Superfund Research Program

Molecular Mechanisms of Bacterial Metal Redox Transformations

Project Leader: Bradley M. Tebo (Oregon Health and Science University)
Grant Number: P42ES010337
Funding Period: 2000-2010

Project-Specific Links

Final Progress Reports

Year:   2009  2004 

This research addresses microbial processes that show promise for the bioremediation of heavy metal contamination; specifically researchers study the molecular mechanisms by which bacteria form Mn oxides and sequester heavy metals or reduce toxic hexavalent chromium (Cr(VI)) to insoluble Cr(III) hydroxides.

Previously, researchers reported the isolation of a calcium-binding heme peroxidase as the Mn(II) oxidase enzyme from the bacteria Aurantimonas manganoxydans SI85-9A1 and Erythrobacter sp. SD21. Researchers named this protein MopA and have continued to characterize its activity. Mn(II) oxidation in outer membrane wash cell fractions was stimulated by the addition of peroxide and calcium and proceeds through an Mn(III) intermediate. To study the role of MopA in this reaction, researchers have begun efforts to purify recombinant, intein/chitin-binding domain (CBD)-tagged MopA from E. coli. Early results indicated that the purified MopA catalyzes the reaction of Mn(II) to Mn(III), leaving open the possibility of a second enzyme in the outer membrane wash responsible for the reaction of Mn(III) to Mn(IV). However, even though the CBD tag is removed from the recombinant protein by intein-mediated cleavage after purification, the tag may still interfere with protein folding, resulting in altered activity in the purified protein. Therefore, researchers are also investigating the feasibility of purifying over-expressed untagged MopA from E. coli.

Researchers have undertaken experiments to identify the enzymes active in Mn(II) oxidation in the environment using the Columbia river estuary and plume as a model system. Researchers collected large volume water samples, concentrated them using tangential flow filtration and assayed for Mn(II) oxidation activity. Mn(II) oxidation assays revealed activity only in the protein fractions associated with the outer membrane, consistent with localization studies done with the model Mn oxidizers A. manganoxydans SI85-9A1 and Erythrobacter SD21. The proteins in these fractions were separated using size exclusion chromatography and the active fractions were concentrated and analyzed using tandem mass spectrometry (MS/MS). Among the proteins identified in the active fractions were a putative hemolysin-type Ca-binding heme peroxidase and a putative MCO suggesting that both types of enzymes could be involved in Mn(II) oxidation in the environment. The presence of the mopA gene in the estuarine samples was also detected via quantitative PCR. Although researchers don’t know what proportion of these mopA homologues encode a Mn(II) oxidase, these results are consistent with a heme-peroxidase mechanism of Mn(II) oxidation in the estuary. These results also suggest that expression of mopA or the MopA protein itself may be a good biomarker for environmental Mn oxidation and consequent metal sequestration.

Previous work identified the Mn(II) oxidase in Bacillus sp. spores as the multicopper oxidase MnxG. Similar to the researcher’s experiments with MopA, work is underway to purify recombinant MnxG using the intein/CBD system. In the case of Bacillus sp. MB-7, the small protein MnxF copurified with MnxG during isolation of the Mn(II) oxidase activity. Therefore, researchers are also generating plasmid constructs that will allow us to over-express and purify MnxF along with MnxG. The purified recombinant proteins will be used to study the mechanism of Mn(II) oxidation at the molecular level.