Date of Award
Doctor of Philosophy in Biological and Environmental Sciences
Cell and Molecular Biology
Cell & Molecular Biology
Jodi L Camberg
Bacterial cell division and the decision to divide is a process that requires a wide variety of factors. In bacterial cells, such as the widely studied Escherichia coli, a large protein complex, termed the Z-ring, assembles at midcell to enable division. The Z-ring is subject to many regulators which govern the spatiotemporal positioning of the ring. The Z-ring is made up of the eukaryotic tubulin homolog FtsZ, which polymerizes to recruit division proteins to the midcell. FtsZ polymers are tethered to the cytoplasmic membrane by the actin homolog FtsA. Together, FtsZ and FtsA act as the first step in the pathway to recruit at least 30 cell division proteins to the septum, 12 of which are essential, to constrict one cell into two identical progeny cells. In the natural environment, cells are not continually dividing as they do under laboratory conditions; they encounter conditions of stress such as nutrient deprivation, pH fluctuation, and oxygen limitation, among others. Some cells can enter a non-replicative, or dormant state, which provides them with survival advantages in the face of environmental stresses and antibiotic treatment.
In Manuscript I, we review the roles that the bacterial cell division proteins FtsZ and FtsA play in bacterial fission. We discuss FtsZ-ring structure and function in the overall context of the bacterial divisome complex. We further highlight the mechanisms of modulating FtsZ abundance and polymerization state. Additionally, we review the importance of interactions of FtsA with late-stage cell division proteins for division. We then discuss how FtsA polymerization and interaction with adenosine triphosphate (ATP) impact the division process.
In Manuscript II, we investigate FtsA polymerization. We first show that FtsA containing a mutation near the bound magnesium in the active site is defective for in vivo function as well as ATP hydrolysis and polymerization on the lipid surface in vitro. Next, we show that purified FtsA lacking the C-terminal membrane targeting sequence (MTS) that anchors it to the cytoplasmic membrane [FtsA(DMTS)], polymerizes in solution in an ATP-dependent manner. We further demonstrate that unassembled FtsZ destabilizes FtsA(DMTS) polymers, and FtsZ polymers coassemble with FtsA(DMTS) filaments. These results lead us to a model in which FtsZ and FtsA regulate each other’s oligomeric state to facilitate division.
In Manuscript III, we further investigate how mutations in the FtsA active site impair function in vivo and disrupt ATP hydrolysis, lipid remodeling, and magnesium dependent phospholipid release. We find that purified FtsA(A188V), which is associated with temperature-sensitive growth in vivo, is significantly defective for robust ATP hydrolysis and lipid remodeling.
In Manuscript IV, we investigate cell division regulation in a strain of Escherichia coli associated with urinary tract infections (UTIs). This uropathogenic E. coli (UPEC) strain can enter a dormant, or quiescent, state where proliferation is halted. We discover that the cell division protein ZapE, and the transcriptional regulator IhfB are essential for maintenance of this quiescent state via a mini transposon genetic screen. Notably, we show that quiescent UPEC cells are tolerant to antibiotic treatment. We determine that the TCA cycle intermediate, succinyl-CoA, is limited during quiescence and that dormant UPEC has slowed overall metabolism. Lastly, we report that ZapE interacts with cell division proteins and metabolic enzyme components, indicating that it may act to facilitate communication between division and metabolism in UPEC, and this interaction is conserved in other E. coli strains, including strains that do not enter a quiescent state.
In conclusion, this work has provided valuable insight into the mechanism of bacterial cell division through FtsA polymerization and ATP interaction. Additionally, this work has furthered our knowledge pertaining to how the decision to divide may be regulated in the UPEC antibiotic tolerant dormant state.
Morrison, Josiah, "BIOCHEMICAL AND GENETIC MECHANISMS UNDERPINNING ESCHERICHIA COLI CELL CYCLE AND GROWTH TRANSITIONS" (2023). Open Access Dissertations. Paper 1525.