Understanding the molecular determinants and regulation of toxin activity in bacteria

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Research on biofilms, bacterial communities that coat our teeth when we sleep, has been ongoing for decades. However, even today, the only certain way to remove biofilms is by mechanical force, i.e. a toothbrush. While brushing teeth is routine, removal of biofilms from ships, pipes, medical devices etc., is exceedingly difficult. Formation of biofilms is one of the major survival mechanisms utilized by bacteria, but knowledge of how it occurs is rudimentary. Bacterial persisters, a genetically identical sub-population of quiescent cells that express protein toxins and exhibit multidrug tolerance, are at the core of biofilm formation and in enabling bacteria adapt to changing environmental conditions. Gene pairs known as toxin-antitoxin (TA) systems have emerged as key components of this adaptation process. This project will determine how toxins block bacterial growth by discovering how they inactivate substrates, how they are inhibited by antitoxins and how bacteria remove toxins in order to exit persistence. In addition to helping discover novel approaches for controlling biofilms, the project will provide research training opportunities for students and STEM education opportunities for high school students through implementation of the Protein Science Workshop in the UA BIOTECH outreach project.Many TA toxins, including MqsR, the focus of this proposal, are ribonucleases (RNases). In order to control biofilm formation, an understanding of how RNase toxins regulate bacterial growth and arrest must be obtained. The research will determine how MqsR recognizes and cleaves mRNA substrates. Preliminary data suggest that this information will lead to a novel mechanism used by RNases to digest mRNA. The project will also identify how antitoxin MqsA inhibits MqsR activity, facilitating development of new approaches for toxin inhibition/modulation. Finally, the molecular determinants that direct MqsA and MqsR proteolysis and turnover in bacteria will be determined. These will define protein recognition sites that can be used to control TA turnover in cells, and provide new insights into the mechanisms used by bacterial proteases to recognize their endogenous substrates. An integrated structural, biochemical and cellular approach will be utilized to address these fundamental questions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.