Identification of abiotic and biotic reductive dechlorination in a chlorinated ethene plume after thermal source remediation by means of isotopic and molecular biology tools.

Thermal tetrachloroethene (PCE) remediation by steam injection in a sandy aquifer led to the release of dissolved organic carbon (DOC) from aquifer sediments resulting in more reduced redox conditions, accelerated PCE biodegradation, and changes in microbial populations. These changes were documented by comparing data collected prior to the remediation event and eight years later. Based on the premise that dual C-Cl isotope slopes reflect ongoing degradation pathways, the slopes associated with PCE and TCE suggest the predominance of biotic reductive dechlorination near the source area. PCE was the predominant chlorinated ethene near the source area prior to thermal treatment. After thermal treatment, cDCE became predominant. The biotic contribution to these changes was supported by the presence of Dehalococcoides sp. DNA (Dhc) and Dhc targeted rRNA close to the source area. In contrast, dual C-Cl isotope analysis together with the almost absent VC (13)C depletion in comparison to cDCE (13)C depletion suggested that cDCE was subject to abiotic degradation due to the presence of pyrite, possible surface-bound iron (II) or reduced iron sulphides in the downgradient part of the plume. This interpretation is supported by the relative lack of Dhc in the downgradient part of the plume. The results of this study show that thermal remediation can enhance the biodegradation of chlorinated ethenes, and that this effect can be traced to the mobilisation of DOC due to steam injection. This, in turn, results in more reduced redox conditions which favor active reductive dechlorination and/or may lead to a series of redox reactions which may consecutively trigger biotically induced abiotic degradation. Finally, this study illustrates the valuable complementary application of compound-specific isotopic analysis combined with molecular biology tools to evaluate which biogeochemical processes are taking place in an aquifer contaminated with chlorinated ethenes.

The impact of bioaugmentation on dechlorination kinetics and on microbial dechlorinating communities in subsurface clay till.

A molecular study on how the abundance of the dechlorinating culture KB-1 affects dechlorination rates in clay till is presented. DNA extracts showed changes in abundance of specific dechlorinators as well as their functional genes. Independently of the KB-1 added, the microbial dechlorinator abundance increased to the same level in all treatments. In the non-bioaugmented microcosms the reductive dehalogenase gene bvcA increased in abundance, but when KB-1 was added the related vcrA gene increased while bvcA genes did not increase. Modeling showed higher vinyl-chloride dechlorination rates and shorter time for complete dechlorination to ethene with higher initial concentration of KB-1 culture, while cis-dichloroethene dechlorination rates were not affected by KB-1 concentrations. This study provides high resolution abundance profiles of Dehalococcoides spp. (DHC) and functional genes, highlights the ecological behavior of KB-1 in clay till, and reinforces the importance of using multiple functional genes as biomarkers for reductive dechlorination.

Field evaluation of biological enhanced reductive dechlorination of chloroethenes in clayey till.

The performance of enhanced reductive dechlorination (ERD) for in situ remediation of cis-1,2-dichloroethene (cDCE) and vinyl chloride in clayey till was investigated in a pilot test. A dilute groundwater solution containing emulsified soybean oil and Dehalococcoides bacteria was injected into a sand-filled hydraulic fracture. Fermentation of the ERD solution caused the establishment of a dechlorinating bioactive zone in the fracture within 1 month of injection. By 148 days, all the cDCE in the fracture was dechlorinated to ethene. Analysis of a clay core from Day 150 indicated that electron donor and fermentation products diffused from the fracture at least 10 cm into clay and that stimulated dechlorination occurred in the clay in the presence of Dehalococcoides (7.9.10(4) cells g(-1)). Comparison of chloroethene profiles in the Day 150 core to modeled diffusion profiles indicated degradation occurred in a bioactive zone extending approximately 5 to 6 cm into the clay matrix. These data suggest that a bioactive zone established in a sand-filled fracture can expand into the adjacent clayey till matrix and facilitate mass transfer from the matrix to the bioactive zone. These findings offer promise for ERD and support further development of methods for deploying ERD in clayey till and other low-permeability deposits.

Evidence of ultraviolet type mutations in xeroderma pigmentosum melanomas.

