During cell division, Gram-negative bacteria must coordinate inner membrane (IM) 4 invagination, peptidoglycan (PG) synthesis and cleavage and outer membrane (OM) 5 constriction. The OM constriction remains largely enigmatic and the nature of this 6 process, passive or active, is under debate. The proton-motive force (PMF) 7 dependent Tol-Pal system performs a network of interactions within these three 8 compartments. Here we confirm that the trans-envelope Tol-Pal complex 9 accumulates at constriction site in Escherichia coli. We show that the IM complex 10 composed of TolA, TolQ and TolR recruits the OM complex TolB-Pal to the septum, 11 in an energy-dependent process. Pal recruitment then allows its binding to PG and 12 subsequently, OM constriction. Our results provide evidence that the constriction of 13 the OM is an energized process. 14
Protein function is generally dependent on its subcellular localisation. In Gram-negative bacteria such as Escherichia coli, a protein can be targeted to five different compartments: the cytoplasm, the inner membrane, the periplasm, the outer membrane and the extracellular medium. Different approaches can be used to determine the protein localisation within a cell such as in silico identification of protein signal sequences and motifs, electron microscopy and immunogold labelling, optical fluorescence microscopy, and biochemical technics. In this chapter, we describe a simple and efficient method to isolate the different compartments of Escherichia coli by a fractionation method and to determine the presence of the protein of interest. For inner membrane proteins we propose a method to discriminate between integral and peripheral membrane proteins.
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