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FtsZ dynamics and the regulation of division site selection by the MinCD division inhibitor in Bacillus subtilis

  • Author(s): Gregory, James Alan
  • et al.
Abstract

Bacillus subtilis and Escherichia coli regulate division site selection through two overlapping systems that together restrict FtsZ-ring assembly to midcell. The first system is nucleoid occlusion, which is mediated by the nonspecific DNA binding protein Noc and SlmA in B. subtilis and E. coli respectively. Nucleoid occlusion prevents assembly of the Z-ring across the nucleoid. The second system prevents division at the cell poles and is mediated by MinC and MinD (MinCD). In the absence of MinCD, division occurs at the cell poles, resulting in anucleate minicells in both E. coli and B. subtilis. The cellular localization of MinCD in E. coli and B. subtilis is differentially regulated by the unrelated proteins MinE and DivIVA respectively. B. subtilis DivIVA sequesters MinCD to the cell poles, where it interacts with FtsZ to prevent Z-ring assembly. E. coli MinE promotes the pole-to -pole oscillation of MinCD that results in a time averaged concentration of MinCD that is highest at the cell poles. The drastic difference between the localization of MinCD in E. coli and B. subtilis led me to reinvestigate the localization of B. subtilis MinCD using new methods. I constructed a transposon that I call TAGIT (Tn5 Assisted Gene Insertion Technology) to transcriptionally fuse gfp to minCD. TAGIT randomly inserts gfp into a target gene and allows for the rapid identification of in-frame insertions. I utilized TAGIT to construct a library of gfp -minCD insertions, from which I isolated a fully functional MinC-GFP that was subsequently integrated at the native chromosomal locus in B. subtilis in order to maintain wild type regulation of minCD. I then carried out time-lapse epifluorescence microscopy, TIRFM (Total Internal Reflection Fluorescence Microscopy), and SI (Structured Illumination) microscopy of MinC-GFP and FtsZ- GFP to study their cellular localization in growing cells. I propose a new model of MinC function; MinC prevents FtsZ structures that assemble immediately after cytokinesis at the new cell pole from supporting cell division. The highest concentration of MinC is found at midcell during the last stages of cytokinesis. MinC is then released from the division site thereby placing it in the same region of the cell at the same time that aberrant FtsZ structures assemble. There it disrupts the lateral interactions between FtsZ protofilaments, which promotes FtsZ to relocalize to the midcell, thus ensuring proper placement of the division site

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