Grant: Discovery Grants Program - Individual

Hypothesis. We hypothesize that members of bacterial genus, Lactobacillus, have developed the ability to acquire specific genetic elements in order to sequester heavy metals to which they are exposed in the environment. Background and Objectives.Lactobacilli are Gram positive facultative anaerobic or microaerophilic rod-shaped bacteria notable for their use in the production of a range of foods, including yogurt, cheese, sauerkraut and pickles as well as silage for animal feed. Of the 180 or so species of Lactobacillus, the L. casei and L. rhamnosus species are of particular interest as they are ubiquitous in fermented foods and a number of strains have proved effective as probiotics. We are intrigued by their adaptability to grow in various foods and survive in conditions within the body and we wondered if they cope with the presence of heavy metals known to contaminate the food chain and the intestine. We have discovered that sequestration and detoxification of mercury does indeed occur in lactobacilli, and appears to be associated with a characterized three-gene reverse trans-sulfuration pathway involved in cysteine metabolism in L. casei DN114-226, We also discovered that resistance to mercury can be induced in L. rhamnosus DN 116-060 after incubation in sub-inhibitory mercury concentrations; the lactobacilli have both a decreased lag growth period and higher minimum inhibitory concentrations when grown in Hg2+. The screening uncovered only this strain, and one other related L. rhamnosus strain possessing a rare horizontally acquired element encoding a minimal mercury resistance operon imparting inducible resistance to mercury by volatilizing the metal. Research aim and objectives The aim of our program is to understand how Lactobacillus acts as a heavy metal detoxifying agent. The five year program designed for two graduate students each year has three objectives:1. Verify the genetic elements that play a role in active and passive mercury resistance and detoxication. 2. Determine how exposure to mercury affects L. rhamnosus DN116-060 functionality 3. Test the concept in a murine model. Using Lactobacillus rhamnosus DN116-060, L. casei DN114-226, and probiotic L. rhamnosus GR-1 as a control and gene recipient, all due to their ability to propagate in food and characteristics of interest, we will use knock-outs and gene insertion to optimize the ability of lactobacilli to bind to mercury. We will perform studies that provide insight into the mechanisms of these actions, including altering genetics and surface properties of lactobacilli to modify the active passive sequestration of mercury, respectively, and doing transcriptomic analysis to understand functionality. A cell culture and mouse model will be used for in vivo experiments. Significance and Future Studies: Heavy metal poisoning is common in certain parts of the world, and here in Canada exposure levels vary and remain a cause for concern. If beneficial bacteria can be utilized to reduce adsorption of mercury through foods, this could have an impact on morbidity and mortality.