SummaryThe Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. Transmembrane domains of TolQ, TolR and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. TolB and the central domain of TolA interact in vitro with the outer membrane porins. In this study, both genetic and biochemical analyses were carried out to analyse the links between TolB, Pal and other components of the cell envelope. It was shown that TolB could be cross-linked in vivo with Pal, OmpA and Lpp, while Pal was associated with TolB and OmpA. The isolation of pal and tolB mutants disrupting some interactions between these proteins represents a first approach to characterizing the residues contributing to the interactions. We propose that TolB and Pal are part of a multiprotein complex that links the peptidoglycan to the outer membrane. The Tol-Pal proteins might form transenvelope complexes that bring the two membranes into close proximity and help some outer membrane components to reach their final destination.

The excC mutants of Escherichia coli are hypersensitive to drugs such as cholic acid and release periplasmic proteins into the extracellular medium. A 1884 bp fragment carrying the excC gene was isolated and sequenced. It contains the 3' end of the tolB gene which maps at min 17 on the E. coli map and an open reading frame which encodes the 18,748 Da ExcC protein. The protein is composed of a hydrophobic region of 22 residues and displayed an overall hydrophilic configuration. It was shown that the ExcC protein is indeed the PAL (peptidoglycan-associated lipoprotein) described by Mizuno (1979). The pal gene had not yet been characterized on the E. coli linkage map since no obvious phenotype could be identified for mutations in this gene. A topologic analysis of the PAL protein using PAL-PhoA translational fusions showed that PAL is associated with the outer membrane only by its N-terminal moiety. The carboxy-terminal part of the protein is necessary for correct interaction of PAL with the peptidoglycan layer.

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