Positive control of cell division: from new concepts to molecular detail

In bacteria that divide by binary fission, cell division starts with the polymerization of the tubulin homologue FtsZ at mid-cell to form a cell division scaffold (the Z-ring), which subsequently recruits the other components of the cell division machinery (divisome). The filamentous antibiotic-producing streptomycetes are ideally suited for the study of cell division, as it is the only organism where cell division is not essential for growth, and up to 100 septa are simultaneously formed during sporulation, requiring an unparallelled complex coordination of septum-site localization, peptidoglycan synthesis and DNA segregation. We discovered that the SsgA-like proteins (SALPs), which are only found in sporulating actinomycetes, help orchestrate developmental cell division in streptomycetes. The textbook view on the control of prokaryotic cell division starts from the principle of negative check points that prevent incorrect positioning of the Z-ring. However, in a project studying the septum-localization complex in Streptomyces, we surprisingly discovered that cell division is positively controlled in this organism, via the direct recruitment of FtsZ by the membrane-associated SsgB. SsgB is a component of the divisome, and hence - again in contrast to the canonical views - is the first example of a divisome component that localizes prior to FtsZ. In turn, the localization of SsgB is mediated through the orthologous SsgA. This system implies the evolution of an entirely new concept of Z-ring control. These new discoveries provide an ideal starting point. The aim of this proposal is to work out these novel concepts, by obtaining detailed insight into the way SsgB and FtsZ interact, and to unravel the upstream mechanisms that control the localization of SsgA and SsgB. An important question to address is, is the activation of cell division (SsgA>SsgB>FtsZ) governed by single proteins or multiprotein complexes? Our recent identification of a tripartite complex of SsgB, a membrane sensory kinase (SepK) and FtsZ suggests that a multicomponent complex may exist analogous to the γ-tubulin ring complex (γTuRC; among others part of the mitotic spindle) in eukaryotes, which ensures the initial recruitment of tubulin subunits and protects the microtubules from collapsing. The possible existence of such an FtsZ-filament recruiting complex (FIRC) will be addressed. The key objectives of this proposal are: [1] Uncovering the mechanisms that control the localization of SsgA, SsgB and FtsZ. SsgAB recruit FtsZ, but how are these proteins themselves localized? And are more proteins involved in the recruitment of FtsZ? Novel interaction partners for SsgA, SsgB and FtsZ will be identified using two-hybrid screening and pull-down assays, and studied by functional and mutational analysis. Localization and in vivo interactions between the partners will be imaged using advanced in vivo imaging techniques, including live imaging in aerial hyphae, bifluorescence complementation (BiFC) to analyse multiple protein interactions, and FRET-FLIM imaging to quantify protein distances and interaction strengths in vivo. To obtain detailed insight into the differences between mutants devoid of (developmental) cell division (ssgA, ssgB, ftsZ) and strains with hyper-division following overexpression of SsgA, transcriptome analysis and advanced proteomics will be used. Finally, SsgA and SsgB interfere with cell division in E. coli, and using two-hybrid screening we will identify conserved interaction partners in this unicellular organism. [2] Obtaining molecular and structural insight in the interaction between SsgB and FtsZ. We recently published the structure of SsgB and the structure of SsgA is underway (collaboration with the PSI:Biology consortium). Through this proposal the structure of the SsgB-FtsZ complex will be resolved. A mutant library will be created for SsgB (similar to the one we produced previously for SsgA), and the mutant variants will be screened for loss-of-function and in a two-hybrid system for changes in interaction with FtsZ (and other partners, such as SepK). The scientific relevance of this project is two-fold. Firstly, we will provide molecular insight into an entirely new concept of cell division control that is novel in comparison to what is known from any known living system. We will also establish how FtsZ filament assembly is initiated, and will trace parallels in unicellular bacteria. This will have major impact on developmental biology in the broadest sense. Secondly, several novel techniques will be introduced (BiFC) or developed futher (live imaging, FRET-FLIM) for developmental microbiology, which will help to further advance the field. The proposal connects very well to many other research programmes within Leiden University and in particular the new Cell Observatory, where developmental biology is a leading theme.