This section has changed only slightly from the original proposal. The ubiquity of bacteria in nature attests to their tremendous adaptability. E. coli must routinely respond to external stress in the form of temperature change, nutrient deprivation, the presence of a drug, or a change in external solute concentration (osmotic stress). In this project we study protein transport (diffusion) and the organization of proteins, DNA, and ribosomes within the cytoplasm of live cells using fluorescence microscopy. To create a sudden osmotic stress, we subject E. coli cells to an increase in external salt concentration (plasmolysis). Alternatively, we can allow cells to gradually adapt to growth in high salt concentration. In the adapted cells, the nucleoid (chromosomal DNA) remains expanded, and diffusion of GFP remains facile. In the plasmolyzed cells, the nucleoid compacts (shrivels to a much smaller volume), and diffusion of GFP is severely hindered. The ability of proteins to diffuse through the plasmolyzed cytoplasmic space may determine the cell's ability to recover from osmotic shock and resume growth and division. Remarkably little is known about how to extend kinetics and thermodynamic results from dilute solutions to the crowded, complex environment of the cytoplasm. We hypothesize a two-domain model (nucleoids and cytoplasmic periphery) in which the spatial distribution of many globular proteins depends on the detailed structure of the nucleoid, especially on its porosity to proteins of different size and charge. The mean axial diffusion coefficient is then a weighted average over time spent within the nucleoid vs the periphery. We will measure the spatial distribution of nucleoids and ribosomes and the diffusion coefficient of a range of proteins in the cytoplasm of live E. coli both in normal growth and as a function of osmotic stress. This will greatly clarify the impact of macromolecular crowding and confinement on protein diffusion. A novel single-cell flow device will measure the time dependence of protein diffusivity and of nucleoid and cytoplasmic size and shape in the same cell, before and after plasmolysis. Calculations based on simple physical models and constrained by experimental measurements will provide a much better understanding of the segregation of ribosomes from the nucleoids and of the partitioning of globular proteins between the nucleoids and the peripheral cytoplasm based on size, charge, and DNA-binding propensity. In the longer term, our methods can be extended to the study of time-dependent drug effects on cytoplasmic organization and protein diffusion at a new level of detail.
Diffusion of proteins in the complex environment of the cytoplasm and the periplasm of E. coli is essential to their survival, growth, and recovery from stress. By applying state-of-the- art, quantitative imaging methods, we will directly observe how proteins move and how they are distributed in live cells, leading to a much better general understanding of many cellular processes. In the long term, the methods we develop are well suited to detailed, time-resolved studies of the effects of drugs or starvation on cytoplasmic structure and function.
| Bakshi, Somenath; Siryaporn, Albert; Goulian, Mark et al. (2012) Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells. Mol Microbiol 85:21-38 |
| Mondal, Jagannath; Bratton, Benjamin P; Li, Yijie et al. (2011) Entropy-based mechanism of ribosome-nucleoid segregation in E. coli cells. Biophys J 100:2605-13 |
| Sochacki, Kem A; Barns, Kenneth J; Bucki, Robert et al. (2011) Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37. Proc Natl Acad Sci U S A 108:E77-81 |
| Bakshi, Somenath; Bratton, Benjamin P; Weisshaar, James C (2011) Subdiffraction-limit study of Kaede diffusion and spatial distribution in live Escherichia coli. Biophys J 101:2535-44 |
| Bratton, Benjamin P; Mooney, Rachel A; Weisshaar, James C (2011) Spatial distribution and diffusive motion of RNA polymerase in live Escherichia coli. J Bacteriol 193:5138-46 |
| Sochacki, Kem A; Shkel, Irina A; Record, M Thomas et al. (2011) Protein diffusion in the periplasm of E. coli under osmotic stress. Biophys J 100:22-31 |
