Role of lipopolysaccharide in assembly of Escherichia coli outer membrane proteins OmpA, OmpC, and OmpF

Abstract

Selection was performed for resistance to a phage, Ox2, specific for the Escherichia coli outer membrane protein OmpA, under conditions which excluded recovery of ompA mutants. All mutants analyzed produced normal quantities of OmpA, which was also normally assembled in the outer membrane. They had become essentially resistant to OmpC and OmpF-specific phages and synthesized these outer membrane porins at much reduced rates. The inhibition of synthesis acted at the level of translation. This was due to the presence of lipopolysaccharides (LPS) with defective core oligosaccharides. Cerulenin blocks fatty acid synthesis and therefore that of LPS. It also inhibits synthesis of OmpC and OmpF but not of OmpA (C. Bocquet-Pages, C. Lazdunski, and A. Lazdunski, Eur. J. Biochem. 118:105-111, 1981). In the presence of the antibiotic, OmpA synthesis and membrane incorporation remained unaffected at a time when OmpC and OmpF synthesis had almost ceased. The similarity of these results with those obtained with the mutants suggests that normal porin synthesis is not only interfered with by production of mutant LPS but also requires de novo synthesis of LPS. Since synthesis and assembly of OmpA into the outer membrane was not affected in the mutants or in the presence of cerulenin, association of this protein with LPS appears to occur with outer membrane-located LPS.It remains unknown how proteins of the outer membrane of gram-negative bacteria are sorted to this membrane. We are studying this question with the 325-residue OmpA protein (7) of Escherichia coli (18,19,25,26) and have asked whether non-ompA mutants exist which might no longer incorporate the OmpA protein, and possibly others, into the outer membrane. Toward this end we have performed selections for resistance to an OmpA-specific phage under conditions which exclude the appearance of mutations in the ompA gene. Paradoxically, almost all mutants recovered so far possessed an unaltered OmpA protein, present in the outer membrane, and greatly reduced quantities of the outer membrane porins OmpC and OmpF, which have nothing to do with infection by this phage. Here we show that these mutants produced defective lipopolysaccharides (LPS). These and some other results shed some light on the role of LPS in the membrane assembly of these three proteins. MATERIALS AND METHODSBacterial strains, phages, and growth conditions. The ompA+ derivative of strain UH203 (19) is lac supF recA proA (or proB) rpsL(F' lacIq lacZAMJS proAB+). Its secA derivative, strain UH204, has the same characteristics but, in addition, is leu:: TnJO and secA(Ts5l) (17,44). The construction of a non mutant (49) in strain UH203 ompA+ was started with a precursor of this strain, JC6650 pro his (15). Phage P1 was grown on strain P400 (which is his' non [57]) and used to transduce the former strain to his'; the non allele is cotransduced at a frequency of -30% and was identified by the inability of strains carrying it to give rise to mutants resistant to phage T7,(49). The his+ non strain was made recA by t...

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