The Role of Mg2+ Transport in Bacterial Heat Tolerance

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Heat tolerance of Salmonella enterica, a pathogen important to food safety, is dramatically enhanced by mutations that result in high-level synthesis of two proteins that transport magnesium ions across bacterial cell membranes. By determining which cellular components are protected at high temperature as a consequence of these single DNA nucleotide mutations, this project will provide a unique opportunity to identify the heat sensitive target(s) that are responsible for loss of viability at high temperature. Results obtained in this study will provide insights into the causes of thermosensitivity not only in Salmonella but also in other bacteria. Success in the construction of thermotolerant varieties of bacteria would provide a simple approach to the engineering of improved strains that can be used for the synthesis of biofuels and other chemicals whose production or isolation would be enhanced at higher temperatures. Also, because high-temperature treatment is the most widely used and cost-effective means for inactivating food pathogens, analysis of the regulation of thermotolerance could provide insights into the development of more efficient ways to ensure microbiological food safety. Funding by the NSF will also enable the PI to continue the training of scientists at all levels from high school through postdoctoral in the integration of genetics, molecular biology, and genomics to address important problems in bacterial physiology.The goals of the proposed research are to test whether the overproduction of two Mg2+ transporters (MgtA and MgtB) will confer increased thermotolerance on other bacteria besides Salmonella and to elucidate the mechanism by which increased synthesis of MgtA and MgtB confers enhanced thermotolerance. The project has the following Specific Aims: 1) Test the hypothesis that overproduction of Mg2+ transporters provides a general method for the engineering of thermotolerant derivatives of bacteria by carrying out such constructions in 3 Gram negative species, a halophile, and in a Gram positive bacterium. 2) Test the hypothesis that the chr (constitutively heat resistant) mutations impart increased thermotolerance by protecting the stability of cellular proteins at high temperature. These experiments will also probe whether denaturation of proteins could be an important cause of cell death at high temperature. 3) Test the hypothesis that the chr mutations result in enhanced heat resistance by protecting membranes at high temperature. These experiments will determine whether the chr mutations decrease membrane leakage at high temperature or stabilize membranes by altering lipid composition. 4) Test whether the chr mutations confer enhanced heat tolerance by inducing thermoprotective genes. RNAseq will be used to probe the effect of the chr mutations on global transcriptomes. Results of these studies will be published in peer-reviewed journals and presented at regional and national scientific conferences.