Structural and molecular basis for bacterial chromosome organisation, segregation, and maintenance

Jalal, Adam (2021) Structural and molecular basis for bacterial chromosome organisation, segregation, and maintenance. Doctoral thesis, University of East Anglia.

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Abstract

Proper chromosome segregation is essential in all living organisms. In most bacteria, faithful chromosome segregation is mediated by the ParABS system, consisting of the ATPase protein ParA, the CTPase and DNA -binding protein ParB, and a centromere -like parS DNA. ParB nucleates on parS before binding to adjacent non-specific DNA (spreading) to form a multimeric nucleoprotein complex. ParA in turn powers the movement of the ParB-DNA nucleoprotein complex to each daughter cell. In Firmicutes, another ParB -like protein, Noc, nucleates onto NBS sites before spreading to form the membrane-associated nucleoprotein complex that physically occludes the assembly of the divisome. However, the molecular basis of ParB/Noc nucleation and spreading is unclear. Here, I elucidate the molecular basis for the DNA-binding specificity of ParB and Noc. I show that specificity is encoded by four residues at the protein-DNA interface, and a combination of permissive and specificity-switching mutations is required to reprogram their DNA-binding specificity. Next, I demonstrate that cytidine triphosphate (CTP) facilitates ParB escape from the parS site, enabling ParB to spread by sliding along the non-specific DNA. Using X-ray crystallography, I elucidate the structural basis for the transition of ParB from the nucleating state to the spreading state. Furthermore, I show that, C. crescentus ParB does not condense non-specific DNA in vitro in absence of CTP. Engineered C. crescentus ParB variants with enhanced DNA condensation activity failed to display any increase in spreading ability in vivo. Overall, I propose that ParB functions primarily as a CTP-dependent molecular switch. Finally, I report Noc as the first example of a CTPase enzyme whose membrane-binding activity is directly regulated by CTP. By in vitro reconstitution and X-ray crystallography, I show that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation. My findings suggest that CTP switches may be more widespread in biology than previously appreciated.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	18 Oct 2021 15:14
Last Modified:	01 Sep 2022 01:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/81753
DOI:

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