Desulfovibrio desulfuricans strainG20 and a Cytochrome c3Mutant Barbara J. Giles, Kate E. Hart, Judy D. Wall Department of Biochemistry, University of Missouri-Columbia Abstract WT and CycA- MutantGrowth on Pyruvate Sulfate The versatility of SRB energy generating processes makes the many environmental impacts of these bacteria possible. A “hydrogen-cycling” model was proposed over twenty years ago to explain how these bacteria may augment energy generation during respiration. Cytochrome c3 (type 1 tetraheme cytochrome c3) is a key to the proposed route of electron flow. A mutant of Desulfovibrio desulfuricans G20 that lacks functional cyt c3 produces a significant portion of the reductant generated from lactate oxidation as hydrogen. The absence of cyt c3 apparently blocks re-oxidation of that hydrogen. The lack of cyt c3 eliminates respiration of sulfate with pyruvate as electron acceptor, restricting growth on pyruvate to fermentation. Neither sulfate nor fumarate is used as a terminal electron acceptor when this tetraheme cytochrome is absent. These data support a “hydrogen-cycling” model for respiration of pyruvate with sulfate; however, the hydrogen atoms may be carried by formate, a possibility yet to be explored. WT and CycA-MutantFermentative Growth on Pyruvate Possible pathways for electrons generated by pyruvate fermentation are indicated by “dashed” lines. The green box covers those routes which are impaired when there is no functional cytochrome c3. PFOR Pyruvate Ferredoxin Oxidoreductase; QMO Quinone-interacting Membrane-bound Oxidoreductase; HMC High Molecular weight Cytochrome; Dsr Dissimilatory Sulfite Reductase Conclusions • Cytochrome c3 is a requirement for respiration of pyruvate with sulfate as the electron acceptor. In the absence of c3, only pyruvate fermentation is possible. • 2. Cells lacking cytochrome c3 can still respire with electrons from lactate. • 3. Delivery of electrons to internal cytoplasmic electron acceptors, such as sulfate or organic acids, is dependent upon the presence of cytochrome c3. This is seen by the generation of significant amounts of succinate during pyruvate fermentation by WT as compared to the CycA- mutant. WT and CycA- MutantGrowth on Lactate Sulfate HPLC Analysis of Culture Filtrates Comparison of WT and CycA- Mutant Growth on Pyruvate and Pyruvate Sulfate Acetate Fumarate Succinate Pyruvate Formate This research was funded through the Department of Energy. Acetate Succinate Pyruvate Lactate Formate Minutes An Aminex HPX-87H HPLC column was used to separate, identify and quantitate the organic compounds present in filtrates of fermentatively grown cultures. 5 mM H2SO4 was the mobile phase.