Assembly and regulation of the bacterial divisome - SUMMARY Bacteria use the cell division pathway for proliferation and colonization of environmental niches and in host organisms, where colonization is closely linked to virulence. Although division in bacteria is highly conserved and essential, we only have an elementary understanding of ‘how’ the process unfolds. A more detailed view would provide fundamental, molecular knowledge to improve the future design of novel antibiotics. In this study, we address several major knowledge gaps in division, including how complexes assemble at the division site, how information is transmitted to the periplasm to initiate cell wall synthesis, and how the dynamic interactions among division proteins are coordinately regulated. Early in division, a protein structure with ring-like architecture, called the Z-ring, assembles at midcell. This ring comprises the essential division proteins FtsZ and FtsA. FtsZ polymers coalesce at this site, tethered to the cytoplasmic membrane by a direct interaction with the membrane-associated actin homolog FtsA, thus forming the dynamic Z-ring. To understand ring formation, we will elucidate the molecular determinants that regulate co-assembly of FtsA-FtsZ copolymers at the membrane. We will determine how FtsA recruits FtsZ to phospholipids, identify parameters that regulate the interaction between FtsA and phospholipids, and investigate dynamic conformational transitions in FtsA that underpin the interactions. The mid-to-late stage division protein FtsN is recruited to FtsA at midcell, and recent models suggest that FtsN subsequently releases from FtsA to engage proteins in the periplasm and promote cell wall synthesis. To elucidate the details of ordered FtsN interactions during division, we will use a combination of biochemical and biophysical approaches and live cell direct imaging of FtsN wild type and mutant proteins. FtsN is a bitopic inner membrane protein, and we will also characterize the purified protein in vitro, assemble complexes to quantify binding to FtsA and peptidoglycan, and generate a map of the FtsA-FtsN binding site. Finally, in a new direction, we will evaluate crosstalk between FtsN, ZapE (‘FtsZ-interacting protein E’), and succinate dehydrogenase (SDH). FtsN and ZapE engage SdhC, a component of the tricarboxylic acid (TCA) cycle located at the membrane. We will identify which regions of ZapE and FtsN are important for binding to SdhC, perform colocalization experiments in live dividing cells to monitor septal recruitment of SdhC, and probe regulation of TCA cycle metabolism in vitro and in vivo. This work will reveal, for the first time in E. coli, the interplay of division proteins with the TCA cycle, a key pathway in central carbon metabolism. Together, these studies will advance our biochemical understanding of the early steps of the cell division pathway, develop new platforms to evaluate interactions, mutant proteins, and potential antibiotics in vitro, and elucidate the role of cell division proteins in regulating dicarboxylate metabolism, which is critical for antibiotic tolerance by pathogenic strains of E. coli.
