Barbara J. Rapp-Giles

Uranium Reduction by Desulfovibrio desulfuricans Strain G20 and a Cytochrome c3 Mutant

ABSTRACT Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.

Complete Genome Sequence and Updated Annotation of Desulfovibrio alaskensis G20

Desulfovibrio alaskensis G20 (formerly Desulfovibrio desulfuricans G20) is a Gram-negative mesophilic sulfate-reducing bacterium (SRB), known to corrode ferrous metals and to reduce toxic radionuclides and metals such as uranium and chromium to sparingly soluble and less toxic forms. We present the 3.7-Mb genome sequence to provide insights into its physiology.

Cytochrome c 3 Mutants of Desulfovibrio desulfuricans

ABSTRACT To explore the physiological role of tetraheme cytochromec3 in the sulfate-reducing bacteriumDesulfovibrio desulfuricans G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion. The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful. The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities. Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant. Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium. These data support a role for tetraheme cytochromec3 in the electron transport pathway from pyruvate to sulfate or sulfite in D. desulfuricans G20.

New Model for Electron Flow for Sulfate Reduction in Desulfovibrio alaskensis G20

ABSTRACT To understand the energy conversion activities of the anaerobic sulfate-reducing bacteria, it is necessary to identify the components involved in electron flow. The importance of the abundant type I tetraheme cytochrome c 3 (TpIc 3) as an electron carrier during sulfate respiration was questioned by the previous isolation of a null mutation in the gene encoding TpIc 3, cycA, in Desulfovibrio alaskensis G20. Whereas respiratory growth of the CycA mutant with lactate and sulfate was little affected, growth with pyruvate and sulfate was significantly impaired. We have explored the phenotype of the CycA mutant through physiological tests and transcriptomic and proteomic analyses. Data reported here show that electrons from pyruvate oxidation do not reach adenylyl sulfate reductase, the enzyme catalyzing the first redox reaction during sulfate reduction, in the absence of either CycA or the type I cytochrome c 3:menaquinone oxidoreductase transmembrane complex, QrcABCD. In contrast to the wild type, the CycA and QrcA mutants did not grow with H2 or formate and sulfate as the electron acceptor. Transcriptomic and proteomic analyses of the CycA mutant showed that transcripts and enzymes for the pathway from pyruvate to succinate were strongly decreased in the CycA mutant regardless of the growth mode. Neither the CycA nor the QrcA mutant grew on fumarate alone, consistent with the omics results and a redox regulation of gene expression. We conclude that TpIc 3 and the Qrc complex are D. alaskensis components essential for the transfer of electrons released in the periplasm to reach the cytoplasmic adenylyl sulfate reductase and present a model that may explain the CycA phenotype through confurcation of electrons.

Genetics and Genomics of Sulfate Respiration in Desulfovibrio

Bacteria that have evolved to use sulfate as a terminal electron acceptor must commit to spending energy for sulfate activation before there is a return on the investment allowing net energy gain. How sulfate is used and how electron flow is controlled have provided challenging topics for research for many years. Having the complete genome sequences of several of these bacteria is a monumental step in the elucidation of these questions. This information has provided the tools for determining the quantity of transcripts for genes under defined growth conditions, not just the relative changes in transcripts in two growth conditions. A comparison of the hybridization signal of messenger RNA with that of genomic DNA with oligonucleotide microarrays of all open reading frames reveals the differences in steady-state levels of transcripts for each gene. Growth of Desulfovibrio vulgaris Hildenborough on defined medium with lactate as a carbon and reductant source and with sulfate as the electron acceptor has been examined by this procedure for levels of gene expression. Relative functional importance was inferred from the levels of gene transcription, in spite of the recognized limitations of this interpretation. Not surprisingly, genes encoding established functions for sulfate reduction were highly expressed. However, the high molecular mass c-type cytochrome genes thought to encode a most important transmembrane electron conduit for sulfate reduction were expressed at quite low levels.

Genes and Genetic Manipulations of Desulfovibrio

These examples of observations of sequences leave more questions than answers but provide intriguing hints that the metabolism of this important group of bacteria is versatile and complex.