Celik Gulsoy, I. | bioRxiv Science

bioRxiv2024

Divisome minimization shows that FtsZ and SepF can form an active Z-ring, and reveals BraB as a new cell division influencing protein in Bacillus subtilis

One of the aims of synthetic biology is the construction of a minimal cell. Since bacteria are the simplest life forms, they are the preferred blueprint for such a cell. In this study, we tried to find the minimal set of proteins required for division of a cell wall containing cell, using the bacterial model system Bacillus subtilis. Bacterial cytokinesis begins with polymerization of the tubulin homologue FtsZ at midcell into the Z-ring, which recruits the late cell division protein that synthesize the division septum. Assembly of FtsZ is carefully regulated, involving a dozen conserved cell division proteins. These proteins are not essential, but removing more than one is in many cases lethal. We made use of known suppressor mutations to find a gene deletion route that eventually enabled us to remove eight conserved cell division proteins: ZapA, MinC, MinJ, UgtP, ClpX, Noc, EzrA and FtsA. Only FtsZ and its membrane anchor SepF appeared to be required for Z-ring formation. Interestingly, SepF is also the FtsZ anchor in archaea, and both proteins may date back to the Last Universal Common Ancestor (LUCA). Curiously, viability was not greatly affected by the multiple deletions, although the frequency of cell division was considerably reduced. However, genome sequencing exposed the accumulation of several suppressor mutations, and revealed an unexpected cell division regulation function for the branched chain amino acid transporter BraB. The implications of these findings for the role of SepF in cell division, and the construction of a minimal cell division machinery are discussed.

microbiology↗

bioRxiv2019

Control of septum thickness by a large protein ring

Gram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored to the cell membrane by two proteins, FtsA and SepF. We have isolated SepF homologues from different bacterial species and found that they all polymerize into large protein rings with diameters varying from 19 to 41 nm. Importantly, these values correlated well with the thickness of their septa. To test whether ring diameter determines septal thickness, we tried to construct different SepF chimeras with the purpose to manipulate the diameter of the SepF protein ring. This was indeed possible and confirmed that the conserved core domain of SepF determines ring diameter. Importantly, when SepF chimeras with a smaller diameter were expressed in the bacterial host Bacillus subtilis, the thickness of its septa also became smaller. These results strongly support a model in which septal thickness is controlled by curved molecular clamps formed by SepF polymers attached to the leading edge of nascent septa. This also implies that the intrinsic shape of a protein polymer can function as a mould to shape the cell wall. Significance StatementMany bacteria form a thick cell wall and divide by forming a cross wall. How they control the thickness of their cell wall and cross wall is unknown. In this study we show that in these bacteria the cell division protein SepF forms very large protein rings with diameters that correspond to the diameter of their cross walls. Importantly, when we reduced the diameter of SepF rings in the bacterial host Bacillus subtilis the cross wall also became thinner. These results provide strong evidence that a large protein ring can function as a mould to control the thickness of the cell wall that divides these bacterial cells.

microbiology↗