Laura A. Vanderberg

In vivo screening of haloalkane dehalogenase mutants

Haloalkane dehalogenase (Dh1A) from Xanthobacter autotrophicus GJ10 catalyzes the dehalogenation of short chain primary alkyl halides. Due to the high Km and low turnover, wild type Dh1A is not optimal for applications in bioremediation. We have developed an in vivo screen, based on a colorimetric pH indicator, to identify Dh1A mutant with improved catalytic activity. After screening 50,000 colonies, we identified a Dh1A mutant with a lower pH optimum. Sequence analysis of the mutant revealed a single substitution, alanine 149 to threonine, which is located close to the active site of Dh1A. Replacement of alanine 149 via site-directed mutagenesis with threonine, serine or cysteine retained the mutant phenotype. Other substitutions at position 149 show little or no activity.

Actinide (Pu, U) interactions with aerobic soil microbes and their exudates: Fundamental chemistry and effects on environmental behavior

To understand the environmental behavior of metals we must consider a tremendous range of phenomena, from simple individual reactions, such as ligand complexation and solubility equilibria, to quite complicated and collective processes, such as metal-mineral-microbial interactions. Because of pressing contamination problems at DOE sites and the paucity of relevant actinide chemistry knowledge, research is needed in this entire range of science. The determination and evaluation of key thermodynamic data for actinide species and the development of geochemical, hydrological, and environmental transport models are progressing. In contrast, we know almost nothing about how actinides interact with microorganisms. Ubiquitous microorganisms can absorb, reduce, oxidize, solubilize, or precipitate actinides, thereby affecting their speciation, solubility, bioavailability, and migration. These effects are due to both direct and indirect interactions, such as sorption to the cell wall and reaction with microbial bypr...

Environmental Actinide Mobility: Plutonium and Uranium Interactions with Exopolysaccharides and Siderophores of Aerobic Soil Microbes

Research efforts into understanding the effect of siderophores and capsules on actinide speciation and resultant environmental behavior are ongoing. Investigations on both siderophores have progressed. The redox chemistry of Pu(VI) and Pu(V) with DFO under acidic conditions was examined by UV-Vis and NMR spectroscopies. Pu(VI) was instantly reduced to Pu(V) by DFO and DFO cleavage resulted. Neither DFO nor the cleavage product appeared to coordinate with Pu(V) in the pH range examined. Growth of S. pilosus for cellular translocation experiments was optimized and experiments to determine DFO-mediated uranium uptake by S. pilosus are underway. OFS siderophore(s) production has been optimized and product identification is underway. Siderophore-like molecules were positive for the Arnow assay and had UV absorbance bands at 250nm and 315nm, suggesting catecholates-type molecules. Siderophores were produced with C13 - C16 as carbon source in AM-1 salts medium with 1.0mM iron. Siderophore accumulation was slowest on C13 due to the slower growth of OFS on this substrate. The C16 concentration in the culture influenced siderophore production, with highest levels at 1.0% and 2.0%.