Resistance of solid-phase U(VI) to microbial reduction during in situ bioremediation of uranium-contaminated groundwater

Speciation of solid-phase uranium in uranium-contaminated subsurface sediments undergoing uranium bioremediation demonstrated that although microbial reduction of soluble U(VI) readily immobilized uranium as U(IV), a substantial portion of the U(VI) in the aquifer was strongly associated with the sediments and was not microbially reducible. These results have important implications for in situ uranium bioremediation strategies.     

U(VI) Reduction by Diverse Outer Surface c-Type Cytochromes of Geobacter sulfurreducens

Early studies with Geobacter sulfurreducens suggested that outer-surface c-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of G. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface c-type cytochromes of G. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ± 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ± 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that G. sulfurreducens requires outer-surface c-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.     

Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (kcat= 8.3 s(-1); Km= 14.1 and 4.3 microM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (Ki= 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K+ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.     

Detection and quantification of Geobacter lovleyi strain SZ: implications for bioremediation at tetrachloroethene- and uranium-impacted sites

Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 x 10(6) and 1 x 10(4) 16S rRNA gene copies per mul of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per mul of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 x 10(7) +/- 0.1 x 10(7) per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.     

Dynamics of microbial community during bioremediation of phenanthrene and chromium(VI)-contaminated soil microcosms

The combined effect of phenanthrene and Cr(VI) on soil microbial activity, community composition and on the efficiency of bioremediation processes has been studied. Biometer flask systems and soil microcosm systems contaminated with 2,000 mg of phenanthrene per kg of dry soil and different Cr(VI) concentrations were investigated. Temperature, soil moisture and oxygen availability were controlled to support bioremediation. Cr(VI) inhibited the phenanthrene mineralization (CO₂ production) and cultivable PAH degrading bacteria at levels of 500–2,600 mg kg⁻¹. In the bioremediation experiments in soil microcosms the degradation of phenanthrene, the dehydrogenase activity and the increase in PAH degrading bacteria counts were retarded by the presence of Cr(VI) at all studied concentrations (25, 50 and 100 mg kg⁻¹). These negative effects did not show a correlation with Cr(VI) concentration. Whereas the presence of Cr(VI) had a negative effect on the phenanthrene elimination rate, co-contamination with phenanthrene reduced the residual Cr(VI) concentration in the water exchangeable Cr(VI) fraction (WEF) in comparison with the soil microcosm contaminated only with Cr(VI). Clear differences were found between the denaturing gradient gel electrophoresis (DGGE) patterns of each soil microcosm, showing that the presence of different Cr(VI) concentrations did modulate the community response to phenanthrene and caused perdurable changes in the structure of the microbial soil community.     

Global transcriptional profiling of Shewanella oneidensis MR-1 during Cr(VI) and U(VI) reduction

Whole-genome DNA microarrays were used to examine the gene expression profile of Shewanella oneidensis MR-1 during U(VI) and Cr(VI) reduction. The same control, cells pregrown with nitrate and incubated with no electron acceptor, was used for the two time points considered and for both metals. U(VI)-reducing conditions resulted in the upregulation (> or = 3-fold) of 121 genes, while 83 genes were upregulated under Cr(VI)-reducing conditions. A large fraction of the genes upregulated [34% for U(VI) and 29% for Cr(VI)] encode hypothetical proteins of unknown function. Genes encoding proteins known to reduce alternative electron acceptors [fumarate, dimethyl sulfoxide, Mn(IV), or soluble Fe(III)] were upregulated under both U(VI)- and Cr(VI)-reducing conditions. The involvement of these upregulated genes in the reduction of U(VI) and Cr(VI) was tested using mutants lacking one or several of the gene products. Mutant testing confirmed the involvement of several genes in the reduction of both metals: mtrA, mtrB, mtrC, and menC, all of which are involved in Fe(III) citrate reduction by MR-1. Genes encoding efflux pumps were upregulated under Cr(VI)- but not under U(VI)-reducing conditions. Genes encoding proteins associated with general (e.g., groL and dnaJ) and membrane (e.g., pspBC) stress were also upregulated, particularly under U(VI)-reducing conditions, pointing to membrane damage by the solid-phase reduced U(IV) and Cr(III) and/or the direct effect of the oxidized forms of the metals. This study sheds light on the multifaceted response of MR-1 to U(VI) and Cr(VI) under anaerobic conditions and suggests that the same electron transport pathway can be used for more than one electron acceptor.     

