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A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21(Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r 2 = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 102 CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 ± 2)% over the range of population densities investigated (102 to 108 CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.
A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21(Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r 2 = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 102 CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 ± 2)% over the range of population densities investigated (102 to 108 CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.
Perennial wildfires in Africa and other continents contribute an estimated 8 x 10 5 kg of mercury to the global atmosphere with a residence time of approximately one year. This phenomenon changes the flux of biologically available mercury in natural microbial communities where enzymatic actions, including mercuric reductase and organomercurial lyase activities, underpin the biogeochemical cycling of mercury with repercussions for human exposure to toxic forms of the element. To elucidate the impact of episodic mercury bioavailability on the response of microbial communities, the expression of microbial proteins and nucleic acids in environmental strains of Pseudomonas species were evaluated under various concentrations of mercury ranging from 0 to 500 µM. Routine cultivation of Pseudomonas aeruginosa PU21 containing the 142.5 kb plasmid Rip64 in medium containing 100 µg of Hg ++ /ml (500 µM) exhibited a prolonged lag phase survived by hyper-resistant cells able to grow in medium containing 200 µg of Hg ++ /ml. Nucleic acid analyses showed a distinct mutation in the merA gene encoding for mercuric reductase activity in cells able to grow at elevated mercury concentrations. A similar mutation was detected in the merR locus which serves as the regulator of the mer operon. Mutations were not detected in merC which encodes for a hydrophobic membrane-associated protein implicated in active mercury transport. Protein profiles of cells grown with elevated mercury concentrations were associated with a stable increase in the production of specific polypeptides. In addition, the survival and genetic response of naturally-occurring mercury resistant bacteria inoculated into contaminated environmental samples were monitored in microcosm experiments over a 30 day period. The results suggest that sudden exposure to high concentrations of mercury either decimates the bacterial population or selects for hyper-resistant strains with high level of constitutive expression of active proteins, including mercuric reductase. Methyl mercury was observed to cause a higher level of induction for mercuric reductase than the specific substrate, inorganic mercury. The selection of hyper-resistant strains is potentially useful for biotechnological strategies to control the bioavailability of mercury, and thereby potentially reducing the re-uptake of mercury into vegetation in regions frequently subjected to wildfires.
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