Analysis of chromosome segregation and its coordination with cell division in alphaproteobacteria

Chromosome replication and segregation as well as their coordination are indispensable for the propagation of life. In bacteria, these processes are so far studied in morphologically simple and well-established model organisms. In this study, we shed light on chromosome segregation, cell division an...

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Bibliographic Details
Main Author: Pulpetta, Revathi Lakshmi
Contributors: Thanbichler, Martin (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:English
Published: Philipps-Universität Marburg 2023
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Summary:Chromosome replication and segregation as well as their coordination are indispensable for the propagation of life. In bacteria, these processes are so far studied in morphologically simple and well-established model organisms. In this study, we shed light on chromosome segregation, cell division and their coordination in alphaproteobacteria. We use two model organisms, the stalked budding bacterium Hyphomonas neptunium and the well-studied model organism Caulobacter crescentus. Both bacteria possess a unique cell cycle wherein flagellated swarmer cells that are replication incompetent develop into replication- competent stalked cells. In the case of the dumb-bell shaped species H. neptunium, new offspring is generated by budding at the distal end of its stalk, making it necessary to translocate a copy of its chromosome through the stalk to the future daughter cell. This happens in a unique two-step process reminiscent of eukaryotic chromosome segregation, wherein the first step, the segregation within the mother cell, occurs in a manner dependent on the ParABS DNA partitioning system, like in its close relative C. crescentus. The duplicated origin stays at the stalked pole and then later, in response to an unknown trigger, initiates the second step of segregation through the stalk. In this study, we systematically analyse the dynamics of chromosome replication of H. neptunium and its coordination with segregation through the stalk. We find out that the replication of more than half of the chromosome is finished before the second step of segregation is initiated. This study opens up several questions such as the reason behind the pause between the two segregation steps and the potential players and the mechanism involved in the coordination of these steps. H. neptunium possesses several ParA homologues. Since the DNA partitioning ATPase ParA and ParA-like proteins are known to be involved in chromosome segregation, we proceed to investigate the novel ParA-like protein HNE_0708 as a potential candidate involved in the coordination of DNA segregation. Interestingly, we find in this study that this homologue belongs to a separate sub-family of ParA-like proteins. Apart from that, this protein contains a TIR domain, which is known to be involved in protein-protein interaction in bacteria. Like ParA, this homologue shows ATPase activity and binds DNA non- specifically. Apart from that, the lack of this protein leads to a characteristic stalk-bulging phenotypic defect and functional defects in chromosome replication and segregation. Thus, HNE_0708 potentially co-ordinates cell morphogenesis with chromosome replication, segregation and potentially, cell division. As a prime example of a system coordinating cell division and chromosome segregation in alphaproteobacteria, we further study the well- established model species C. crescentus. The DNA partitioning protein ParB of C. crescentus interacts with canonical ParA to bring about chromosome segregation and it interacts with the ParA-like protein MipZ to bring about division site placement, thus coordinating chromosome segregation with cell division. The interaction between ParB and MipZ is crucial for the robust placement of the division site, as this interaction primarily stimulates the dimerisation of MipZ monomers, which effectively block the formation of the cytokinetic FtsZ ring in their vicinity. In this study, we systematically analyse this interaction by biochemical methods. We find out that the C-terminal domain of ParB dimers interacts with the C-terminal region of MipZ in both its monomeric and dimeric form. We also conclude that the C-terminal region of ParB is necessary and sufficient for its interaction with MipZ in vitro through a series of biochemical experiments, in which we tested this interaction by constructing chimeric ParB variants as well as using a C-terminal fragment of ParB to recruit MipZ in vitro. Together, these findings shed light on the interaction between the complex machinery bacteria employ to regulate and coordinate chromosome segregation and cell division.
DOI:10.17192/z2023.0088