Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Prahathees Eswara, Ph.D.

Committee Member

Lindsey Shaw, Ph.D.

Committee Member

Wenqi Yu, Ph.D.

Committee Member

Yu Chen, Ph.D.

Keywords

Bacillus subtilis, Cell division, Staphylococcus aureus

Abstract

There are two big drivers motivating studies into bacterial cell division. The first is a desire to understand life around us. One of the defining characteristics of life is the ability for a cell to grow and divide, and without in-depth knowledge of this process, we cannot truly understand the complexities that allow for life on this planet. The second motivator is to identify new drug targets in the ongoing fight against antimicrobial resistance. Many infectious organisms have become resistant to commonly used antibiotics, and the bacterial cell division machinery is a largely untapped essential process with many potential therapeutic targets. Work carried out over the past several decades elucidated several complex processes that coordinate cell division. However, there are still large gaps in our understanding of these processes, especially in important human pathogens where many of the characterized systems are absent or function differently. Staphylococcus aureus is one bacterium where the cell division machinery functions differently than the functions characterized in the model organisms. To investigate novel aspects of S. aureus cell division, we chose to study an essential cell division protein, GpsB, which is conserved in bacteria of the Firmicutes phylum. GpsB in S. aureus was previously shown to uniquely interact with FtsZ, a tubulin homolog that marks the division site. In this work, we first describe a novel role for GpsB in cell envelope decoration that is distinct from its role in regulating FtsZ, by interacting with the wall teichoic acid export protein TarG. We also show that a 3-residue motif at the end of the N-terminal domain to be necessary for this interaction. Next, we investigate the molecular details that allow for FtsZ-GpsB interaction. Through solving the crystal structure of GpsB, we discover that there is a hinge formation in the N-terminal domain caused by the insertion of 3 residues that is not seen in other GpsB homologs. We further show the flexibility provided by the hinge region is important for the physiological function of GpsB. We elucidate that the end of the C-terminal region of S. aureus FtsZ contains the GpsB recognition motif and show that the residues in this motif are critical for the GpsB-FtsZ interaction. Additionally, we note that PBP4 also contains the GpsB recognition sequence and show PBP4 is an interaction partner of GpsB. Finally, we investigate the impact of Ser/Thr phosphorylation on GpsB activity. Previous work showed that GpsB is phosphorylated and identified phosphorylated residues. We examine the role of GpsB phosphorylation by using genetic mutations of stk and stp that encode a Ser/Thr kinase and phosphatase respectively. We also confirm the phenotypes using phosphomimetic and phosphoablative mutations of GpsB. We find that phosphorylation leads to inactivation of GpsB function and speculate reversible phosphorylation and dephosphorylation allows for finetuning GpsB activity during the cell cycle. As a whole, this research provides new insight into the molecular mechanisms governing the function of the S. aureus cell division protein GpsB and identifies GpsB as a potential antibiotic target in the continued fight against multi-drug resistant staphylococcal infections.

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