The dramatic decrease in solubility accompanying the reduction of U(VI) to U(IV), producing the insoluble mineral uraninite, has been viewed as a potential mechanism for sequestration of environmental uranium contamination. In the past 15 years, it has been firmly established that a variety of bacteria exhibit this reductive capacity. To obtain an understanding of the microbial metal metabolism, to develop a practical approach for the acceleration of in situ bioreduction, and to predict the long-term fate of environmental uranium, several aspects of the microbial process have been experimentally explored. This review briefly addresses the research to identify specific uranium reductases and their cellular location, competition between uranium and other electron acceptors, attempts to stimulate in situ reduction, and mechanisms of reoxidation of reduced uranium minerals.

Nitrate is an inhibitor of sulfate-reducing bacteria (SRB). In petroleum production sites, amendments of nitrate and nitrite are used to prevent SRB production of sulfide that causes souring of oil wells. A better understanding of nitrate stress responses in the model SRB, Desulfovibrio vulgaris Hildenborough and Desulfovibrio alaskensis G20, will strengthen predictions of environmental outcomes. Nitrate inhibition of SRB has historically been considered to result from the generation of small amounts of nitrite, to which SRB are quite sensitive. Here we explored the possibility that nitrate might inhibit SRB by a mechanism other than through nitrite inhibition. We found that nitrate-stressed D. vulgaris cultures grown in lactate-sulfate conditions eventually grew in the presence of high concentrations of nitrate, and their resistance continued through several subcultures. Nitrate consumption was not detected over the course of the experiment, suggesting adaptation to nitrate. With high-throughput genetic approaches employing TnLE-seq for D. vulgaris and a pooled mutant library of D. alaskensis, we determined the fitness of many transposon mutants of both organisms in nitrate stress conditions. We found that several mutants, including homologs present in both strains, had a greatly increased ability to grow in the presence of nitrate but not nitrite. The mutated genes conferring nitrate resistance included the gene encoding the putative Rex transcriptional regulator (DVU0916/Dde_2702), as well as a cluster of genes (DVU0251-DVU0245/Dde_0597-Dde_0605) that is poorly annotated. Follow-up studies with individual D. vulgaris transposon and deletion mutants confirmed high-throughput results. We conclude that, in D. vulgaris and D. alaskensis, nitrate resistance in wild-type cultures is likely conferred by spontaneous mutations. Furthermore, the mechanisms that confer nitrate resistance may be different from those that confer nitrite resistance.

Progress in the genetic manipulation of the Desulfovibrio strains has provided an opportunity to explore electron flow pathways during sulfate respiration. Most bacteria in this genus couple the oxidation of organic acids or ethanol with the reduction of sulfate, sulfite, or thiosulfate. Both fermentation of pyruvate in the absence of an alternative terminal electron acceptor, disproportionation of fumarate and growth on H₂ with CO₂ during sulfate reduction are exhibited by some strains. The ability to produce or consume H₂ provides Desulfovibrio strains the capacity to participate as either partner in interspecies H₂ transfer. Interestingly the mechanisms of energy conversion, pathways of electron flow and the parameters determining the pathways used remain to be elucidated. Recent application of molecular genetic tools for the exploration of the metabolism of Desulfovibrio vulgaris Hildenborough has provided several new datasets that might provide insights and constraints to the electron flow pathways. These datasets include (1) gene expression changes measured in microarrays for cells cultured with different electron donors and acceptors, (2) relative mRNA abundances for cells growing exponentially in defined medium with lactate as carbon source and electron donor plus sulfate as terminal electron acceptor, and (3) a random transposon mutant library selected on medium containing lactate plus sulfate supplemented with yeast extract. Studies of directed mutations eliminating apparent key components, the quinone-interacting membrane-bound oxidoreductase (Qmo) complex, the Type 1 tetraheme cytochrome c₃ (Tp1-c₃), or the Type 1 cytochrome c₃:menaquinone oxidoreductase (Qrc) complex, suggest a greater flexibility in electron flow than previously considered. The new datasets revealed the absence of random transposons in the genes encoding an enzyme with homology to Coo membrane-bound hydrogenase. From this result, we infer that Coo hydrogenase plays an important role in D. vulgaris growth on lactate plus sulfate. These observations along with those reported previously have been combined in a model showing dual pathways of electrons from the oxidation of both lactate and pyruvate during sulfate respiration. Continuing genetic and biochemical analyses of key genes in Desulfovibrio strains will allow further clarification of a general model for sulfate respiration.

Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c ₃ could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c ₃ 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 c ₃ 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.

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes. For a few bacteria, it is well known how they can make all 20 of the standard amino acids (the building blocks of proteins). For many other bacteria, their genome sequence implies that there are gaps in these biosynthetic pathways, so that the bacteria cannot make all of the amino acids and would need to take up some of them from their environment instead. But many bacteria can grow in minimal media (without any amino acids) despite these apparent gaps. We studied 10 bacteria with predicted gaps in amino acid biosynthesis that nevertheless grow in minimal media. Most of these gaps were spurious, but 11 of the gaps were genuine and could not be explained by current knowledge. Using high-throughput genetics, we systematically identified genes that were required for growth in minimal media and identified the biosynthetic genes that fill 9 of the 11 gaps. We hope that this approach can be applied to many more bacteria and will eventually allow us to accurately predict the nutritional requirements of a bacterium from its genome sequence.