Cloning of a mer operon from Naturally Occurring Aquatic Bacteria

Methylmercury is a toxic substance which accumulates in fish and other biota and causes toxicological problems when consumed by humans. Certain mercury resistant bacteria are able to convert the highly toxic methylmercury to a much less toxic form. Several mer operons, a group of genes which encode this function, have been cloned and characterized. However, they have been isolated and cloned from clinical isolates, and these may not be adequate models for studying mercury resistance in nature. The purpose of this project is to isolate and clone the mer operon from a natural isolate to be used as a tool for studying mercury detoxification in aquatic habitats. DNA from various natural isolates was digested with restriction endonucleases to identify the mer operon on a smaller fragment of DNA. These restriction fragments will be probed with a DNA fragment which is complementary to the mer operon. After locating the operon on a restriction fragment, it will be cloned into a cloning vector and characterized by restriction mapping. This project will be beneficial because it provides a model for mercury resistance in natural isolates that can be used for studies on mercury detoxification in an environmental setting. To date, the genomic DNA from strains L016, MB2, and PA07 has been isolated, digested with six different restriction enzymes (Fig. 2), and run on an agarose gel. These gels have been blotted onto a nylon membrane with the Southern blot technique and are ready for hybridization. The fragment that will be used for the hybridization probe has been identified from the EcoR I/Ava II digest. Introduction Mercury is a toxic heavy metal that has caused numerous cases of human toxicity throughout history. It is found in several forms, both organic and inorganic, in the environment. The elemental state (Hg°) is volatile, causing few problems with toxicity. An ionic form of Hg, Hg[II 2], is found in aquatic environments and can be methylated to the methylmercury (CH3 Hg) by bacteria. Methylmercury is highly toxic and even lethal to humans. Humans are currently affected most by it after consumption of fish that have high levels of accumulated methylmercury. There are certain bacteria (mercury resistant) that have the ability to demethylate this toxic form into the much less toxic Hg[II]. Hg[II] is then enzymatically reduced to Hg° (Nazaret et al, 1994). This resistance mechanism has been studied in an attempt to understand the potential mechanisms of environmental mercury detoxification. There are two forms of bacterial mercury: broad and narrow spectrum. Broad spectrum resistance is the ability to detoxify both inorganic and organic mercury CLONING OF A MER OPERON compounds. Narrow spectrum resistance refers to the detoxification of only inorganic mercury (Gilbert and Summers, 1988). This study focuses on broad spectrum resistance because this can detoxify methylmercury. The genes that encode for mercury resistance (mer operon) encode several proteins with various functions (Fig 1). The merT and merP gene products transport mercury in the cell. The merB gene product encodes organomercurial lyase which cleaves the carbon-mercury bond in organomercurials producing Hg[II]. The merA gene product (mercuric reductase) then reduces Hg[II] to the volatile Hg° which is released from the cell. The merR gene encodes a regulatory protein that represses the operon in the absence of mercury (Brown et al, 1986). This purpose of this project is to isolate and clone the mer operon from a natural isolate to be used as a tool for studying mercury detoxification in aquatic habitats. This will be a much more appropriate model for these studies than the current mer operon that was isolated from clinical isolates.