| Listeria monocytogenes is a gram-positive food-borne pathogen that is notably resistant to osmotic stress and can grow at refrigerated temperatures. The two most commonly used methods of food preservation, namely, salting and refrigeration, are ineffective in keeping Listeria from growing because the organism has transport systems that actively accumulate stress-protective solutes such as glycine betaine and carnitine. The focus of this research was to determine the mechanism of chill and osmotic stress resistance conferred by one of three major membrane-bound transport systems for compatible solutes in L. monocytogenes, glycine betaine porter II, or Gbu. To examine Gbu transport system, three Gbu proteins (GbuA, GbuB, and GbuC) were overexpressed then purified in E. coli and L. monocytogenes.;GbuC, the substrate binding protein, was found to be postranslationally processed to attach lipids at cysteine residue found in the N-terminal end of the protein. The composition of these lipids is temperature-dependent and varies according expression host strains. Mutation experiments in vivo, in which the cysteine residue was replaced by alanine to make such processing null, showed that the mutation had detrimental effect on both growth rate and transport rate of glycine betaine and carnitine in L. monocytogenes.;This research has shown that Gbu transport is coupled with ATP hydrolysis and the oligomeric state of ATPase subunit, GbuA. ATP caused the majority of GbuA to dimerize, whereas ADP caused GbuA to monomerize. This change in the oligomeric state of GbuA within the catalytic cycle represents a possible mechanism for the transfer of the chemical energy of ATP hydrolysis to mechanical energy involved in transport. |
