SpoIIIE/FtsK are membrane-anchored, ATP-fuelled, directional motors responsible for chromosomal segregation in bacteria. Directionality in these motors is governed by interactions between specialized sequence-recognition modules (SpoIIIE-c/ FtsK-c) and highly skewed chromosomal sequences (SRS/KOPS). Using a new combination of ensemble and single-molecule methods, we dissect the series of steps required for SRS localization and motor activation. First, we demonstrate that SpoIIIE/DNA association kinetics are sequence independent, with binding specificity being uniquely determined by dissociation. Next, we show by single-molecule and modelling methods that hexameric SpoIIIE binds DNA non-specifically and finds SRS by an ATP-independent target search mechanism, with ensuing oligomerization and binding of SpoIIIE-c to SRS triggering motor stimulation. Finally, we propose a new model that provides an entirely new interpretation of previous observations for the origin of SRS/KOPS-directed translocation by SpoIIIE/FtsK.

Milk sphingomyelin (MSM) and cholesterol segregate into domains in the outer bilayer membrane surrounding milk fat globules. To elucidate the morphology and mechanical properties of theses domains, supported lipid bilayers with controlled molar proportions of MSM, dioleoylphosphatidylcholine (DOPC) and cholesterol were produced in buffer mimicking conditions of the milk aqueous phase. Atomic force microscopy imaging showed that (i) for T < 35 °C MSM segregated in gel phase domains protruding above the fluid phase, (ii) the addition of 20 mol % cholesterol resulted in smaller and more elongated l(o) phase domains than in equimolar MSM/DOPC membranes, (iii) the MSM/cholesterol-enriched l(o) phase domains were less salient than the MSM gel phase domains. Force spectroscopy measurements furthermore showed that cholesterol reduced the resistance of MSM/DOPC membrane to perforation. The results are discussed with respect to the effect of cholesterol on the biophysical properties of lipid membranes. The combination of AFM imaging and force mapping provides unprecedented insight into the structural and mechanical properties of milk lipid membranes, and opens perspectives for investigation of the functional properties of MSM domains during milk fat processing or digestion.