LExEn: Mercury Resistance in Archaea

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This project is investigating the relationship between mercury and archaea. Mercury is an extremely toxic heavy metal mined from cinnabar deposits but is otherwise rare. Most bacterial prokaryotes employ the mer genes to achieve mercury resistance. The mer genes are highly conserved, widely distributed, and frequently transmitted among bacteria. Surprisingly, there are no reports of mercury resistance among archaeal prokaryotes. To investigate this question studies have been undertaken on a naturally occurring mercury-rich geothermal environment distinguished by sulfhydryl deficiency and oxidative excess. Mercury resistant archaea were cultured from Pool 3-4, a cinnabar hot springs (pH 1.7, 78 degrees C, 2 mg/liter mercury) located in the Mojave Desert. Pool 3-4 is strongly influenced by seasonal rainfall with concomitant changes in pool solute levels, particularly mercury. Efforts in this project are focused on one isolate identified as Sulfolobus solfataricus. Maintenance of archaeal mercury resistance in this isolate requires selective pressure. Co-segregation of mercury resistance and a 13 kb endogenous plasmid indicates that resistance is plasmid encoded. A mercury adaptive response was identified which is accompanied by transient induction of a 25 kDa protein. The first part of this LExEn project focuses on archaeal mercury resistance and adaptation. Enzyme assays for mercury reduction and volatilization will test for presence of a bacterial mer-type mechanism. Characterization of the endogenous plasmid will enable identification of possible mer orthologous sequences. Regions critical for mercury resistance will be identified using plasmid deletion derivatives for genetic transfer studies with mercury sensitive recipients. Identification of the mercury induced 25 kDa protein will help clarify the mechanism of toxicity. The relationship between mercury resistance and adaptation will focus on expression patterns of the 25 kDa protein. The second part of the project focuses on the origin and potential transfer of mercury resistance genes. Phylogenetic analysis of cultured and uncultured microbes from Pool 3-4 will be completed and used to create specific fluor-labled probes for community structure analysis. Measurements of community composition will establish the consequences of seasonal variation in mercury levels. Archaeal mercury resistance gene probes will be used to track distribution and transfer of mercury resistance among members of the Pool 3-4 community coinciding with peak mercury concentrations. The anticipated results should distinguish possible origins of archaeal mercury resistance and establish an understanding of the mechanism of mercury toxicity in archaea. This information will extend knowledge about life in extremely oxidizing environments and contribute to the genetic manipulation of archaeal hyperthermophiles.