Modulation of Bacterial Cell Division by (p)ppGpp

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PROJECT SUMMARY/ABSTRACT In bacteria, cell size positively correlates with nutrient availability and negatively correlates with levels of the key nutritional signaling molecules pppGpp and ppGpp (abbreviated (p)ppGpp). (p)ppGpp is produced in response to environmental nutrient limitation and functions primarily to inhibit biosynthesis and slow growth. In the model organism Escherichia coli, (p)ppGpp modulates cell physiology at both the transcriptional and post- transcriptional levels through interactions with RNA polymerase (RNAP) and 56 additional cellular targets. However, the mechanism by which (p)ppGpp contributes to regulation of cell size is not fully understood. The balance between cell division and elongation is a major determinant of size in rod-shaped bacteria. Several pieces of evidence suggest that (p)ppGpp contributes to cell size in part by modulating the balance between these two processes. Increases in (p)ppGpp levels suppress the heat sensitivity of conditional cell division mutants and leads to resistance to mecillinam, an antibiotic targeting the elongation machinery (elongasome). These data suggest that (p)ppGpp positively affects activity of the cell division machinery (divisome). Strains lacking (p)ppGpp ((p)ppGpp0) are ~30% longer than wild-type cells and frequently filament. These phenotypes are not recapitulated in RNAP mutants defective for (p)ppGpp binding, suggesting that (p)ppGpp contributes to cell size through a post-transcriptional interaction with one of its other binding partners. I hypothesize that (p)ppGpp indirectly promotes divisome assembly and activation via interaction with its target proteins. To illuminate the molecular basis of (p)ppGpp mediated changes in divisome and elongasome activity, I propose two complementary aims. In Aim 1, I will characterize the effects of alterations in intracellular (p)ppGpp concentration on production, assembly, and activation of the cell division machinery. In Aim 2, I will screen for (p)ppGpp binding proteins that are required to increase cell length. I will then determine the effect of these proteins on the transcription, translation, assembly, and activity of divisome components (Sub-aim 2b) and, in Sub-aim 2c, determine the mechanism by which candidate proteins modulate cell division. The expected contribution of the proposed work is an enhanced understanding of the mechanisms by which (p)ppGpp modulates bacterial physiology. This contribution is significant because (p)ppGpp is a key component of environmental adaptation throughout the bacterial kingdom. This proposal will also enhance our understanding of (p)ppGpp’s role in intrinsic resistance to the clinically important β-lactam antibiotics, which target components of the divisome and elongasome. In addition, this F32 fellowship will provide me with opportunities to learn new techniques in microscopy and biochemistry, explore new conceptual avenues, and obtain additional professional training that will prepare me for a career as a professor and independent investigator.