To look for a direct role of ultraviolet radiation (UV) exposure in cutaneous melanoma induction, we studied xeroderma pigmentosum (XP) patients who have defective DNA repair resulting in a 1000-fold increase in melanoma risk. These XP melanomas have the same anatomic distribution as melanomas in the general population. We analyzed laser capture microdissection samples of skin melanomas from XP patients studied at the National Institutes of Health. The tumor suppressor gene PTEN was sequenced and analyzed for UV-induced mutations. Samples from 59 melanomas (47 melanomas in situ and 12 invasive melanomas) from 8 XP patients showed mutations in the PTEN tumor suppressor gene in 56% of the melanomas. Further, 91% of the melanomas with mutations had 1 to 4 UV type base substitution mutations (occurring at adjacent pyrimidines) (P < 0.0001 compared to random mutations). We found a high frequency of amino-acid-altering mutations in the melanomas and demonstrated that these mutations impaired PTEN function; UV damage plays a direct role in induction of mutations and in inactivation of the PTEN gene in XP melanomas including in situ, the earliest stage of melanoma. This gene is known to be a key regulator of carcinogenesis and therefore these data provide solid mechanistic support for UV protection for prevention of melanoma.

Concurrent ethene generation and growth of Dehalococcoides containing vinyl chloride reductive dehalogenase genes during an enhanced reductive dechlorination field demonstration.

Dehalococcoides bacteria that produce catabolic vinyl chloride (VC) reductive dehalogenase enzymes have been implicated as a requirement for successful biological dechlorination of VC to ethene in groundwater systems. Therefore, the functional genes in Dehalococcoides that produce VC reductase (e.g., vcrA) may be important biomarkers for predicting and monitoring the performance of bioremediation systems treating chloroethenes via enhanced reductive dechlorination (ERD). As part of an ERD field demonstration, 45 groundwater samples were analyzed for vcrA using quantitative PCR. The demonstration delivered lactate continuously via groundwater recirculation over 201 days to an aquifer contaminated with cis-1,2-dichloroethene (cDCE, approximately 150 microM) and VC (approximately 80 microM). Ethene (approximately 4 microM) and Dehalococcoides containing vcrA (average concentration of 4 x 10(3) gene copies L(-1)) were detected a priori in the demonstration plot; however, aquifer materials in a bench treatability test were able to dechlorinate cDCE with only a 4-month lag period. Given the short (7-month) schedule for the field demonstration, the field plot was bioaugmented on Day 69 with a mixed culture (KB-1) that included Dehalococcoides containing vcrA. Stimulated ethene generation commenced within four weeks of donor addition. Ethene concentrations increased until Day 145, and reached maximum concentrations of 10-25 microM. Concentrations of vcrA increased concurrently with ethene production until Day 145, and plateaued thereafter at 10(7) to 10(8) gene copies L(-1). These results indicate simultaneous growth of Dehalococcoides containing vcrA and ethene generation in an ERD field application. The quantitative increase in concentrations of Dehalococcoides containing vcrA at this site provides further evidence that the vcrA gene is an effective biomarker for field-scale ERD systems.

Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the effect of therapeutic kinase inhibitors on cell survival.

The abilities of mutated active RAS proteins to modulate cell survival following exposure to ionizing radiation and small molecule kinase inhibitors were examined. Homologous recombination in HCT116 cells to delete the single allele of K-RAS D13 resulted in a cell line that exhibited an approximately 75% reduction in basal extracellular signal-regulated kinase 1/2, AKT, and c-jun-NH2-kinase 1/2 activity. Transfection of cells lacking K-RAS D13 with H-RAS V12 restored extracellular signal-regulated kinase 1/2 and AKT activity to basal levels but did not restore c-jun-NH2-kinase 1/2 phosphorylation. In cells expressing H-RAS V12, radiation caused prolonged intense activation of AKT. Inhibition of H-RAS V12 function, blockade of phosphatidylinositol 3-kinase (PI3K) function using small interfering RNA/small-molecule inhibitors, or expression of dominant-negative AKT abolished radiation-induced AKT activation, and radiosensitized these cells. Inhibition of PI3K function did not significantly radiosensitize parental HCT116 cells. Inhibitors of the AKT PH domain including perifosine, SH-(5, 23-25) and ml-(14-16) reduced the plating efficiency of H-RAS V12 cells in a dose-dependent fashion. Inhibition of AKT function using perifosine enhanced radiosensitivity in H-RAS V12 cells, whereas the SH and ml series of AKT PH domain inhibitors failed to promote radiation toxicity. In HCT116 H-RAS V12 cells, PI3K, PDK-1, and AKT were membrane associated, whereas in parental cells expressing K-RAS D13, only PDK-1 was membrane bound. In H-RAS V12 cells, membrane associated PDK-1 was phosphorylated at Y373/376, which was abolished by the Src family kinase inhibitor PP2. Inhibition of PDK-1 function using the PH domain inhibitor OSU-03012 or using PP2 reduced the plating efficiency of H-RAS V12 cells and profoundly increased radiosensitivity. OSU-03012 and PP2 did not radiosensitize and had modest inhibitory effects on plating efficiency in parental cells. A small interfering RNA generated against PDK1 also radiosensitized HCT116 cells expressing H-RAS V12. Collectively, our data argue that molecular inhibition of AKT and PDK-1 signaling enhances the radiosensitivity of HCT116 cells expressing H-RAS V12 but not K-RAS D13. Small-molecule inhibitory agents that blocked stimulated and/or basal PDK-1 and AKT function profoundly reduced HCT116 cell survival but had variable effects at enhancing tumor cell radiosensitivity.

