Mechanistic characterization of in vitro reconstituted bacterial cell division subcomplexes – MecaDiv

Bacterial cell division is a temporally and spatially regulated process coordinated by a multi-protein complex called the divisome. The assembly of the divisome is initiated and organized by a highly conserved bacterial protein, the bacterial tubulin homologue FtsZ, which polymerizes to form a dynamic ring structure (Z-ring) that marks the site of cell division. Following ring assembly, FtsZ recruits structural and accessory proteins in an ordered manner to form the functional cell division machinery and to build a new cell wall. The precise molecular mechanisms by which the assembly and regulation of the bacterial cell division machinery is achieved remains elusive, even in extensively studied model organisms such as Escherichia coli, Bacillus subtilis or Caulobacter crescentus. While genetic and biochemical techniques have identified many interactions amongst cell division proteins, the overall structure and dynamics of the divisome as a (large) multi-protein complex are still completely unknown. Furthermore, although the general scheme for divisome assembly and function seems to be widely conserved in bacteria, important species-specific differences exist – most likely to satisfy different cell morphologies, growth modes and cell wall composition. This is particularly true in Corynebacteriales, the suborder of Actinobacteria including important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae and Corynebacterium diphtheriae. Corynebacteriales are Gram +ve diderm bacteria with a complex cell wall and a polar elongation mode. In this large phylum, many of the well characterized divisome regulators are missing from the genomes.

This proposal aims to provide an in-depth understanding of the Corynebacteriales core divisome architecture and function, using Corynebacterium glutamicum as a model organism. The major objective is to understand how protein-protein interactions within the divisome link the intracellular status of the cell to the extracellular cell wall machinery, via transmembrane interactions during bacterial cell division. Such a goal raises two major challenges. First, several corynebacterial divisome components, which might be important to stabilize or render functional larger multi-protein complexes, are yet to be identified and functionally characterized. And second, not only in Corynebacteriales but also in well-studied bacteria such as E. coli or B. subtilis, we are still very far from a precise mechanistic understanding of divisome assembly and function, even though most core divisome members have been identified, general functions have been assigned and several individual X-ray structures (often truncated mutants lacking the transmembrane domains) are available. The main working hypothesis of this project is that we will require structural information of stable full-length multi-protein subcomplexes of the divisome to understand the detailed mechanisms by which the intricate network of protein-protein interactions is coupled to conformational changes and mechano-transductive transmembrane communication to regulate and activate the PG machinery at the division plane. We will use high-end MS interactomics methods to identify missing divisomal components, biophysical, biochemical and cell biological techniques to characterize them, and integrative structural biology approaches to determine high resolution structures of subcomplexes to understand their assembly mechanisms, enzymatic activities, and regulation. To overcome the challenges intrinsic to structure determination of potentially flexible transmembrane (multi-)protein complexes we will develop tools aiming at stabilizing favored conformations of these complexes in solution.