Anaerobic reduction of europium by a Clostridium strain as a strategy for rare earth biorecovery

The biorecovery of europium (Eu) from primary (mineral deposits) and secondary (mining wastes) resources is of interest due to its remarkable luminescence properties, important for modern technological applications. In this study, we explored the tolerance levels, reduction and intracellular bioaccumulation of Eu by a site-specific bacterium, Clostridium sp. 2611 isolated from Phalaborwa carbonatite complex. Clostridium sp. 2611 was able to grow in minimal medium containing 0.5 mM Eu3+. SEM-EDX analysis confirmed an association between Eu precipitates and the bacterium, while TEM-EDX analysis indicated intracellular accumulation of Eu. According to the HR-XPS analysis, the bacterium was able to reduce Eu3+ to Eu2+ under growth and non-growth conditions. Preliminary protein characterization seems to indicate that a cytoplasmic pyruvate oxidoreductase is responsible for Eu bioreduction. These findings suggest the bioreduction of Eu3+ by Clostridium sp. as a resistance mechanism, can be exploited for the biorecovery of this metal.

Organohalide-respiringDesulfolunaspecies isolated from marine environments

The genusDesulfolunacomprises two anaerobic sulfate-reducing strains,D. spongiiphilaAA1⊤andD. butyratoxydansMSL71⊤of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third member of this genus from marine intertidal sediment, designedD. spongiiphilastrain DBB. All threeDesulfolunastrains harbour three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes. Brominated but not chlorinated aromatic compounds were dehalogenated by all three strains. TheDesulfolunastrains maintained OHR in the presence of 20 mM sulfate or 20 mM sulfide, which often negatively affect OHR. Strain DBB sustained OHR with 2% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR (RT-qPCR) revealed differential induction ofrdhAgenes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed differential expression ofrdhA1with 1,4-dibromobenzene, and revealed a possible electron transport chain from lactate dehydrogenases and pyruvate oxidoreductase to RdhA1 via menaquinones and either RdhC, or Fix complex (electron transfer flavoproteins), or Qrc complex (Type-1 cytochrome c3:menaquinone oxidoreductase).

Disruption of the Operon Encoding Ehb Hydrogenase Limits Anabolic CO2 Assimilation in the Archaeon Methanococcus maripaludis

ABSTRACT Methanococcus maripaludis is a mesophilic archaeon that reduces CO2 to methane with H2 or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ehb. To determine the role of Ehb, a deletion in the ehb operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessary to restore growth to nearly wild-type levels, suggesting that Ehb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ehb in anabolic CO2 assimilation in methanococci.

Enzymes Associated with Reductive Activation and Action of Nitazoxanide, Nitrofurans, and Metronidazole in Helicobacter pylori

ABSTRACT Nitazoxanide (NTZ) is a redox-active nitrothiazolyl-salicylamide prodrug that kills Helicobacter pylori and also many anaerobic bacterial, protozoan, and helminthic species. Here we describe development and use of a spectrophotometric assay, based on nitroreduction of NTZ at 412 nm, to identify H. pylori enzymes responsible for its activation and mode of action. Three enzymes that reduce NTZ were identified: two related NADPH nitroreductases, which also mediate susceptibility to metronidazole (MTZ) (RdxA and FrxA), and pyruvate oxidoreductase (POR). Recombinant His-tagged RdxA, FrxA, and POR, overexpressed in nitroreductase-deficient Escherichia coli, each rapidly reduced NTZ, whereas only FrxA and to a lesser extent POR reduced nitrofuran substrates (furazolidone, nitrofurantoin, and nitrofurazone). POR exhibited no MTZ reductase activity either in extracts of H. pylori or following overexpression in E. coli; RdxA exhibited no nitrofuran reductase activity, and FrxA exhibited no MTZ reductase activity. Analysis of mutation to rifampin resistance (Rifr) indicated that NTZ was not mutagenic and that nitrofurans were only weakly mutagenic. Alkaline gel DNA electrophoresis indicated that none of these prodrugs caused DNA breakage. In contrast, MTZ caused DNA damage and was strongly mutagenic. We conclude that POR, an essential enzyme, is responsible for most or all of the bactericidal effects of NTZ against H. pylori. While loss-of-function mutations in rdxA and frxA produce a Mtzr phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.

Metabolic Pathways for Cytotoxic End Product Formation from Glutamate- and Aspartate-Containing Peptides byPorphyromonas gingivalis

ABSTRACT Metabolic pathways involved in the formation of cytotoxic end products by Porphyromonas gingivalis were studied. The washed cells of P. gingivalis ATCC 33277 utilized peptides but not single amino acids. Since glutamate and aspartate moieties in the peptides were consumed most intensively, a dipeptide of glutamate or aspartate was then tested as a metabolic substrate of P. gingivalis. P. gingivalis cells metabolized glutamylglutamate to butyrate, propionate, acetate, and ammonia, and they metabolized aspartylaspartate to butyrate, succinate, acetate, and ammonia. Based on the detection of metabolic enzymes in the cell extracts and stoichiometric calculations (carbon recovery and oxidation/reduction ratio) during dipeptide degradation, the following metabolic pathways were proposed. Incorporated glutamylglutamate and aspartylaspartate are hydrolyzed to glutamate and aspartate, respectively, by dipeptidase. Glutamate is deaminated and oxidized to succinyl-coenzyme A (CoA) by glutamate dehydrogenase and 2-oxoglutarate oxidoreductase. Aspartate is deaminated into fumarate by aspartate ammonia-lyase and then reduced to succinyl-CoA by fumarate reductase and acyl-CoA:acetate CoA-transferase or oxidized to acetyl-CoA by a sequential reaction of fumarase, malate dehydrogenase, oxaloacetate decarboxylase, and pyruvate oxidoreductase. The succinyl-CoA is reduced to butyryl-CoA by a series of enzymes, including succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and butyryl-CoA oxidoreductase. A part of succinyl-CoA could be converted to propionyl-CoA through the reactions initiated by methylmalonyl-CoA mutase. The butyryl- and propionyl-CoAs thus formed could then be converted into acetyl-CoA by acyl-CoA:acetate CoA-transferase with the formation of corresponding cytotoxic end products, butyrate and propionate. The formed acetyl-CoA could then be metabolized further to acetate.