title: Molecular Mechanism of Chromosome Segregation by the E. coli Min system
creator: Klauss, Linda Elisabeth
subject: 570
subject: 570 Life sciences
description: Cell division is one of the most essential processes underlying life. Of importance is certainly the equal partitioning of the genetic material into the two daughter cells termed chromosome segregation. It is still debated if entropic forces alone are sufficient to fulfill this task, or whether additional dedicated protein machineries play an active role in this process.      The E. coli Min system, consisting of the proteins MinC, MinD and MinE, is well known for its function in defining mid-cell and directing there the FtsZ-ring, which marks the position of the future division site. The Min system is characterized by a pole-to-pole oscillation, corresponding to a time-averaged bipolar protein distribution. It has been discovered in our lab that MinD is able to directly bind DNA in vitro and in vivo. This led to the proposal of a Brownian Ratchet-like model for chromosome segregation, in which membrane-bound MinD provides DNA tethering sites and biases the diffusion of the duplicated chromosomes in the direction of the poles.  However, the molecular details of this mechanism were still lacking.     Here I used several in vitro and in vivo assays to understand better how MinD binds to the DNA and to clarify what the role of other proteins, such as MinC, MinE and FtsZ, is.  Specifically, I performed ChIP-Seq to study the genome-wide binding of MinD in cells with and without the endogenous Min system and found that MinD does not associate to specific chromosomal macrodomains. This supports the notion that the Min system assists the segregation of the chromosomal bulk. Furthermore, analysis of the transcriptome revealed that the Min system does not function as a global transcriptional regulator.     Using electrophoretic mobility shift assays (EMSAs) with purified proteins, I established that the MinC-MinD complex has a much higher affinity for DNA than the individual proteins. Lipid vesicles coated with MinC-MinD complexes could tether plasmid DNA to the lipid-associated pellet in co-sedimentation assays. These findings advocate for a refined model in which the “working unit” in chromosome segregation in vivo is not MinD alone, but rather the MinC-MinD complex. MinE is able to dissociate the DNA from the MinC-MinD complex, suggesting a role for this protein in the termination of the transient membrane tethering of the DNA by the Min system in vivo.     Using rational mutagenesis of MinC and MinD, I could pinpoint several residues that are important for the binding and discovered the critical role of the N-terminal domain of MinC.     To clarify whether the recently reported MinC-MinD co-polymers are needed for DNA-binding, I performed in vitro assays mixing wild type MinC with the MinCR133A mutant that allows complex formation between a MinD dimer and a MinC-MinCR133A heterodimer, but not the formation of the co-polymers. Interestingly, in this case, DNA-binding was reduced by 75%.     Finally, since in vivo the Min system interacts with FtsZ for its function in mid-cell placement, I studied whether FtsZ could interfere with DNA-binding by MinC-MinD using in vitro assays. I found that the binding is not affected unless FtsZ is present at very high concentrations. These data suggest that the presence of FtsZ in the cell, away from the Z-ring, does not interfere with DNA-binding by the MinC-MinD complex.     Taken together, the data I collected during my doctoral work contributed to our understanding of the molecular mechanism of DNA-binding by the Min system. More comprehensive studies in live cells are required to study this mechanism in vivo in more detail. Future work is needed to unambiguously determine the surface of the MinC-MinD complex used for direct DNA-binding.
date: 2018
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/23229/1/Linda%20Klauss_Molecular%20mechanism%20of%20chromosome%20segregation%20by%20the%20E.%20coli%20Min%20system.pdf
identifier: DOI:10.11588/heidok.00023229
identifier: urn:nbn:de:bsz:16-heidok-232299
identifier:   Klauss, Linda Elisabeth  (2018) Molecular Mechanism of Chromosome Segregation by the E. coli Min system.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/23229/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng