Rod-shaped bacteria often divide with high precision at midcell to produce two equally sized daughter cells. The positioning of the division machinery in Escherichia coli and Bacillus subtilis is spatially regulated by two inhibitory systems, the nucleoid occlusion and the Min system. The current models suggest that the target of the inhibitory mechanism is the cytoskeletal element FtsZ and that the concerted action of nucleoid occlusion and Min are necessary for correct placement of the division machinery. However, recent advances show that at least the Min system also ensures that division occurs only once in a cell cycle and might act downstream of FtsZ assembly.
We have recently identified a new division site selection protein in B. subtilis, MinJ. MinJ serves as a bridge between DivIVA and MinD. Strikingly, we observed that MinJ localization is dynamic and one central function of the Min system is that it acts to prevent re-initiation of cytokinesis at the sites of division rather than simply preventing the formation of new septa close to the cell poles. MinJ interacts with several membrane integral division proteins and seems to couple the inhibitory effect of MinCD to the membrane-integral parts of the divisome.
Figure 1: Cell division in Bacillus subtilis is studied using light and electron microscopy. Several staining techniques and the use of fluorecently labeled proteins enable us to detect subcellular localization of proteins and compartment specific gene expression. (A) Cell division starts with polymerization of FtsZ (green) between the segregated nucleoids (blue). The cell membrane is stained with nile red (red). (B) Expression of GFP under control of a sF specific promoter within the prespore compartment. The prespore is formed by an asymetric cell division close to one pole of the cell. (C) Transmission EM image of a dividing B. subtilis cell. Note the inwardly growing cell wall, which is typical for Gram positive bacteria. (D) Sites of active peptidoglycan synthesis were stained with a fluorescent antibiotic. Dividing cells synthesize the majority of peptidoglycan at the division site.
Figure 2: Dynamic control of cytokinesis. (A) In E. coli an oscillating Min System spatially organizes divisome (red) placement. MinCD (blue) complexes are released from the membrane by MinE (green) triggered ATP hydrolysis (orange). (B) In B. subtilis MinCD is recruited to the cell poles and division sites by a DivIVA/MinJ (green) complex. Protein dynamics are indicated by arrows.
Biological membranes are the essential barrier that enables cellular life. One third of all proteins are membrane proteins and help to organize the transfer of information and material from the cytoplasm to the environment. Amazingly, the phospholipid bilayer is highly organized in space and time. Proteins and lipids are not uniformly distributed but often clustered into micro-compartments.
We are trying to understand how the cytoplasmic membrane of B. subtilis cells is organized and how proteins affect membrane organization and rearrangements. We focus our research on two proteins, the bacterial dynamin DynA and a bacterial flotillin YuaG. These proteins are amazing homologs of their eukaryotic counterparts and our research shows that DynA mediates membrane fusion and flotillin is involved in membrane organization. The latter is important to create membrane microdomains that are essential for correct functioning of membrane protein machineries such as the secretion system.
Figure 3: DynA is able to tether and fuse membranes. (A) Tethering of 0.2 mg mL-1 NBD-PE labeled E. coli liposomes in the presence of 2 µM DynA. (B) Aggregation of liposomes in the presence of 0.2 µM protein as measured by turbidity change at 350 nm. The inset shows nile red stained fusion products. Liposomes (400 nm) were fused and subsequently treated with proteinase K to remove the DynA coat. (C) Cartoon showing FRET based lipid mixing (FBLM) experiments to unravel membrane fusion. (D) Experimental results of a FBLM assay using NBD-PE / Rh-PE labeled donor liposomes and non-fluorescent acceptor liposomes in the presence of 0.2 µM DynA and the indicated MgSO4 concentrations.
Figure 4: Structural models of DynA D1 and D2 domain. Structural models of the DynA D1 (A) and D2 (B) domain were modeled using the I-Tasser platform (Roy et al., 2010). The structure of the N. punctiforme BDLP (2J68) has been used as template. Shown are aligned structures of DynA-D1 (red) and DynA-D2 (orange) with BDLP (cyan). Residues that were shown to be essential for lipid interactions of BDLP are indicated in yellow (Low et al., 2009). Similar residues in DynA that likely contribute to lipid-binding have been highlighted in blue. Note the absence of a paddle-like domain in the D2 domain of DynA.