Investigating the Role of Divisome Protein FtsK as an Essential Checkpoint During Bacterial Cell Division
Bacterial cell division is an essential and fundamental process required to sustain life. In Escherichia coli, division requires the recruitment and assembly of approximately thirty soluble and membrane-bound proteins, ten of which are essential (FtsZ, FtsA, ZipA, FtsK, FtsQ/B/L, FtsW/I, FtsN). Complete assembly of this macromolecular complex relies on formation of a dynamic ring-like structure known as the Z-ring. Following initial formation and stabilization of the Z-ring, cells must complete segregation of the bacterial chromosome and remodel the cell envelope to allow septum formation. One key modulator linking early (Z-ring) and late (cell envelope remodeling) division complexes is the essential protein FtsK. FtsK is a bifunctional transmembrane protein that coordinates chromosome segregation with its C-terminus (FtsKC) and cell division with its membrane-anchored N-terminal domain (FtsKN). Although the structure and function of FtsKN during division is unclear, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation can begin. In this capacity, we hypothesize that FtsK must modulate septum formation during division through the formation of dynamic and essential protein interactions with both the Z-ring and late stage division machinery. Drawing on advanced molecular techniques and imaging technologies, this thesis refines the membrane topology of FtsKN using site-directed fluorescence labeling, and elucidates several protein interaction partners that are critical for its role as an essential division checkpoint. Our revised topology revealed a novel functional periplasmic loop of FtsKN that, when mutated, produces cellular voids. We extensively characterized this novel cell division defect by fluorescence microscopy and high-resolution transmission electron microscopy, which exposed a novel role for FtsKN in linking cell envelope septation events. In addition, UV cross-linking and a genomic suppressor screen each uncovered potential interaction partners of FtsKN involved in both cell elongation and division. Two of these proteins, rare lipoprotein A (RlpA) and FtsA, were confirmed as direct FtsKN protein interactors by in vitro pull-down assays. Together, these findings provide critical evidence on how FtsKN may mediate the transition between cell elongation and septation in E. coli, and significantly advances our understanding of what is necessary for bacteria to replicate and survive.