Promoting uranium immobilization by the activities of microbial phophatases Page: 1 of 3
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Promoting uranium immobilization by the activities of microbial phophatases
Sobecky, Patricia A.
Georgia Institute of Technology
RESULTS TO DATE: Patricia Sobecky and Martial Taillefert - Progress Report
The following is a summary of progress in our project ?Promoting uranium immobilization by the activities
of microbial phosphatases? during the first funded period of the project.
I. The first objective of this project is to determine the relationship of phosphatase activity to metal
resistance in subsurface strains and the role of lateral gene transfer (LGT) in dissemination of nonspecific
acid phosphatase genes. Nonspecific acid phosphohydrolases are a broad group of secreted microbial
phosphatases that function in acidic-to-neutral pH ranges and utilize a wide range of organophosphate
substrates. We have previously shown that P043- accumulation during growth on a model
organophosphorus compound was attributable to the overproduction of alkaline phosphatase by
genetically modified subsurface pseudomonads [Powers et al. (2002) FEMS Microbiol. Ecol. 41:115-123].
During this report period, we have extended these results to include indigenous metal resistant
subsurface microorganisms cultivated from the Field Research Center (FRC), in Oak Ridge Tennessee.
To address this objective we have been conducting phenotypic screening of phosphatase activities of
metal resistant (e.g., lead resistant) FRC isolates from our existing culture collection. Phosphatase
phenotypes are tested on TPMG medium that provides a visible color change during growth. FRC
isolates exhibiting (positive) phosphatase phenotypes are subsequently selected for further molecular-
based characterizations (e.g., zymograms). We subsequently tested phosphate liberation of select FRC
isolates (lead resistant; Pbr) shown to exhibit cell surface and/or extracellular phosphatase activity. FRC
isolates assayed for phosphate liberation were obtained from the background reference site (AG38), Area
2 (AB44) and Area 3 (U26, X43, Y4, Y9 and Y29). Batch experiments to determine whether or not FRC
isolates could hydrolyze and release inorganic phosphate from organophosphorus substrates were
conducted with numerous strains including several lead resistant Bacillus sp.Y4, Y9-602 and Y29
isolates. During the 168-hr incubation, the lead resistant isolates hydrolyzed approximately 50% of the
organophosphorus compound (glycerol-3-phosphate; G3P). In contrast, a genetically modified control
strain, Pseudomonas veronii, containing a constitutively expressed alkaline phosphatase hydrolyzed 72%
of the G3P. Interestingly, strains that were determined to be lead resistant yet exhibiting phosphatase
positive phenotypes (e.g., strains AG38, AB44, U26 and X43) hydrolyzed <0.5% of the exogenous G3P.
Further studies of lead resistant and lead sensitive FRC strains will focus on determining if phosphatase
activity is mediated by alkaline phosphatases, non-specific acid phosphatases or polyphosphates.
The analysis of a subset of aerobic heterotrophic FRC isolates cultured from radionuclide and metal
contaminated FRC soils and uncontaminated (control) sediments indicated a higher percentage of
isolates exhibiting phosphatase phenotypes (i.e., in particular those surmised to be P043--irrepressible)
relative to isolates from the background reference site. A high percentage of strains which exhibited such
putatively P043--irrepressible phosphatase phenotypes were also shown to be resistant to the heavy
metals lead and cadmium. During this report period, a gram-negative FRC strain, Y9, identified by 16S
rDNA to be a Rhanella sp., has been shown, in preliminary experiments, to promote the precipitation of
uranium from solution. We are also in the process of developing and optimizing primer sets that will be
suitable for amplifying microbial acid phosphatases of the known molecular classes [i.e., classes A, B and
C]. Primers (20-mer) sets have been developed and are in the process of being tested with control strains
and FRC strains from contaminated soils. Studies to determine the tolerance of the FRC strains to
uranium have also been initiated during this report period. Cell viability assays were conducted to
determine U tolerance using concentrations ranging from 20 microM to 200 microM uranyl acetate during
pH 4.0 incubation. These conditions were designed to reflect the in situ pH and U concentrations reported
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Sobecky, Patricia A. Promoting uranium immobilization by the activities of microbial phophatases, report, June 1, 2005; Atlanta, Georgia. (digital.library.unt.edu/ark:/67531/metadc884387/m1/1/: accessed April 26, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.