Date of Award

2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

John Cox

Committee

Jason Rosch; Marko Radic; Michael Whitt; Scot Ouellette

Keywords

chlamydia, divisome, elongasome, FtsK, MreB, XerD

Abstract

In evolving to obligate intracellular dependence, Chlamydia trachomatis serovar L2 (Ct), has eliminated several gene products essential for cell division in most other bacteria, including FtsZ, the central coordinator of divisome assembly. In the absence of FtsZ, we show that divisome assembly in Ct is initiated by FtsK, a chromosomal translocase. Chlamydial FtsK forms discrete foci at the septum and at the base of the progenitor mother cell, and our data indicate that FtsK foci at the base of the mother cell mark the location of nascent divisome complexes that form at the site where a daughter cell will emerge in the next round of division. The divisome in Ct has a hybrid composition, containing elements of the divisome and elongasome from other bacteria, and FtsK is recruited to nascent divisomes prior to the other chlamydial divisome proteins assayed, including the PBP2 and PBP3 transpeptidases, and MreB and MreC. Knocking down FtsK prevents divisome assembly in Ct and inhibits cell division and septal peptidoglycan (PG) synthesis. We further show that MreB does not function like FtsZ and serves as a scaffold for the assembly of the Ct divisome. Rather, MreB is one of the last proteins recruited to the chlamydial divisome, and it is necessary for the formation of septal PG rings. Our recent studies have demonstrated the critical role of localized cardiolipin (CL) synthesis in coordinating cell division in Ct. CL is concentrated at the poles and septum, where it promotes the recruitment of MreB during polarized cell division. The lipid’s unique structure helps induce membrane curvature and facilitates divisome protein assembly, making it essential for proper spatial organization of septal PG synthesis and MreB localization. This localized CL synthesis is required for MreB to support the formation of septal PG rings. Our studies further illustrate the critical function of chlamydial FtsK in coordinating divisome assembly and PG synthesis in this obligate intracellular bacterial pathogen. However, the mechanism that specifies the site of divisome assembly in Ct is unclear. In E. coli, the transient interaction of FtsK with XerD stimulates XerCD dependent recombination at the chromosomal dif site to decatenate chromosomes that become interlinked by homologous recombination during DNA replication. We show here chlamydial FtsK colocalizes with XerD during cell division. In addition, CRISPRi which uses a crRNA to target an inducible and defective Cas12 enzyme (dCas12) to a specific chromosomal sequence, has shown that chlamydial FtsK also colocalizes with a chromosomal sequence immediately adjacent to the dif site during cell division. However, when CRISPRi was used to target dCas12 to the XerD binding sequence at the chlamydial dif site, FtsK did not colocalize with Cas12 and cell division was blocked. Our data indicate that the site of divisome assembly in Ct is dependent upon the interaction of FtsK with XerD bound to the chromosomal dif site.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0009-0007-4178-244X

DOI

10.21007/etd.cghs.2024.0683

Additional Files
2024-023-Harpring ETD Declaration of Authorship.pdf (377 kB)
Declaration of Authorship
Download

Share

COinS