Soluble human core 2 beta6-N-acetylglucosaminyltransferase C2GnT1 requires its conserved cysteine residues for full activity.

Human UDP-GlcNAc: Galbeta1-3GalNAc- (GlcNAc to GalNAc) beta1,6-GlcNAc-transferase (C2GnT1) is a member of a group of beta6-GlcNAc-transferases that belongs to CAZy family 14. One of the striking features of these beta6-GlcNAc-transferases is the occurrence of nine completely conserved cysteine residues that are located throughout the catalytic domain. We have expressed the soluble catalytic domain of human C2GnT1 in insect cells, and isolated active enzyme as a secreted protein. beta-Mercaptoethanol (beta-ME) and dithiothreitol (DTT) were found to stimulate the enzyme activity up to 20-fold, indicating a requirement for a reduced sulfhydryl for activity. When the enzyme was subjected to nonreducing PAGE, the migration of the protein was identical to the migration in reducing gels, demonstrating the absence of intermolecular disulfide bonds. This suggested that the monomer is the active form of the enzyme. Sulfhydryl reagents such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) inactivated the enzyme, and the inactivation was partially prevented by prior addition of donor or acceptor substrate and by sulfhydryl reducing agents. We therefore investigated the role of all nine conserved cysteine residues in enzyme stability and activity by site-directed mutagenesis where individual cysteine residues were changed to serine. All of the mutants were expressed as soluble proteins. Seven of the Cys mutants were found to be inactive, while C100S and C217S mutants had 10% and 41% activity, respectively, when compared to the wild-type enzyme. Wild-type and C217S enzymes had similar K(M) and V(max) values for acceptor substrate Galbeta1-3GalNAcalpha-p-nitrophenyl (GGApnp), but the K(M) value for UDP-GlcNAc was higher for C217S than for the wild-type enzyme. In contrast to wild-type enzyme, C217S was not stimulated by reducing agents and was not inhibited by sulfhydryl specific reagents. These results suggest that Cys-217 is a free sulfhydryl in active wild-type enzyme and that Cys-217, although not required for activity, is in or near the active site of the protein. Since seven of the mutations were totally inactive, it is likely that these seven Cys residues play a role in maintaining an active conformation of soluble C2GnT1 by forming disulfide bonds. These bonds are only broken at high concentrations of disulfide reducing agents.

Phylogenetic analysis of bacterial populations in an anaerobic microbial consortium capable of degrading saturation concentrations of tetrachloroethylene.

An anaerobic microbial consortium able to biodegrade saturation levels of perchloroethylene (PCE) in a column containing a source zone of PCE was examined phylogenetically to determine microbial community structure and spatial variation in relation to the PCE source. The consortium was comprised of at least 34 members with 7 organisms sharing affiliations with known respiratory or cometabolic dechlorinators. Seven other organisms had their closest phylogenetic relative detected in other environments containing chlorinated compounds. Based on denaturing gradient gel electrophoresis, significant Bacteria were Dehalococcoides ethenogenes, Shewanella putrefaciens, and an Acetobacterium species. Spatial variations in community structure of the consortium relative to the PCE source zone were observed. A Pseudomonas species was predominant in a zone 30 cm from the PCE source. A Methanothrix species was predominant at points over 85 cm from the source zone. A Trichlorobacter species was detected where PCE concentrations were highest, up to 85 cm from the PCE source, whereas D. ethenogenes was ubiquitous to over 128 cm from the PCE source.

Monitoring gene expression in mixed microbial communities by using DNA microarrays.

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10(5) cells/ml was clearly detected against a background of 10(8) cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10(6) cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.