Characterizing the Interactions Between Mg2+ and a Periplasmic Lipoprotein Involved in Mg2+ Homeostasis in Salmonella enterica

Abstract

The ability to acquire Mg2+ is needed for certain bacteria to cause disease due to the nutrient's importance in physiological processes. Pathogenic bacteria have proteins which help them to adapt to environments where Mg2+ is in low supply. We have identified a periplasmic lipoprotein found in Salmonella enterica, a bacteria that is a major contributor to food‐borne illness, that allows the pathogen to survive in low Mg2+ environments. The Salmonella lipoprotein of 19.7 kDa monomeric molecular weight consists of a signal peptide and a single domain of unknown function that is conserved among enterobacteria. The purpose of this research project is to determine if the protein binds Mg2+ and identify the amino acid residues that are required for the protein to function properly. We have developed a protocol to overproduce and purify the recombinant lipoprotein. Protein variants are created using site directed mutagenesis to replace residues thought to be critical for binding Mg2+. To test for whether a particular residue is required for the ability of this lipoprotein to promote growth in a low Mg2+ environment, growth of Salmonella expressing these protein variants in low Mg2+ is being assayed. This physiological assay will indicate which residues are required for the physiological function of this protein in Mg2+ homeostasis. A fluorescence assay that utilizes Mg2+ sensor 8‐hydroxyquinoline‐5‐sulfonic acid (HQS) has provided evidence that the lipoprotein is sequestering Mg2+ from solution. We observed a change in HQS fluorescence when the protein is present, indicating that HQS and the protein compete for Mg2+. The results from the HQS biochemical assay, combined with our site directed mutagenesis experiments, will provide a model for how the protein functions. The results of the project may allow for a further understanding of how Salmonella and other bacteria adapt to their environment and maintain virulence in harsh conditions.Support or Funding InformationThis research is supported by an UW‐La Crosse Undergraduate Research and Creativity Grant (TD) and a UW‐La Crosse Faculty Research Grant (JM).