Summary In E. coli and other bacteria, MinD, along with MinE and MinC, rapidly oscillates from one pole of the cell to the other controlling the correct placement of the division septum. MinD binds to the membrane through its amphipathic C-terminal α-helix. This binding, promoted by ATP-induced dimerization, may be further enhanced by a consequent attraction of acidic phospholipids and formation of a stable proteolipid domain. In the context of this hypothesis we studied changes in dynamics of a model membrane caused by MinD binding using membrane-embedded fluorescent probes as reporters. A remarkable increase in membrane viscosity and order upon MinD binding to acidic phospholipids was evident from the pyrene and DPH fluorescence changes. This viscosity increase is cooperative with regards to the concentration of MinD-ATP, but not of the ADP form, indicative of dimerization. Moreover, similar changes in the membrane dynamics were demonstrated in the native inverted cytoplasmic membranes of E. coli, with a different depth effect. The mobility of pyrene-labeled phosphatidylglycerol indicated formation of acidic phospholipid-enriched domains in a mixed acidic-zwitterionic membrane at specific MinD/phospholipid ratios. A comparison between MinD from E. coli and N. gonorrhea is also presented.

DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein’s domain III, rather than confined to a particular sequence. We propose a new binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication.

Summary MinD, a well-conserved bacterial amphitropic protein involved in spatial regulation of cell division, has a typical feature of reversible binding to the membrane. MinD shows a clear preference for acidic phospholipids organized into lipid domains in bacterial membrane. We have shown that binding of MinD may change the dynamics of model and native membranes (see accompanying paper [1]). On the other hand, MinD dimerization and anchoring could be enhanced on preexisting anionic phospholipid domains. We have tested MinD binding to model membranes in which acidic and zwitterionic phospholipids are either well-mixed or segregated to phase domains. The phase separation was achieved in binary mixtures of 1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol] (SOPG) with 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC) or 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG) and binding to these membranes was compared with that to a fluid mixture of SOPG with 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC). The results demonstrate that MinD binding to the membrane is enhanced by segregation of anionic phospholipids to fluid domains in a gel-phase environment and, moreover, the protein stabilizes such domains. This suggests that an uneven binding of MinD to the heterogeneous native membrane is possible, leading to formation of a lipid-specific distribution pattern of MinD and/or modulation of its temporal behavior.

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