Assembly of bacterial cytoplasmic protein complexes measured by LRET

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DESCRIPTION (provided by applicant): Bacterial cytoplasm contains remarkable ordered structures that are linked to pathogenicity and virulence. Actin-like filaments, made of the protei MreB, span the length of bacterial rods. Organelle-like bacterial microcompartments (BMCs), bounded by geometric protein shells, sequester volatile or toxic reaction intermediates. Cogwheel shaped tubules in many gram-negative pathogens help secrete toxins into targeted cells. Only limited information is available about the assembly and disassembly of these structures in vivo. Luminescence resonance energy transfer (LRET) will be used to test proposed models for actin-like filament assembly and bacterial microcompartment and tubule structure and dynamics. Recently it was discovered that Escherichia coli takes up luminescent terbium ions under conditions where these ions bind to genetically encoded sites without toxicity to the cells. The proposed experiments will exploit this finding to study molecular structures in live cells. MreB and the microcompartment shell proteins EutL and EutM will be tagged with tetraCys sequences, which will subsequently bind fluorescein arsenical to generate LRET acceptors. Terbium will be coordinated by a lanthanide-binding tag (LBT) on a cytoplasmic protein that will serve as the LRET donor. Because of the long excited state lifetimes of lanthanides, LRET is sensitive to the diffusion rates of donor and acceptor molecules. When the acceptors are released from ordered structures into the cytoplasm, a large increase in LRET should be detected, and when the acceptors are removed from the cytoplasm into ordered structures, a large decrease in LRET should be detected. This will provide spectroscopic signals of structure assembly and disassembly that can be followed during cell division and metabolic induction. Terbium uptake will also be measured in Salmonella enterica and Vibrio cholerae. If terbium uptake similar to E. coli is observed, studies will also be done on assembly of S. enterica Pdu microcompartments and V. cholerae type VI secretory system tubules. The results of these experiments will provide fundamental information about how bacterial actin-like filaments, microcompartments, and tubules are assembled in live cells. This information will aid future targeting of these structures to weaken the pathogenic effects of enterobacteria.