Molecular and Cellular Mechanisms of Bacterial Cell Wall Constriction and Chromosome Organization

Project Summary In this funding period, we will investigate the molecular and cellular mechanisms underlying the spatiotemporal organization and regulation of two essential bacterial cellular processes, cell wall constriction and chromosome organization, using E. coli as a model organism. Cell wall constriction is the concurrent new septal cell wall synthesis and old septal cell wall degradation, which leads to the formation of two new cell poles and the separation of two daughter cells. Chromosome organization is the three-dimensional arrangement of the chromosomal DNA, which impacts all aspects of genomic processes, including replication, repair, and transcription. Past studies have identified many key molecular players involved in these two processes. However, we still lack fundamental knowledge about how the two processes are spatiotemporally organized and regulated in living cells. We will address this knowledge gap by probing the molecular compositions, dynamics, functions, and regulations of key players of the two processes. We are uniquely positioned for these tasks because of our extensive expertise in single-molecule imaging and bacterial cell biology, which enables us to directly link a single protein molecule’s moving dynamics and localizations in live cells with its function and activity. For the cell wall constriction project, we will identify the molecular composition of the core septal cell wall synthase complex, molecular mechanisms leading to the highly processive septal cell wall synthesis of two complementary synthase systems, and the septal localization and dynamics of cell wall hydrolases. Our overall goal is to elucidate the spatiotemporal coordination between septal cell wall synthesis and hydrolysis, which must be balanced to ensure robust cell wall constriction without lesions during cell division. For the chromosome organization project, we will investigate the roles of a highly conserved nucleoid-associated protein HU, small noncoding RNAs, and DNA supercoiling in compacting the genome. Our overall goal is to elucidate the underlying principles of bacterial chromosome organization, which will impact our understanding of how genomic processes occur in a realistic cellular environment.