DNA translocases and origin region segregation in B. subtilis
Specialized mechanisms involved in chromosome segregation, septum placement, and chromosome dimer resolution contribute to the maintenance of chromosome integrity throughout the cell cycle. The first part of this work focuses on the investigation of DNA translocases in the Gram positive model organ...
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|Summary:||Specialized mechanisms involved in chromosome segregation, septum placement, and chromosome dimer resolution contribute to the maintenance of chromosome integrity throughout the cell cycle. The first part of this work focuses on the investigation of DNA translocases in the Gram positive model organism Bacillus subtilis, which move the chromosomes away from the division plane by directed DNA transport. SpoIIIE is a membrane-integral translocase that also acts during sporulation, while SftA is associated with the division septum by an unknown mechanism and was reported to be a soluble protein in vitro. The solubility of SftA in vivo was proven with cell fractionation experiments, and the part of the protein that serves for septal targeting was determined through the localization of different truncations of fluorescently labeled SftA, which helped narrow down the stretch of amino acids responsible for targeting the protein to the septum. Expression in a Eukaryotic heterologous system revealed an interaction between SftA and FtsA, but not FtsZ. Further evidence was provided by single molecule tracking experiments whereby the fraction of soluble SftA molecules increased in an FtsA depletion background as compared to a wild type background. Concomitant investigation on the single molecule level of SpoIIIE, a membrane associated DNA translocase in B. subtilis, and PfkA, a soluble phosphofructokinase, revealed a different behavior of the two translocases (SftA and SpoIIIE): SftA has a septal bound fraction, and a small soluble fast moving fraction,comparable in diffusion coefficient to the exclusively soluble PfkA tracked under the same conditions. SpoIIIE is much slower in comparison, and even its “fast” moving fraction is much slower than that of SftA. It seems to move slowly along the membrane with no specific enrichment at the septum, even after Mitomycin C (MMC) treatment. The second part of this work focused on the count of nucleoids, origin, and terminus of replication regions under conditions of fast and slow growth, with or without the induction of double strand breaks with MMC. B. subtilis seemed to be predominantly diploid, with a fraction of polyploid and monoploid cells which changed depending on the growth conditions. Replication of the origin proximal regions increased after DNA damage induction, as was shown by the increase in the number of origins during the time of DNA repair, while the number of termini remained constant. Time lapse experiments of the segregation of the tagged origin regions revealed that the movement is best described as directed diffusion, but seems to be quite robust and continues, though slower, after MMC treatment or treatment with Ciprofloxacin which blocks the topoisomerase IV|
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