Brady D. Lee

Screening of Cyanobacterial Species for Calcification

Species of cyanobacteria in the genera Synechococcus and Synechocystis are known to be the catalysts of a phenomenon called “whitings”, which is the formation and precipitation of fine‐grained CaCO3 particles. Whitings occur when the cyanobacteria fix atmospheric CO2 through the formation of CaCO3 on their cell surfaces, which leads to precipitation to the ocean floor and subsequent entombment in mud. Whitings represent one potential mechanism for CO2 sequestration. Research was performed to determine the ability of various strains of Synechocystis and Synechococcus to calcify when grown in microcosms amended with 2.5 mM HCO3‐ and 3.4 mM Ca2+. Results indicated that although all strains tested have the ability to calcify, only two Synechococcus species, strains PCC 8806 and PCC 8807, were able to calcify to the extent that a CaCO3 precipitate was formed. Enumeration of the cyanobacterial cultures during testing indicated that cell density did not appear to have a direct effect on calcification. Factors that had the greatest effect on calcification were CO2 removal and subsequent generation of alkaline pH. Whereas cell density was similar for all strains tested, differences in maximum pH were demonstrated. As CO2 was removed, growth medium pH increased and soluble Ca2+ was removed from solution. The largest increases in growth medium pH occurred when CO2 levels dropped below 400 ppmv. Research presented demonstrates that, under the conditions tested, many species of cyanobacteria in the genera Synechocystis and Synechococcus are able to calcify but only two species of Synechococcus were able to calcify to an extent that led to the precipitation of calcium carbonate.

Application of Molecular Techniques To Elucidate the Influence of Cellulosic Waste on the Bacterial Community Structure at a Simulated Low-Level-Radioactive-Waste Site

ABSTRACT Low-level-radioactive-waste (low-level-waste) sites, including those at various U.S. Department of Energy sites, frequently contain cellulosic waste in the form of paper towels, cardboard boxes, or wood contaminated with heavy metals and radionuclides such as chromium and uranium. To understand how the soil microbial community is influenced by the presence of cellulosic waste products, multiple soil samples were obtained from a nonradioactive model low-level-waste test pit at the Idaho National Laboratory. Samples were analyzed using 16S rRNA gene clone libraries and 16S rRNA gene microarray (PhyloChip) analyses. Both methods revealed changes in the bacterial community structure with depth. In all samples, the PhyloChip detected significantly more operational taxonomic units, and therefore relative diversity, than the clone libraries. Diversity indices suggest that diversity is lowest in the fill and fill-waste interface (FW) layers and greater in the wood waste and waste-clay interface layers. Principal-coordinate analysis and lineage-specific analysis determined that the Bacteroidetes and Actinobacteria phyla account for most of the significant differences observed between the layers. The decreased diversity in the FW layer and increased members of families containing known cellulose-degrading microorganisms suggest that the FW layer is an enrichment environment for these organisms. These results suggest that the presence of the cellulosic material significantly influences the bacterial community structure in a stratified soil system.

Uranium exerts acute toxicity by binding to pyrroloquinoline quinone cofactor

Uranium as an environmental contaminant has been shown to be toxic to eukaryotes and prokaryotes; however, no specific mechanisms of uranium toxicity have been proposed so far. Here a combination of in vivo, in vitro, and in silico studies are presented describing direct inhibition of pyrroloquinoline quinone (PQQ)-dependent growth and metabolism by uranyl cations. Electrospray-ionization mass spectroscopy, UV-vis optical spectroscopy, competitive Ca(2+)/uranyl binding studies, relevant crystal structures, and molecular modeling unequivocally indicate the preferred binding of uranyl simultaneously to the carboxyl oxygen, pyridine nitrogen, and quinone oxygen of the PQQ molecule. The observed toxicity patterns are consistent with the biotic ligand model of acute metal toxicity. In addition to the environmental implications, this work represents the first proposed molecular mechanism of uranium toxicity in bacteria, and has relevance for uranium toxicity in many living systems.

Removal of low concentrations of carbon tetrachloride in compost-based biofilters operated under methanogenic conditions

Research was performed to demonstrate the removal of carbon tetrachloride (CT) using compost biofilters operated under methanogenic conditions. Biofilters were operated at an empty-bed residence time of 2.8 minutes using nitrogen as the atmosphere. Hydrogen and carbon dioxide were supplied as an electron donor and carbon source, respectively, during acclimation of the bed medium microbes. Once methanogenesis was demonstrated, CT flow to the biofilter was established. Biofilters were operated over a CT concentration range from 20 to 700 ppbv for 6 months. Bed medium microbes were able to remove up to 75% of the inlet CT. At excessively high CT concentrations (> 500 ppmv), methane production and hydrogen utilization by the bed medium microbes appeared to be inhibited. CT removal by the biofilter decreased when the hydrogen supply was removed from the biofilter inlet, indicating that hydrogen acted as the electron donor for reductive dechlorination. The removal efficiency and relatively low empty bed residence times demonstrated by these laboratory-scale biofilters indicate that anaerobic biofiltration of CT may be a feasible full-scale process.

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites. Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.

The effect of uranium on bacterial viability and cell surface morphology using atomic force microscopy in the presence of bicarbonate ions.

Past disposal practices at nuclear production facilities have led to the release of liquid waste into the environment creating multiple radionuclide plumes. Microorganisms are known for the ability to interact with radionuclides and impact their mobility in soils and sediments. Gram-positive Arthrobacter sp. are one of the most common bacterial groups in soils and are found in large numbers in subsurface environments contaminated with radionuclides. This study experimentally analyzed changes on the bacteria surface at the nanoscale level after uranium exposure and evaluated the effect of aqueous bicarbonate ions on U(VI) toxicity of a low uranium-tolerant Arthrobacter oxydans strain G968 by investigating changes in adhesion forces and cell dimensions via atomic force microscopy (AFM). Experiments were extended to assess cell viability by the Live/Dead BacLight Bacterial Viability Kit (Molecular Probes) and quantitatively illustrate the effect of uranium exposure in the presence of varying concentrations of bicarbonate ions. AFM and viability studies showed that samples containing bicarbonate were able to withstand uranium toxicity and remained viable. Samples containing no bicarbonate exhibited deformed surfaces and a low height profile, which, in conjunction with viability studies, indicated that the cells were not viable.

Iodate Reduction by Shewanella oneidensis Does Not Involve Nitrate Reductase

ABSTRACT Microbial iodate (IO3−) reduction is a major component of iodine biogeochemical cycling and is the basis of alternative strategies for remediation of iodine-contaminated environments. The molecular mechanism of microbial IO3− reduction, however, is not well understood. In several microorganisms displaying IO3− and nitrate (NO3−) reduction activities, NO3− reductase is postulated to reduce IO3− as alternate electron acceptor. In the present study, whole genome analyses of 25 NO3−-reducing Shewanella strains identified various combinations of genes encoding one assimilatory (cytoplasmic Nas) and three dissimilatory (membrane-associated Nar and periplasmic Napα and Napβ) NO3− reductases. Shewanella oneidensis was the only Shewanella strain whose genome encoded a single NO3− reductase (Napβ). Terminal electron acceptor competition experiments in S. oneidensis batch cultures amended with both NO3− and IO3− demonstrated that neither NO3− nor IO3− reduction activities were competitively inhibited by the presence of the competing electron acceptor. The lack of involvement of S. oneidensis Napβ in IO3− reduction was confirmed via phenotypic analysis of an in-frame gene deletion mutant lacking napβA (encoding the NO3−-reducing NapβA catalytic subunit). S. oneidensis ΔnapβA was unable to reduce NO3−, yet reduced IO3− at rates higher than the wild-type strain. Thus, NapβA is required for dissimilatory NO3− reduction by S. oneidensis, while neither the assimilatory (Nas) nor dissimilatory (Napα, Napβ, and Nar) NO3− reductases are required for IO3− reduction. These findings provide the first genetic evidence that IO3− reduction by S. oneidensis does not involve nitrate reductase and indicate that S. oneidensis reduces IO3− via an as yet undiscovered enzymatic mechanism.

Conceptual Model of Iodine Behavior in the Subsurface at the Hanford Site

The fate and transport of 129I in the environment and potential remediation technologies are currently being studied as part of environmental remediation activities at the Hanford Site. A conceptual model describing the nature and extent of subsurface contamination, factors that control plume behavior, and factors relevant to potential remediation processes is needed to support environmental remedy decisions. Because 129I is an uncommon contaminant, relevant remediation experience and scientific literature are limited. Thus, the conceptual model also needs to both describe known contaminant and biogeochemical process information and to identify aspects about which additional information needed to effectively support remedy decisions. this document summarizes the conceptual model of iodine behavior relevant to iodine in the subsurface environment at the Hanford site.

Assessment of Hexavalent Chromium Natural Attenuation for the Hanford Site 100 Area

Hexavalent chromium (Cr(VI)) plumes are present in the 100 Area at the Hanford Site. Remediation efforts are under way with objectives of restoring the groundwater to meet the drinking-water standard (48 µg/L) and protecting the Columbia River by ensuring that discharge of groundwater to the river is below the surface-water quality standard (10 µg/L). Current remedies include application of Pump-and-Treat (P&T) at the 100-D, 100-H, and 100-K Areas and Monitored Natural Attenuation (MNA) at the 100-F/IU Area. Remedy selection is still under way at the other 100 Areas. Additional information about the natural attenuation processes for Cr(VI) is important in all of these cases. In this study, laboratory experiments were conducted to demonstrate and quantify natural attenuation mechanisms using 100 Area sediments and groundwater conditions.

Mobility of Source Zone Heavy Metals and Radionuclides: The Mixed Roles of Fermentative Activity on Fate and Transport of U and Cr

Predicting the potential migration of metals and radionuclides from waste pits and trenches will require understanding the effects of carbon and electron flow through these environments. Important aspects of this flow include the physiological activity of cellulolytic and non-cellulolytic fermentative microbial populations, as well as the subsequent activity of metal and radionuclide reducing bacteria. The activity of subsurface fermentative microbial populations is significantly understudied even though these organisms can affect contaminant migration by at least two mechanisms. In the first mechanism, products of the fermentation process can act as chelators for metals and radionuclides increasing their transport through underlying geological media. The second mechanism is the reduction and immobilization of metals and radionuclides since some fermentative bacteria have been shown to directly reduce metals and radionuclides, while their fermentation products can provide carbon and energy for respiratory metal reducing bacteria that can also reduce oxidized metals and radionuclides.