Regulatory Pathways of the Osmotic Stress Response in Bacteria

University Of Delaware, Newark DE

Investigators

Abstract

Bacteria, such as Vibrio, are now being detected worldwide in places where they were never seen before. This is due to Vibrio's ability to cope with changes in water temperature and salinity. It is known that Vibrio are able to respond to different salt levels by altering the level of compatible solutes within the cell. They do this by synthesizing or transporting these solutes into the cell. But, the genetic and molecular basis for how they regulate these processes is unknown. This research tests the hypothesis that regulators which control gene expression in response to quorum sensing, are also involved in responses to changes in salt levels and temperature. Previously, quorum sensing regulators were thought to regulate gene expression only in response to the density of bacterial populations. The idea that quorum sensing regulators are involved in responding to changes in the environment is thus novel and exciting. Graduate students and undergraduates, including representatives recruited from under-represented groups in science, will be trained in molecular genetic techniques as part of this research. In particular, students in an upper level undergraduate class will identify genes involved in salt tolerance. Vibrio species play important roles in the carbon and nutrient cycles in marine and estuarine environments. Many Vibrio species are also serious pathogens of humans and marine animals. Therefore, understanding how bacteria, such as Vibrio, adapt to changes in the environment is critical to understanding their spread and potential impacts on human health and critical marine and estuarine ecosystems. Halophilic Vibrio species have the unusual ability to synthesize both ectoine (from aspartate) and glycine betaine (from choline) and contain multiple transporters for their transport into the cell. Quorum sensing is a mechanism of communication that bacteria employ using autoinducer signals to monitor and respond to changes in population density. Quorum sensing regulators control the expression of 100s of genes. Bioinformatics analysis identified quorum sensing master regulators OpaR and AphA binding sites in the regulatory region of ectoine and glycine betaine biosynthesis and transporter genes. One model for quorum sensing regulation of compatible solute biosynthesis and transport, is that, in order for halophiles to grow at to high population densities, they require high NaCl concentrations, which necessitates the accumulation of compatible solutes. OpaR, which is produced maximally at high population densities, regulates the biosynthesis and transport of compatible allowing cells to grow optimally. In the proposed work, bioinformatics, molecular genetics and biochemical approaches will be used to demonstrate that OpaR (LuxR homologue) and AphA can control the osmotic stress response in halophiles. We will determine the role of these regulators in the low salinity response by analyzing the salinity response transcriptome using RNA-seq. We will develop a course in experimental biology utilizing global insertional mutagenesis to uncover novel targets required for the salinity stress response. This will introduce more undergraduate students to scientific discovery, provide them with opportunities to develop technical skills, and ultimately make them more competitive and career ready.