Ochrobactrum tritici strain 5bvl1 - characterization of a Cr(VI)-resistant and Cr(VI)-reducing strain

Bacterial strain 5bvl1, isolated from a chromium-contaminated wastewater treatment plant and identified as Ochrobactrum tritici, was resistant to a broad range of antibiotics, to Cr(VI), Ni(II), Co(II), Cd(II), and Zn(II), and was able to grow in the presence of 5% NaCl and within the pH range 4-10. Characterization showed that strain 5bvl1 could be considered a halotolerant and alkalitolerant microorganism resistant to high concentrations of Cr(VI). This strain was able to grow aerobically in up to 10 mmolxL(-1) Cr(VI). Cr(VI) resistance was independent of sulphate concentration. Under aerobic conditions strain 5bvl1 was also able to reduce high Cr(VI) concentrations (up to 1.7 mmolxL(-1)). Increasing concentrations of Cr(VI) in the medium lowered the growth rate of strain 5bv11 but the reduction in growth rate could not be directly correlated with the amount of Cr(VI) reduced. Unlike the type strain, which was only able to reduce Cr(VI), strain 5bvl1 was resistant to Cr(VI) and able to reduce it. Moreover, in strain 5bvl1, the rate and extent of Cr(VI)-reduction were higher than in the other strains of the genus Ochrobactrum. Ochrobactrum strain 5bvl1 resists high Cr(VI) concentrations and has a high Cr(VI)-reducing ability, making it a valuable tool in bioremediation.     

Relationship between intramitochondrial citrate and the activity of carbamoyl-phosphate synthase (ammonia).

The possibility of control of the activity of carbamoyl-phosphate synthase (ammonia) (EC 2.7.2.5) in rat-liver mitochondria by variation in the intramitochondrial free Mg2+ concentration has been investigated. Carbamoyl-phosphate synthase activity was measured by coupling the formation of carbamoylphosphate to the synthesis of citrulline in a reaction mixture containing ammonia, bicarbonate, a source of ATP, and ornithine. The synthesis of citrulline was inhibited by lowering the concentration of intramitochondrial free Mg2+. This could be achieved not only by depleting the mitochondria of Mg2+ (by adding the ionophore A23187), but also by increasing the intramitochondrial concentration of citrate. Under various conditions an inverse relationship between the rate of citrulline synthesis and the magnitude of the intramitochondrial concentration of citrate was observed. Inhibition of citrulline synthesis by intramitochondrial citrate could be partly reversed by addition of Mg2+ in the presence of A23187. Possible implications of the regulation of carbamoyl-phosphate synthase (ammonia) activity by intramitochondrial citrate for nitrogen metabolism in the liver are discussed.     

Simultaneous bioremediation of Cr(VI) and lindane in soil by actinobacteria

Environments co-contaminated with metals and organic compounds are difficult to remediate. Actinobacteria is an important group of microorganisms found in soils, with high metabolic versatility and potential for bioremediation. In this paper, actinobacteria were used to remediate soil co-contaminated with Cr(VI) and lindane. Five actinobacteria, tolerant to Cr(VI) and lindane mixture were selected: Streptomyces spp. A5, A11, M7, and MC1, and Amycolatopsis tucumanensis DSM 45259. Sterilized soil samples were inoculated with actinobacteria strains, either individually or as a consortium, and contaminated with Cr(VI) and lindane, either immediately or after 7 days of growth, and incubated at 30 °C during 14 days. All actinobacteria were able to grow and remove both contaminants, the consortium formed by Streptomyces spp. A5, M7, MC1, and A. tucumanensis showed the highest Cr(VI) removal, while Streptomyces sp. M7 produced the maximum lindane removal. In non-sterile soil samples, Streptomyces sp. M7 and the consortium removed more than 40% of the lindane, while Streptomyces sp. M7 demonstrated the greatest Cr(VI) removal. The most appropriate strategy for bioremediation of Cr(VI) and lindane co-contaminated soils would be the inoculation with Streptomyces sp. M7.     

Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor

The mechanisms for Fe(III) oxide reduction in Geobacter species are of interest because Fe(III) oxides are the most abundant form of Fe(III) in many soils and sediments and Geobacter species are prevalent Fe(III)-reducing microorganisms in many of these environments. Protein abundance in G. sulfurreducens grown on poorly crystalline Fe(III) oxide or on soluble Fe(III) citrate was compared with a global accurate mass and time tag proteomic approach in order to identify proteins that might be specifically associated with Fe(III) oxide reduction. A total of 2991 proteins were detected in G. sulfurreducens grown with acetate as the electron donor and either Fe(III) oxide or soluble Fe(III) citrate as the electron acceptor, resulting in 86% recovery of the genes predicted to encode proteins. Of the total expressed proteins 76% were less abundant in Fe(III) oxide cultures than in Fe(III) citrate cultures, which is consistent with the overall slower rate of metabolism during growth with an insoluble electron acceptor. A total of 269 proteins were more abundant in Fe(III) oxide-grown cells than in cells grown on Fe(III) citrate. Most of these proteins were in the energy metabolism category: primarily electron transport proteins, including 13 c-type cytochromes and PilA, the structural protein for electrically conductive pili. Several of the cytochromes that were more abundant in Fe(III) oxide-grown cells were previously shown with genetic approaches to be essential for optimal Fe(III) oxide reduction. Other proteins that were more abundant during growth on Fe(III) oxide included transport and binding proteins, proteins involved in regulation and signal transduction, cell envelope proteins, and enzymes for amino acid and protein biosynthesis, among others. There were also a substantial number of proteins of unknown function that were more abundant during growth on Fe(III) oxide. These results indicate that electron transport to Fe(III) oxide requires additional and/or different proteins than electron transfer to soluble, chelated Fe(III) and suggest proteins whose functions should be further investigated in order to better understand the mechanisms of electron transfer to Fe(III) oxide in G. sulfurreducens.     

A procedure for the analysis of citrate synthase (E.C. 4.1.3.7) in somatic cell hybrids

An electrophoretic procedure has been developed which is capable of resolving mouse and human forms of citrate synthase (E.C. 4.1.3.7) on cellulose acetate strips. This has been used to demonstrate the presence of human citrate synthase in several human-mouse somatic cell hybrids.The work was supported, in part, by a grant from the Medical Research Council.     

Functional groups in the activity and regulation of Escherichia coli citrate synthase

1. Citrate synthase has been purified from Escherichia coli and shown to exist at an equilibrium between three forms: monomer (mol.wt. 57000), tetramer (mol.wt. 230000) and, possibly, octamer. Modification of the enzyme by photo-oxidation and by treatment with specific chemical reagents has been carried out to gain information on the amino acid residues involved in enzymic activity and in the inhibition of activity by NADH and alpha-oxoglutarate. 2. Several photo-oxidizable amino acids appear to be involved in activity. The nature of the pH-dependence of their rates of photo-oxidation with Methylene Blue suggests that these are histidines, a conclusion supported by the greater rate of photo-inactivation with Rose Bengal and the destruction of activity by diethyl pyrocarbonate. 3. The participation of histidine at the alpha-oxoglutarate effector site is indicated by photo-oxidation and the participation of cysteine at the NADH effector site suggested by photo-oxidation is confirmed by the desensitization to NADH produced by treatment with 5,5'-dithiobis-(2-nitrobenzoate). Inactivation of the enzyme after modification with this reagent suggests the additional involvement of cysteine in catalytic activity. 4. Amino acid analyses of native and photo-oxidized enzyme are consistent with these conclusions. 5. Modification with 2-hydroxy-5-nitrobenzyl bromide indicates the participation of tryptophan in the activity of the enzyme.     

