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Gram-negative bacteria are surrounded by a complex multilayered cell envelope, consisting of an inner and an outer membrane, and separated by the aqueous periplasm, which contains a thin peptidoglycan cell wall. These bacteria employ an arsenal of highly specialized membrane protein machineries to ensure the correct assembly and maintenance of the membranes forming the cell envelope. Here, we review the diverse protein systems, which perform these functions in Escherichia coli, such as the folding and insertion of membrane proteins, the