QUANTITATIVE IN VIVO IN VITRO STUDIES OF CELL PROCESSES

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DESCRIPTION(from applicant's abstract): In this project the principal investigator and colleagues propose to quantify: 1) the major changes in the types and amounts of cytoplasmic and periplasmic solutes and in amounts of cell and compartment water which allow E. coli to grow over a very wide range of external osmolalities; and 2) the accompanying large changes in turgor pressure across the cell wall. By comparison with in vitro effects of solutes, these large changes in concentrations of cytoplasmic solutes and biopolymers should individually perturb most cell processes, but collectively do not. Equally striking are the large changes in amount of cytoplasmic water, which measurements of turgor pressure show are not accompanied by large changes in water activity. The long term goals are: 1) to understand E. coli as a chemical and osmotic system; 2) to relate studies of solute-biopolymer interactions in vitro to solute effects on biopolymer processes in vitro and in vivo; and 3) to understand the global compensation mechanisms by which cell processes are buffered against changes in solute concentrations, and the molecular basis for the stimulatory effect on growth rate of metabolically-inert "osmoprotectant" solutes. The specific aims are: 1) to determine the physiological and biochemical responses of E. coli to the stress of high and low osmolality environments; 2) to quantify the interactions of E. coli osmolytes, other solutes and crowding agents with biopolymers in vitro in order to obtain structural predictions/interpretations of solute effects on biopolymer processes; and 3) to test by quantitative in vivo and in vitro studies the proposal that global compensation mechanisms involving balances between destabilizing effects of accumulated solutes, stabilizing effects of excluded solutes, and macromolecular crowding maintain biopolymer structure and function, allowing cell growth over a wide range of osmolalities at a rate which increases with the amount of free cytoplasmic water. The in vivo studies use standard analytical methods for solutes and a differential radioisotope assay for compartment water; the principal investigator proposes a quantitative application of gene array technology to analyze changes in amounts of individual mRNAs vs. growth osmolality. They use a novel application of osmometry for in vitro studies of solute-biopolymer interactions and use rapid-quench mixing and radioisotope and enzymatic assays to characterize solute effects on biopolymer processes. An understanding of the osmotic behavior of E. coli, the best characterized living system, is of practical as well as fundamental significance, both as a model for volume and osmotic regulation in kidney and many other eucaryotic cells and in combating both osmotically-induced virulence and tolerance of pathogenic strains of E. coli and Salmonella typhimurium to desiccation, heat, peroxide and urea.