LIPOPOLYSACCHARIDES AND DIVALENT-CATIONS ARE INVOLVED IN THE FORMATION OF AN ASSEMBLY-COMPETENT INTERMEDIATE OF OUTER-MEMBRANE PROTEIN PHOEOF ESCHERICHIA-COLI

To identify the requirements for the biogenesis of outer-membrane prot eins in Gram-negative bacteria, the sorting and assembly of the trimer ic, pore-forming protein PhoE was studied in vitro. Purified lipopolys accharide (LPS) in combination with low amounts of Triton X-100 and di valent cations induced the formation of folded monomers. LPS of deep-r ough strains was far less efficient in the formation of folded monomer s than wild-type LPS was, These folded monomers could be converted int o heat-stable trimers upon addition of outer membranes and higher amou nts of Triton X-100. Trimerization could precede the insertion step. T hese in vitro data suggest that the assembly in vivo proceeds sequenti ally by (i) formation of a folded monomer by interaction with LPS; (ii ) sorting of the folded monomers to assembly sites in the outer membra ne; (iii) trimerization; and (iv) insertion.