Ontogenetic changes in citrate synthase and lactate dehydrogenase activity in the jumping muscle of the American locust (Schistocerca americana)

Intraspecific studies have repeatedly shown that muscle-specific oxidative enzyme activities scale negatively with body mass while muscle-specific glycolytic enzyme activities scale positively. However, most of these studies have not included juveniles. In this study, we examined how citrate synthase (CS, EC 2.3.3.1) and lactate dehydrogenase (LDH; EC 1.1.1.27) activity in the jumping muscle of Schistocerca americana grasshoppers varied with ontogeny across a 40-fold increase in body size. In contrast to the pattern observed when adult conspecifics are compared, we show that jumping muscle CS activity increased more than 2-fold from 2nd instars to adults, while jumping muscle LDH activity increased more than 5-fold. The increased LDH activity in older grasshoppers supports previous data that older grasshoppers have a reduced jumping endurance. The increased CS activity with age may help older grasshoppers efficiently produce aerobic ATP to bend cuticular springs for energy storage before a jump or alternatively recover from anaerobic metabolism after jumping. Metabolic changes in S. americana jumping muscle are similar to other developing taxa and highlight the importance of including juveniles within intraspecific studies. When compared to adults, juvenile locomotion may have increased selection pressure because of both greater energetic demands during growth and higher predation rates.     

Dynamics of microbial community composition and function during in situ bioremediation of a uranium-contaminated aquifer

A pilot-scale system was established to examine the feasibility of in situ U(VI) immobilization at a highly contaminated aquifer (U.S. DOE Integrated Field Research Challenge site, Oak Ridge, TN). Ethanol was injected intermittently as an electron donor to stimulate microbial U(VI) reduction, and U(VI) concentrations fell to below the Environmental Protection Agency drinking water standard (0.03 mg liter(-1)). Microbial communities from three monitoring wells were examined during active U(VI) reduction and maintenance phases with GeoChip, a high-density, comprehensive functional gene array. The overall microbial community structure exhibited a considerable shift over the remediation phases examined. GeoChip-based analysis revealed that Fe(III)-reducing bacterial (FeRB), nitrate-reducing bacterial (NRB), and sulfate-reducing bacterial (SRB) functional populations reached their highest levels during the active U(VI) reduction phase (days 137 to 370), in which denitrification and Fe(III) and sulfate reduction occurred sequentially. A gradual decrease in these functional populations occurred when reduction reactions stabilized, suggesting that these functional populations could play an important role in both active U(VI) reduction and maintenance of the stability of reduced U(IV). These results suggest that addition of electron donors stimulated the microbial community to create biogeochemical conditions favorable to U(VI) reduction and prevent the reduced U(IV) from reoxidation and that functional FeRB, SRB, and NRB populations within this system played key roles in this process.     

Influence of thyroid hormone deficiency on the citrate synthase activity in rat brain regions during early postnatal development

The developmental pattern of citrate synthase activity has been studied in the liver and several brain areas of hypothyroid rats during the 4 first weeks of life. While citrate synthase activity in the liver showed a rise during the 2 first weeks of life, different patterns of enzyme activity were found in the brain regions of euthyroid animals. Citrate synthase activity increased in the cerebellum, decreased in the cerebral cortex and did not change significantly in the brain stem during the period studied. In the liver and brain areas, too, a decrease in citrate synthase activity was observed during hypothyroidism. From the 2nd week of birth, the citrate synthase activity in the brain but not in the liver was found to have recovered. The newly elevated citrate synthase activity coincided with a slight increase in thyroid hormone serum levels.