| Microbial cell division typically occurs by the formation of a single septum that dissects the mother cell into two daughter cells. In contrast, the process of sporulation-specific cell division in streptomycetes involves the production of up to around 100 septa that are simultaneously formed in multinucleoid hyphae, requiring an unparallelled complex coordination of septum-site localization, peptidoglycan synthesis and DNA segregation. The septa are produced over the still "randomly" distributed chromosomes, which are then segregated, followed by septum closure. The main question I seek to answer is: how do streptomycetes time and coordinate the production of multiple septa and the concomitant segregation of all the chromosomes? We discovered that the SsgA-like proteins (SALPs, seven homologues (SsgA-G) in Streptomyces coelicolor), which are only found in sporulating actinomycetes, help orchestrate the amazingly complex process of developmental cell division in Streptomycetes. In a previous project we showed that the cell division activator SsgA acts through marking the cell wall at places where re-opening/restructuring of the peptidoglycan is required, such as during spore germination, tip growth, branching and septum formation, and that SsgB and SsgG localise at septum sites and are crucial for correct septum placement and synthesis. Besides ssgA, ssgB and ssgG, in a mutant screen we have just identified a cluster of five new genes that are involved in septum-site localization and again these are unique to spore-forming actinomycetes. Considering that the control proteins known from other eubacteria (Min, Noc, SulA, FtsA, ZipA) are absent and that all members of the Streptomyces septum localization 'complex' (designated SLC) identified so far occur exclusively in sporulating actinomycetes, we believe that the system that ensures the correct timing and localization of multiple cell division requires a large set of proteins specific for these complex microorganisms. The aim of this proposal is to establish the extent of the SLC, to provide insight into which proteins are involved, how they depend on each other and to provide a functional analysis of the individual components. This will enable us to paint a detailed picture of how actinomycete-specific proteins direct the localization of the universally conserved divisome during sporulation of streptomycetes. The five newly discovered slc genes together with the previously studied ssg genes form an ideal basis for this project. The specific questions we will address are: [1] Which genes are involved in localization of the sporulation septa? Comparative proteome and microarray analyses will be used to identify genes/proteins that are specifically expressed at the time coinciding with the onset of sporulation and mutants of the most promising genes will be made using the efficient transposon-based knockout-cosmid library available from Dr. Paul Dyson (Swansea, UK). These mutants will be analysed by advanced microscopy to identify true SLC-related genes. In addition a novel flow-cytometry-based method developed in our group will be used to compare the expression profiles of young and sporulating aerial hyphae (proteomes and if feasible microarrays), to obtain more refined expression data. [2] Is the SLC truly a complex and (how) do the proteins functionally depend on each other? Individual SLC proteins will be tagged using fluorescent translational fusions (e.g. to GFP and RFP) and their localizations studied. Their functional and spatial dependence on other SLC proteins will be studied by expressing fluorescently labelled SLC proteins in slc mutants (incl. in already available ssgABG single and double mutants) and study their localization, and interactions will be analysed in vivo using FRET-FLIM and in vitro by protein interaction studies. The scientific relevance of this project is two-fold. Firstly, analysis of the complex regulation of sporulation-specific cell division is of great interest for developmental microbiology. Secondly, we will further develop a novel approach towards compartment-specific genomics using flow cytometry (FACS cell sorter). 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. |
