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Create knockout mutants in genes known to be highly expressed at low temperatures in Listeria monocytogenes. Determine their effect on growth and other genes expressed at low temperatures. Study the effect of inhibitors of fatty acid biosynthesis and membrane active compounds on growth of L. monocytogenes, particularly at low temperatures.
Allele-inactivated (knockout) mutants will be created by first cloning selected full-length target genes by PCR using L. monocytogenes genomic DNA as a template. Genes will be disrupted using a DNA fragment encoding a kanamycin resistance determinant, and used to transform competent cells. Mutants will be selected due to allelic exchange between the kanamycin interrupted genes and their chromosomal equivalents. The minimum growth inhibitory concentrations of various inhibitors of fatty acid biosynthesis and membrane active compounds will be determined on solid medium at different temperatures.
Listeria monocytogenes is a foodborne bacterium that causes listeriosis, a disease with a high incidence of mortality. Contamination of food with this bacterium leads to costly product recalls. Listeria has an unusual ability to grow at refrigeration temperatures. The objectives of this project are to study the role of various genes known to be highly expressed at low temperatures in the growth of the organism at refrigeration temperatures. This will be accomplished by creating knockout mutants of the genes in question. In addition, the effect of potential control agents, such as inhibitors of fatty acid biosynthesis and membrane-active compounds, on growth, particularly at low temperatures, will be determined.