Single-Molecule Studies of Bacterial Chromosome Organization and Segregation

Abstract

The bacterial chromosome is dramatically compacted into a subcellular structure called nucleoid. Simultaneously, it must be organized to allow the protein machinery for essential cellular processes to gain access to the DNA. Bacteria lack the histones that play a critical role in chromosome compaction in other kingdoms of life; instead they have evolved other mechanisms to compact, organize, and segregate their chromosomes. Particularly, a group of chromosomal architectural proteins called nucleoid-associated proteins (NAPs) and the highly conserved structural maintenance of chromosomes (SMC) condensin complex are two of the major players in bacterial chromosome condensation and organization. Emerging single-molecule techniques, which can detect transient changes in DNA structure with high spatial and temporal resolutions, have contributed significantly to our understanding of how NAPs and SMC family members condense DNA. However, most nanomanipulation methods only measure the change in extension of DNA as a proxy to investigate protein-DNA interactions; they are unable to directly measure protein binding on DNA without fluorescently labeling the protein. In this dissertation, I describe a new approach to simultaneously monitor the association of proteins with DNA and changes in DNA conformation, while bypassing the difficult procedure of labeling proteins. Our method is demonstrated to be a relatively simple yet highly quantitative assay to study any DNA-binding protein in general. The second part of this dissertation focuses on the study of ParB, which is part of the parABS partitioning system required in chromosome segregation for most bacteria. ParB binds specifically to the centromere DNA sequence parS and to adjacent non-specific DNA in a phenomenon called spreading. Previous studies have argued that ParB spreading requires cooperative interactions between ParB dimers including DNA bridging and possible nearest-neighbor interactions. A recent structure of a ParB homolog co-crystalized with parS revealed that ParB dimers form a higher order tetrameric complex. Using this structure as a guide, a series of proposed intermolecular interactions in the Bacillus subtilis ParB was ablated to investigate their effect on spreading. Our results, based on in vivo and in vitro characterizations, demonstrated that a network of both cis and trans interactions between ParB dimers is necessary for spreading.