The participation of the partitioning protein para in the coordination of the bacterial cell cycle in Caulobacter crescentus

Puentes-Rodriguez, Stephanie G.

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https://hdl.handle.net/2142/129258 Copy

Description

Title

The participation of the partitioning protein para in the coordination of the bacterial cell cycle in Caulobacter crescentus

Author(s)

Puentes-Rodriguez, Stephanie G.

Issue Date

2025-04-28

Director of Research (if dissertation) or Advisor (if thesis)

Mera, Paola E

Doctoral Committee Chair(s)

Mera, Paola E

Committee Member(s)

• Imlay, James A
• Kuzminov, Andrei
• Hatoum-Aslan, Asma

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

C. crescentus, ParA, partitioning, chromosome, DNA, cell cycle

Abstract

The maintenance of chromosome after the completion of each cell cycle is a process required for all life propagation. In the bacterium Caulobacter crescentus, chromosome replication initiates at the origin of replication ori, and segregation does not begin until the centromere-like region parS is replicated, coordinating the onset of both replication initiation and segregation. However, our understanding of this exquisite coordination remains limited. We use the model organism C. crescentus in this study due to its dimorphic life cycle that allows separation between its nascent and mature populations, which can then be utilized to study the initial stages of the cell cycle. In C. crescentus, the active segregation of the chromosome is mediated by the tripartite ParABS system. One of its components, the partitioning protein ParA, is an ATPase which forms dimers that bind DNA nonspecifically and are released from the chromosome upon ATP hydrolysis. In this bacterium, the segregation of parS involves steps such as: release from the polar anchoring protein PopZ, initial slow movement to mid-cell and then fast movement to the opposite cell pole through the partitioning protein ParA. In this thesis, we expand on roles for ParA beyond the active segregation of the chromosome. Our data revealed that disrupting the levels of ParA results in cells displaying multiple origins of replication (oriC) in a DnaA-ATP dependent manner. We found these aberrant rounds of replication are also observed when expressing ParA variants deficient in chromosome segregation. We also observed that ParA promoting the initiation of replication also affects cell length, albeit this connection suggests different ParA variants may impact these processes independently of each other. These data suggest that ParA’s ability to impact chromosome replication initiation is an indirect effect from altering the cell cycle, and the connection still needs to be elucidated. We have also observed that the separation of the ParB/parS complex from the cell pole is a highly regulated step that involves ParA’s competing attractions between PopZ and non-specific DNA binding. Interfering with this balance by favoring either PopZ interactions with ParA or DNA binding results in an inability of parS to separate from the cell pole and culminates in cell death. We showed that a ParA variant that localizes with PopZ blocks the ability to release the ParB/parS complex from the cell pole, resulting in cells with multiple ParB/parS loci trapped at the pole, and this accumulation of ParB/parS complexes is dependent on interactions between ParA and PopZ. Together, these data suggest that the initial steps in chromosome separation are tightly controlled. We have also investigated in more depth the interactions between ParA, the partitioning protein ParB, and PopZ to understand the behavior of these proteins that drive chromosome segregation and cell cycle progression. Using a previously established Escherichia coli system, we examined that the ParA gradient can be reconstituted in E. coli using a ParA-ATP monomer and a PopZ unable to interact with ParB, suggesting that ParA’s gradient localization depends on interactions with PopZ. These preliminary data were then observed in C. crescentus, using a strain with native ParA fluorescently tagged to visualize gradient formation, and noted that the ParA gradient may be triggered by overexpression of ParA. Lastly, we also observed that ParA variants that bind ParB or DNA stall the ParB/parS complexes in ΔpopZ cells, and this stalling results in cell death, highlighting the importance of the equilibrium of these proteins for cell cycle progression. Collectively, these preliminary data suggest that the interactions between ParA and PopZ drive ParA localization, although fundamental questions about ParA’s behavior in vivo require further study. Overall, our data provide new insights into the interconnected network required to progress the bacterial cell cycle. Our work reveals that ParA participates in processes upstream of centromere (parS) segregation, such as chromosome replication initiation and parS release from the polar anchoring. These data also shed light into the fundamental questions that remain to be answered about ParA’s behavior in vivo, such as its localization in C. crescentus, and the importance of furthering our understanding of universal processes in cell cycle progression.

Graduation Semester

2025-05

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/129258

Copyright and License Information

Copyright 2025 Stephanie Puentes-Rodriguez

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