Correct Sorting of Lipoproteins into the Inner and Outer Membranes of Pseudomonas aeruginosa by the Escherichia coli LolCDE Transport System.

Abstract

Biogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. In Enterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that in Pseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those from Escherichia coli The P. aeruginosa lipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either the E. coli or P. aeruginosa LolCDE. We further demonstrate that an inhibitor of E. coli LolCDE is active against P. aeruginosa only when expressing the E. coli orthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCE Gram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways of Escherichia coli and Pseudomonas aeruginosa are interchangeable, with the E. coli orthologues correctly sorting the P. aeruginosa lipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified in E. coli as aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.