Characterization and membrane-biogenesis of Streptococcus mutans magnesium transporters

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Project Summary Dental caries is a ubiquitous infectious disease that impacts the quality of life of billions of people worldwide. According to the Surgeon General (2020), ~USD 125 billion dollars are spent annually on related treatments in the USA alone. A major causative agent of dental caries is Streptococcus mutans, which is also associated with infectious endocarditis. The native environment of S. mutans is rich in metal cations including Ca2+, K+, and Mg2+. In fact, Mg2+ is the most abundant divalent metal cation in bacteria and fourth most abundant in vertebrates including humans. Of the total Mg2+ content in the human body, 60‐70% is found in bones and teeth. Therefore, the oral bacterium, S. mutans, is constantly exposed to Mg2+ salts. Magnesium is an important component of toothpastes and dental implants. Despite its abundance and its requirement to support bacterial growth and virulence , Mg2+ homeostasis has not yet been studied in S. mutans, or other oral streptococci. A few isolated studies discussed the significance of supplemental Mg2+ salts for S. mutans biofilm formation and genetic competence, but Mg2 transport is not understood. We will address Mg2+ homeostasis from a novel perspective, where we will not only characterize the transporters, but also study their insertion into the membrane. Membrane localization/insertion is a key requisite for the proper functioning of all membrane proteins. Deletion of putative transporters singly and in combination, followed by measurement of cellular metal content will establish identity of Mg2+ transporters. Compensatory uptake/efflux by other divalent metal cation transporters have been recognized to interfere with Mg2+ homeostasis in other bacteria. Therefore, Mn2+ and Fe2+ transporters are also included in this study. Mutants defective in putative Mg2+ transporter(s) will be evaluated at the level of transcription, cellular metal content, and insertion into membrane. Next, we will use a forward genetic screen to identify gain of function/suppressor mutations in Mg2+‐replete/deplete conditions using mutants defective in Mg2+ transporters, or in mutants lacking membrane biogenesis machinery components that impact Mg2+ homeostasis. We appreciate the significance of proper localization of transporters to their activity; therefore, we will apply the experience/tools/skills of our lab to study that aspect of magnesium homeostasis in S. mutans. Characterization of insertion pathways will involve construction and characterization of combinatorial mutants of membrane biogenesis components with Mg2+ transporters, and phenotypic analysis following that used for characterization of the transport mutants. Molecular cloning, reverse and forward genetics, bioinformatics, and biochemical approaches will be utilized to address the aims of this proposal. Ultimately a better understanding of Mg2+ homeostasis in S. mutans is expected to guide future development of novel anti‐caries strategies.