Stability, placement and interactions of the divisome components in nucleoid-deprived Escherichia coli cells

Resumen

Escherichia coli cell division is carried out by the formation of a septal ring-like complex placed at midcell, the divisome. Its correct placement at midcell is essential for the obtention of two identical viable daughter cells. Nucleoid Occlusion (NO) and MinCDE systems act on the placement of the proto-ring and specifically on the Z-ring. The proto-ring is the first subcomplex formed during the midcell assembly of the divisome, and it is made up of three essential proteins: FtsZ, FtsA and ZipA. Its assembly starts with the formation of an FtsZ-ring like structure (Z-ring), which is stabilized and tethered to the cytoplasmic membrane via ZipA and FtsA. During protoring assembly several modulatory proteins regulate the Z-ring formation, enhancing and stabilizing its formation (e.g. ZapA, ZapB, ZapC or ZapD) or inhibiting it (e.g. ClpX, MinC, SlmA or SulA). The work presented in this thesis is focused on the study of the proto-ring formation and placement using nucleoid free cells, also known as maxicells. In the section 1 of Results, the experimental procedure for the production of maxicells was optimized starting with the conditions given by Sancar et al., (1979). Next to the chromosome degradation, in section 2 a small inventory of the proto-ring and proto-ring associated proteins was described, remarking a significant decrease in the FtsZ levels due to the action of the protease complex ClpXP. The absence of a functional nucleoid occlusion (NO) system led to study the role of the septum site positioning system MinCDE, which maintains its protein levels and the oscillatory movement in maxicells. This role over the FtsZ placement was tested in growing cells and in maxicells using three FtsZ variants: FtsZ+ and two artificially anchored to the inner membrane by the carboxyl-terminal amphipathic membrane targeting sequence of MinD (MinDmts), which allows FtsZ binds to the membrane without the assistance of FtsA and ZipA. In section 3 the effect of ZipA and FtsA over the FtsZ stability was studied and described. A new role for ZipA was found, showing its ability to avoid the FtsZ degradation by the ClpXP protease complex. The globular domain (FZB) was identified as the only region of ZipA necessary for the interaction and protection of FtsZ. These results were not shown either by FtsA or FtsA*, which were also unable to interfere with the FtsZ protection carried out by ZipA. Section 4 describes the interaction between different cell division proteins, by Bimolecular Fluorescent Complementation (BiFC). Up to 35 different pair of proteins combinations were assayed in growing cells, involving proto-ring components (FtsZ, FtsA/FtsA* and ZipA) and FtsZ-effector proteins (ZapA, ZapB, MinC, SulA and SlmA), and identifying six examples of protein-protein interaction, i.e. ZipA-FtsZ, ZipA-ZapA, ZapB-FtsZ, ZapB-ZapA, ZapB-ZipA and ZapB-ZapB. Whereas in maxicells, which do not grow actively and the FtsZ levels are significantly decreased, the ZipA-ZapA or the ZipA-ZapB interaction did not take place; in growing cells under FtsZ depletion only ZipA-ZapB interaction was observed. Therefore we suggest that FtsZ bridges the interaction between ZipA and ZapA, and the ZipA-ZapB interaction does not depend on the FtsZ protein levels but most likely on an active growth of the cell.