Genetic engineering of plants with the bacterial genes mera and merb for the phytoremediation of methylmercury contaminated sediments

Abstract

Methylmercury (CH3Hg + ) is an environmental toxin that biomagnifies in the aquatic food web with severe consequences for humans and wildlife. To reduce the incidence of CH3Hg + , our lab is engineering plants to be used for in situ processing (phytoremediation) of mercury-contaminated sediments. Our ongoing stategy has been to provide plants with the bacterial genes for organomercurial lyase (merB) and mercuric reductase (merA), two enzymes that operate sequentially to convert CH3Hg + to a less hazardous form of mercury, Hg(0). Elemental mercury is volatile and, therefore, can be transpired through plant stomata and diluted in the atmosphere. We used the model system, Arabidopsis thaliana, to test our proposed strategy. Plants containing merA and merB were shown to grow vigorously on levels of methylmercury and phenylmercuric acetate (an alternative substrate for MerB) that are lethal to wild-type plants. An in vivo biochemical assay was used to demonstrate that merA/merB plants absorb organic mercury from a liquid medium and release Hg(0). We also showed that the rate of organic mercury degradation is related to the steady-state concentration of MerB and that this enzyme localizes to the cytoplasm. However, variations in combined MerA and MerB enzyme expression explain only ~40% of the variation in the rate of organic mercury degradation. This suggested that the diffusion of the organic mercury substrate to MerB is inefficient and that organic mercury may be accumulating at the cell wall or in membrane-rich compartments away from the enzyme. A final research objective has been to determine whether the efficiency of organic mercury detoxification can be improved by altering the distribution of MerB. Specifically, we have created new gene constructs containing signal sequences to target MerB to the cell wall and the endoplasmic reticulum. After transforming plants, we analyzed the T2 generation for organic mercury resistance, enzyme expression levels, and rates of organic mercury conversion. Plants in which MerB has been targeted to the secretory pathway appear to be much more efficient at degrading organic mercury than plants that express MerB cytoplasmically. With approximately 10-100X less protein, they achieve resistance levels and rates of degradation comparable to plants with the wild-type merB sequence.